EP4633656A1 - Geklammerte peptide und verfahren dafür - Google Patents

Geklammerte peptide und verfahren dafür

Info

Publication number: EP4633656A1
Authority: EP; European Patent Office
Prior art keywords: composition; agent; independently; group; amino acid
Prior art date: 2022-12-15
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Pending

Application number

EP23904731.9A

Other languages

English (en)

French (fr)

Inventor

Jonathan Barry Hurov

Yaguang Si

Brian Halbert WHITE

Daniel Seungduk LA

Lorenzo Josue Alfaro-Lopez

Paula Cristina Ortet

Sarah Isabelle CAPPUCCI

Zhi Li

John Hanney Mcgee

Martin Robert TREMBLAY

Gregory L. Verdine

Kevin LING

Peicheng Du

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Parabilis Medicines Inc

Original Assignee

Parabilis Medicines Inc

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2022-12-15

Filing date

2023-12-15

Publication date

2025-10-22

2023-12-15 Application filed by Parabilis Medicines Inc filed Critical Parabilis Medicines Inc

2025-10-22 Publication of EP4633656A1 publication Critical patent/EP4633656A1/de

Status Pending legal-status Critical Current

Links

Classifications

- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution

Definitions

Stapled peptides are useful for various applications. For example, as biologically active agents, they can be utilized to modulate various biological functions.
the present disclosure provides powerful technologies (e.g., agents (e.g., those that are or comprise peptides, in many embodiments, stapled peptides), compositions, methods, etc.) for modulating various biological functions.
provided technologies are useful for treating various conditions, disorders or diseases including cancer.
provided technologies are useful for treating colorectal cancer.
provided technologies are useful for treating cancer expressing high levels of Myc.
provided technologies reduce Myc expression levels for extended periods of time as described herein.
the present disclosure provides agents, e.g., stapled peptides that comprise multiple staples. In some embodiments, the present disclosure provides agents, e.g., stapled peptides that comprise three or more staples. In some embodiments, the present disclosure provides agents, e.g., stapled peptides that comprise three or more staples within 10-20 amino acid residues, e.g., 10-15, 11-15, 11-14, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acid residues. In some embodiments, the present disclosure provides agents, e.g., stapled peptides that comprise three or more staples within 11 consecutive amino acid residues.
the present disclosure provides agents, e.g., stapled peptides that comprise three or more staples within 14 consecutive amino acid residues. In some embodiments, within such numbers of amino acid residues there are three staples. In some embodiments, within such numbers of consecutive amino acid residues there are four staples.
provided agents, e.g., stapled peptides have increased rigidity than reference peptides (e.g., unstapled peptides, or stapled peptides having fewer staples (in some embodiments, fewer staples within certain numbers of amino acid residues as described herein), etc.).
provided agents e.g., stapled peptides demonstrate various desired properties and/or activities.
provided agents, e.g., stapled peptides provide improved desired properties and/or activities than reference peptides (e.g., unstapled peptides, or stapled peptides having fewer staples (in some embodiments, fewer staples within certain numbers of amino acid residues as described herein), etc.).
provided technologies comprise designed structural features, e.g., novel amino acid residues, that can provide significantly improved properties and/or activities compared to comparable reference technologies that do not contain such designed structural features.
the present disclosure provides designed amino acids as described herein, whose incorporation into peptide agents, including stapled peptides, can provide significantly improved properties and/or activities such as improved lipophilicity and/or delivery into cells compared to reference amino acids (e.g., Asp).
the present disclosure provides technologies including peptides comprising such designed amino acid residues.
the present disclosure provides stapled peptides comprise such designed amino acid residues. [0006]
the present disclosure provides technologies for modulating one or more functions of beta-catenin.
the present disclosure provides various agents, e.g., peptides, in many instances stapled peptides, that can bind to beta-catenin and modulate its functions. As demonstrated herein, in some embodiments, the present disclosure binds agents that can interact with beta-catenin at a unique set of residues. In some embodiments, a binding site comprises one or more or all of the set of residues.
provided agents interact with one or more of a set of residues that are or correspond to the following residues of SEQ ID NO: 1: A305, Y306, G307, N308, Q309, K312, R342, K345, V346, V349, Q375, R376, Q379, N380, L382, W383, R386, N387, D413, N415, V416, T418, and C419.
provided agents interact with one or more of amino acid residue that are or correspond to A305, Y306, G307, N308, Q309, K312, R342, K345, V346, V349, Q375, Q379, N380, L382, W383, R386, N387, D413, N415, V416, T418, and C419 of SEQ ID NO: 1.
provided agents interact with one or more of amino acid residues that are or correspond to A305, Y306, G307, N308, Q309, K312, K345, V346, V349, Q379, N380, L382, W383, R386, N387, D413, N415, V416, T418, and C419 of SEQ ID NO: 1.
provided agents interact with one or more of amino acid residues that are or correspond to G307, K312, K345, W383, N387, D413, and N415 of SEQ ID NO: 1.
provided agents interact with one or more of amino acid residues that are or correspond to K312, K345, R386 and W383 of SEQ ID NO: 1.
provided agents interact with one or more of a set of residues that are or correspond to the following residues of SEQ ID NO: 1: G307, K312, K345, Q379, L382, W383, N387, N415, and V416. In some embodiments, provided agents interact with all of a set of residues that are or correspond to the following residues of SEQ ID NO: 1: Y306, G307, K312, K345, Q379, L382, W383, N387, N415, and V416.
provided agents interact with all of a set of residues that are or correspond to the following residues of SEQ ID NO: 1: G307, K312, K345, Q379, L382, W383, N387, N415, and V416. In some embodiments, provided agents interact with all of a set of residues that are or correspond to the following residues of SEQ ID NO: 1: Y306, G307, K312, K345, Q379, L382, W383, N387, N415, and V416. In some embodiments, provided agents interact with one or more of amino acid residues that are or correspond to K312, K345 and W383 of SEQ ID NO: 1.
provided agents interact with the amino acid residues that are or correspond to K312, K345 and W383 of SEQ ID NO: 1.
provided technologies can modulate one or more biological processes associated with beta-catenin.
provided agents e.g., stapled peptides, compete with a ligand (e.g., with a member of the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors) for binding to beta-catenin.
TCF/LEF T cell factor/lymphoid enhancer factor
provided agents compete with a ligand for binding to beta-catenin at a particular binding site (e.g., with a member of the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors at the TCF site on beta-catenin).
TCF/LEF T cell factor/lymphoid enhancer factor
provided technologies compete with TCF for interactions with beta-catenin.
binding of provided agents to a beta-catenin site decreases, suppresses and/or blocks binding to beta-catenin by another binding partner (e.g., a kinase).
binding of provided agents blocks binding of beta-catenin by a TCF/LEF family member.
the present disclosure provides agents that can bind to a site of beta-catenin selectively over one of more other binding sites by other ligands (e.g., peptides, proteins, etc.; in some embodiments, a ligand is Axin; in some embodiments, a ligand is Bcl9).
ligands e.g., peptides, proteins, etc.; in some embodiments, a ligand is Axin; in some embodiments, a ligand is Bcl9.
provided technologies modulate one or more beta-catenin functions associated with its interactions with TCF.
provided technologies selectively modulate beta-catenin functions, e.g., functions associated with TCF interactions.
provided technologies selectively modulate beta-catenin functions and do not significantly impact functions that are not associated with beta-catenin (e.g., various functions and/or processes in the Wnt pathway that are not associated with beta-catenin).
provided technologies are useful for inhibiting beta- catenin functions.
provided technologies are usefully for promoting and/or enhancing immune activities, e.g., anti-tumor adaptive immunity. [0008] In some embodiments, provided technologies are useful for preventing or treating various conditions, disorders or diseases including cancer.
the present disclosure provides methods for treating or preventing a condition, disorder or disease associated with beta-catenin, comprising administering to a subject suffered therefrom or susceptible thereto an effective amount of a provided agent or a pharmaceutically acceptable salt thereof.
a condition, disorder or disease is associated with beta-catenin’s interactions with TCF.
a condition, disorder or disease is colorectal cancer.
a subject has one or more mutations, e.g., as described in a model herein.
a cancer comprises overexpression of Myc.
an agent e.g., a staple peptide, is administered as a pharmaceutical composition.
the present disclosure provides pharmaceutical compositions which comprise or deliver a provided agent or a pharmaceutically acceptable salt thereof.
a pharmaceutical composition further comprises a lipid.
a suitable lipid can promote delivery/activities.
an agent is or comprises a peptide.
an agent is or comprises a stapled peptides.
provided agents that can bind beta-catenin comprise one or more designed amino acid residues.
the present disclosure provides a pharmaceutical composition comprising an effective amount of an agent, e.g., a stapled peptide, wherein when the composition is administered to a model as described herein, a result as described herein (e.g., reduction of tumor size, inhibition of tumor growth, etc.) is observed.
the present disclosure provides a pharmaceutical composition comprising an effective amount of an agent, e.g., a stapled peptide, wherein when the composition is administered to a system or a subject, Myc expression is reduced as described herein.
provided technologies e.g., agents, compositions, etc., are capable of reducing level of a Myc transcript.
provided technologies are capable of reducing level of a Myc polypeptide. In some embodiments, provided technologies are capable of reducing level of a c-Myc transcript. In some embodiments, provided technologies are capable of reducing level of a c-Myc polypeptide. In some embodiments, provided technologies are capable of reducing cell proliferation, e.g., proliferation of colorectal cancer cells. In some embodiments, provided technologies are capable of reducing tumor growth, e.g., of colorectal tumors, in subjects, PDX models, etc. In some embodiments, provided technologies are capable of shrinking tumor sizes, e.g., of colorectal tumors, in subjects, PDX models, etc.
cancer cells and tumors comprise mutations as described herein, e.g., APC mutations in colorectal cancer cells and tumors.
cancer cells and tumors contain high levels of Myc expression.
cancer cells and tumors contain high levels of c-Myc transcripts and/or polypeptides.
the present disclosure provides agents that bind to a polypeptide comprising or consisting of SEQ ID NO: 1 (Uniprot ID P35222), or residues 250-450 of SEQ ID NO: 1, or residues 305-419 of SEQ ID NO: 1: Uniprot No.
provided agents specifically interact with one or more residues which are or correspond to residues 305-419 of SEQ ID NO: 1.
provided agents bind to a motif (e.g., a portion of a polypeptide, a domain of a polypeptide, etc.) that comprise one or more residues corresponding to Ala305, Tyr306, Gly307, Asn 308, Gln309, Lys312, Arg342, Lys345, Val346, Val349, Gln375, Arg376, Gln379, Asn380, Leu382, Trp383, Arg386, Asn387, Asp413, Asn415, Val416, Thr418, and Cys419 of SEQ ID NO: 1.
provided agents bind to a motif (e.g., a portion of a polypeptide, a domain of a polypeptide, etc.) that comprise one or more residues corresponding to Ala305, Tyr306, Gly307, Asn 308, Gln309, Lys312, Lys345, Val346, Val349, Gln375, Arg376, Gln379, Asn380, Leu382, Trp383, Arg386, Asn387, Asp413, Asn415, Val416, Thr418, and Cys419 of SEQ ID NO: 1.
a motif e.g., a portion of a polypeptide, a domain of a polypeptide, etc.
an agent binds to a motif comprising one or more of the following residues within SEQ ID NO: 1: Ala305, Tyr306, Gly307, Asn 308, Gln309, Lys312, Arg342, Lys345, Val346, Val349, Gln375, Arg376, Gln379, Asn380, Leu382, Trp383, Arg386, Asn387, Asp413, Asn415, Val416, Thr418, and Cys419.
an agent binds to a motif comprising one or more of the following residues within SEQ ID NO: 1: Ala305, Tyr306, Gly307, Asn 308, Gln309, Lys312, Lys345, Val346, Val349, Gln375, Arg376, Gln379, Asn380, Leu382, Trp383, Arg386, Asn387, Asp413, Asn415, Val416, Thr418, and Cys419.
an agent binds to a motif comprising one or more of the following residues within SEQ ID NO: 1: Ala305, Tyr306, Gly307, Asn 308, Gln309, Lys312, Arg342, Lys345, Val346, Val349, Gln 375, Gln379, Asn380, Leu382, Trp383, Arg386, Asn387, Asp413, Asn415, Val416, Thr418, and Cys419.
an agent binds to a motif comprising one or more of the following residues within SEQ ID NO: 1: Ala305, Tyr306, Gly307, Asn 308, Gln309, Lys312, Lys345, Val346, Val349, Gln379, Asn380, Leu382, Trp383, Arg386, Asn387, Asp413, Asn415, Val416, Thr418, and Cys419.
provided technologies bind to a motif comprising at least 2, 3, 4, 5, or 6 of G307, K312, K345, W383, N387, and N415.
provided technologies bind to a motif comprising at least 2, 3, 4, 5, 6, or 7 of G307, K312, K345, W383, N387, D413, and N415.
provided agents specifically bind to such motifs.
a motif may be referred to as a binding site.
provided technologies selectively bind to such a binding site over an Axin binding site.
provided technologies selectively bind to such a binding site over a Bcl9 binding site.
provided technologies selectively bind to such a binding site over an ICAT binding site.
provided technology binds to such a binding site in a reverse N to C direction compared to TCF.
provided technologies do not bind to Axin binding site of beta- catenin. In some embodiments, provided technologies do not bind to Bcl9 binding site of beta-catenin. In some embodiments, provided technologies do not bind to ICAT binding site of beta-catenin.
Various technologies e.g., crystallography, NMR, biochemical assays, etc., may be utilized to assess interactions with beta-catenin in accordance with the present disclosure.
the provided technology provides an agent, e.g., a stapled peptide, that comprises multiple, e.g., two, three, or more, staples within 10-20, 10-15, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids residues.
a first staple in an agent e.g., a staple peptide
a staple peptide are bonded to amino acid residues at positions i and i+3.
i is an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, i is 1.
a fourth staple in an agent, e.g., a stapled peptide.
an agent e.g., a stapled peptide.
Such a staple may be referred to as a (i, i+3) staple.
an agent e.g., a stapled peptide, comprises a (i, i+2) staple and a (i, i+7) staple.
an agent e.g., a stapled peptide, comprises a (i, i+3) staple and a (i, i+7) staple.
a (i, i+3) staple and (i, i+7) staple are bonded to the same amino acid residue.
a (i, i+3) staple and (i, i+7) staple bond to the same atom.
a (i, i+3) staple and (i, i+7) staple bond to the same alpha carbon atom.
an agent further comprises a third staple.
a third staple is (i, i+4).
a third staple is (i, i+7).
a third staple is not bonded to any of the amino acid residues that are bonded to the first two staples.
an agent further comprises a fourth staple.
a fourth staple is (i, i+4).
a fourth staple is (i, i+7). In some embodiments, a fourth staple is not bonded to any of the amino acid residues that are bonded to the first two staples. In some embodiments, a fourth staple is not bonded to any of the amino acid residues that are bonded to the first third staples.
a provided agent e.g., a peptide agent such as a stapled peptide agent, comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following groups (in some embodiments, from the N to C direction): a first acidic group (e.g., of a first acidic amino acid residue); a second acidic group (e.g., of a second acidic amino acid residue); optionally a third acidic group (e.g., of a third acidic amino acid residue); optionally a hydrophobic group (e.g., of a hydrophobic amino acid residue) a first aromatic group (e.g., of a first aromatic amino acid residue); a second aromatic group (e.g., of a first aromatic amino acid residue); and a third aromatic group (e.g., of a third aromatic amino acid residue).
a first acidic group e.g., of a first acidic amino acid residue
a second acidic group e.g.,
an agent comprises a first and second acidic group and a first, second and third aromatic group.
such an agent additionally comprises a third acidic group (e.g., of a third acid amino acid residue) and/or a hydrophobic group (e.g., of a hydrophobic amino acid residue).
such an agent additionally comprises a third acidic group (e.g., of a third acid amino acid residue) and a hydrophobic group (e.g., of a hydrophobic amino acid residue).
the distance between a first acidic group and a second acidic group is about the distance between the acidic groups of two acidic amino acid residues of a peptide motif, wherein there are two amino acid residues between the two acidic amino acid residues (e.g., if the first acidic amino acid residue is at position N, the second is at position N+3), the distance between a first acidic group and a third acidic group (if present) is about the distance between the acidic groups of two acidic amino acid residues of a peptide motif, wherein there are three amino acid residues between the two acidic amino acid residues (e.g., if the first acidic amino acid residue is at position N, the third is at position N+4), the distance between a first acidic group and a hydrophobic group (if present) is about the distance between the acidic group of an acidic amino acid residue and the hydrophobic group of a hydrophobic amino acid residue of a peptide motif, wherein there are five amino acid residues between the
a first acidic amino acid residue is at position N
a second acidic amino acid residue is at position N+3
a first, second and third aromatic amino acid residue are at positions N+7, N+10 and N+11, respectively.
a first acidic amino acid residue is at position N
a second acidic amino acid residue is at position N+3
a third acidic amino acid residue is at position N+4
a first, second and third aromatic amino acid residue are at positions N+7, N+10 and N+11, respectively.
a first acidic amino acid residue is at position N
a second acidic amino acid residue is at position N+3
a hydrophobic amino acid residue is at position N+6, and a first, second and third aromatic amino acid residue are at positions N+7, N+10 and N+11, respectively.
a first acidic amino acid residue is at position N
a second acidic amino acid residue is at position N+3
a third acidic amino acid residue is at position N+4
a hydrophobic amino acid residue is at position N+6, and a first, second and third aromatic amino acid residue are at positions N+7, N+10 and N+11, respectively.
M is N+7.
N is 1-7.
N is 1, 2, 3, 4, or 5. In some embodiments, N is 1. In some embodiments, N is 2. In some embodiments, N is 3. In some embodiments, N is 4. In some embodiments, N is 5. In some embodiments, M is 8-16. In some embodiments, M is 8. In some embodiments, M is 9. In some embodiments, M is 10. In some embodiments, M is 11. In some embodiments, M is 12. In some embodiments, M is 13. In some embodiments, a peptide motif is an alpha-helical motif wherein each amino acid residue is independently an alpha amino acid residue. In some embodiments, a peptide motif is stapled.
a peptide motif is or comprises an agent described in a Table herein (e.g., I-xxxx wherein xxxx is a number (e.g., I-1, I-10, I-100, I-1000, etc.)).
a first acidic group is of X 2 as described herein
a second acidic group is of X 5 as described herein
a third acidic group (if present) is of X 6 as described herein
a hydrophobic group (if present) is of X 8 as described herein
a first aromatic group is of X 9 as described herein
a second aromatic group is of X 12 as described herein
a third aromatic group is of X 13 as described herein.
a provided agent is a stapled peptide comprising one or more staples.
a provided agent is a stapled peptide comprising two or more staples.
a provided agent is a stapled peptide comprising three or more staples.
a first acidic group when contacted with a beta-catenin polypeptide, a first acidic group interacts with Lys312 and/or Gly307 or amino acid residues corresponding thereto, a second acidic group interacts with Asn387, Trp383 and/or Arg386 or amino acid residues corresponding thereto, a first aromatic group interacts with Lys345 and/or Trp383 or amino acid residues corresponding thereto, a second aromatic group interacts with Trp383 and/or Asn415 or amino acid residues corresponding thereto, and a third aromatic group interacts with Gln379, Leu383, Val416, Asn415 and/or Trp383 or amino acid residues corresponding thereto.
a third acidic group interacts with Asn387, Trp383 and/or Arg386 or amino acid residues corresponding thereto.
a hydrophobic group interacts with Trp383 or an amino acid residue corresponding thereto.
the present disclosure provides an agent which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue.
the present disclosure provides an agent which is or comprises a peptide comprising: [X] p X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 [X] p’ , wherein: each of p15, p16 and p17 is independently 0 or 1; each of p and p’ is independently 0-10; each of X, X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue.
an agent is R N ⁇ [X]pX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 [X 14 ] p 14[X 15 ] p15 [X 16 ] p16 [X 17 ] p17 [X]p’ ⁇ R C , wherein each variable is independently as described herein.
an agent is or comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 [X 14 ] p14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 [X 18 ] p18 [X 19 ] p19 [X 20 ] p20 [X 21 ] p21 [X 22 ] p22 [X 23 ] p 23 , wherein each of p14, p15, p16, p17, p18, p19, p20, p21, p22, and p23 is independently 0 or 1, and each of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X
such a peptide comprises three or more staples. In some embodiments, such a peptide comprises five or more residues suitable for stapling.
the present disclosure provides an agent, wherein the agent is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 ,
the present disclosure provides an agent, wherein the agent is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar
an agent is or comprises a peptide. In some embodiments, an agent is or comprises a stapled peptide. In some embodiments, an agent is a peptide. In some embodiments, an agent is a stapled peptide. In some embodiments, an agent, a peptide, or a stapled peptide has the structure of [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 .
X 1 and X 4 , and/or X 4 and X 11 are independently amino acid residues suitable for stapling, or are stapled, or X 3 and X 10 independently amino acid residues suitable for stapling, or are stapled.
X 1 and X 4 are independently amino acid residues suitable for stapling.
X 1 and X 4 are stapled.
X 4 and X 11 are independently amino acid residues suitable for stapling.
X 4 and X 11 are stapled.
X 1 and X 4 , and X 4 and X 11 are independently amino acid residues suitable for stapling.
a stapled peptide is a stitched peptide comprising two or more staples, some of which may bond to the same backbone atom.
X 1 and X 4 are stapled, and X 4 and X 11 are stapled.
a staple connecting X 1 and X 4 and a staple connecting X 4 and X 11 are bonded to a common backbone atom of X 4 .
a common backbone atom is the alpha-carbon of X 4 .
X 3 and X 10 are independently amino acid residues suitable for stapling. In some embodiments, X 3 and X 10 are stapled.
X 1 and X 3 are independently amino acid residues suitable for stapling. In some embodiments, X 1 and X 3 are stapled. In some embodiments, X 10 and X 14 are independently amino acid residues suitable for stapling. In some embodiments, X 10 and X 14 are stapled. In some embodiments, X 7 and X 10 are independently amino acid residues suitable for stapling. In some embodiments, X 7 and X 10 are stapled. In some embodiments, X 7 and X 14 are independently amino acid residues suitable for stapling. In some embodiments, X 7 and X 14 are stapled. In some embodiments, X 3 and X 7 are independently amino acid residues suitable for stapling.
X 3 and X 7 are stapled.
the present disclosure provides agents that bind to a polypeptide comprising or consisting of residues 305-419 of SEQ ID NO: 1 as described herein.
an agent e.g., a peptide
the present disclosure provides various technologies, e.g., reagents, methods, etc., for preparing, characterizing, assessing and using provided agents and compositions thereof. In some embodiments, the present disclosure provides, e.g., methods, reagents and/or systems for identifying, characterizing and/or assessing provided agents and use thereof (e.g., as therapeutic or diagnostic agents). [0028] In some embodiments, the present disclosure provides pharmaceutical compositions comprising or delivering a provided agent and a pharmaceutical acceptable carrier. In some embodiments, a provided agent is a pharmaceutically acceptable salt form.
a provided composition comprises a pharmaceutically acceptable salt form an agent.
agents are provided as pharmaceutically acceptable salt forms.
the present disclosure provides methods for modulating a property, activity and/or function of beta-catenin, comprising contacting beta-catenin with a provided agent.
the present disclosure provides methods for modulating a property, activity and/or function of beta-catenin in a system comprising or expressing beta-catenin, comprising administering or delivering to a system an effective amount of a provided agent.
an activity or function of beta- catenin is inhibited or reduced.
a property, activity and/or function is associated with beta-catenin/TCF interaction.
the present disclosure provides methods for modulating beta-catenin/TCF interaction, comprising contacting beta-catenin with a provided agent.
the present disclosure provides methods for modulating beta-catenin/TCF interaction in a system comprising or expressing beta-catenin and TCF, comprising administering or delivering to the system an effective amount a provided agent.
interactions between beta-catenin and TCF is reduced.
interactions between beta-catenin and TCF is inhibited.
the present disclosure provides methods for inhibiting cell proliferation, comprising administering or delivering to a population of cells an effective amount of a provided agent. In some embodiments, the present disclosure provides methods for inhibiting cell proliferation in a system, comprising administering or delivering to the system an effective amount of a provided agent. In some embodiments, the present disclosure provides methods for inhibiting cell growth, comprising administering or delivering to a population of cells an effective amount of a provided agent. In some embodiments, the present disclosure provides methods for inhibiting cell growth in a system, comprising administering or delivering to the system an effective amount of a provided agent. In some embodiments, such cell proliferation is beta-catenin dependent. In some embodiments, such cell growth is beta-catenin dependent.
a system is in vitro. In some embodiments, a system is ex vivo. In some embodiments, a system is in vivo. In some embodiments, a system is or comprise a cell. In some embodiments, a system is or comprises a tissue. In some embodiments, a system is or comprises an organ. In some embodiments, a system is or comprises an organism. In some embodiments, a system is an animal. In some embodiments, a system is human. In some embodiments, a system is or comprises cells, tissues or organs associated with a condition, disorder or disease.
a system is or comprises cancer cells, e.g., colorectal cancer cells. In some embodiments, a system comprises a mutation as described herein. In some embodiments, a system overexpresses Myc. [0033] In some embodiments, the present disclosure provides methods for treating conditions, disorders or diseases. In some embodiments, the present disclosure provides methods for treating a condition, disorder or disease, comprising administering or delivering to a subject suffering therefrom an effective amount of an agent of the present disclosure. In some embodiments, a symptom is reduced, removed or prevented. In some embodiments, one or more parameters for assessing a condition, disorder or disease are improved. In some embodiments, survivals of subjects are extended.
a condition, disorder or disease is cancer.
a condition, disorder or disease is associated with beta-catenin.
a condition, disorder or disease is associated with beta-catenin interaction with TCF.
a condition, disorder or disease is associated with Myc overexpression.
a condition, disorder or disease is bladder cancer.
a condition, disorder or disease is endometrial cancer.
a condition, disorder or disease is adrenocortical carcinoma.
a condition, disorder or disease is gastric cancer. In some embodiments, a condition, disorder or disease is lung cancer. In some embodiments, a condition, disorder or disease is melanoma. In some embodiments, a condition, disorder or disease is esophageal cancer. In some embodiments, a condition, disorder or disease is colorectal cancer. In some embodiments, a cancer is liver cancer. In some embodiments, a cancer is prostate cancer. In some embodiments, a cancer is breast cancer. In some embodiments, a cancer is endometrial cancer. [0034] In some embodiments, agents are administered as pharmaceutically compositions that comprise or deliver such agents. In some embodiments, agents are provided and/or delivered in pharmaceutically acceptable salt forms.
an agent in a composition (e.g., a liquid composition of certain pH) an agent may exist in various forms including various pharmaceutically acceptable salt forms.
a provided agent is utilized in combination with a second therapy.
a provided agent is utilized in combination with a second therapeutic agent.
a second therapy or therapeutic agent is administered prior to an administration or delivery of a provided agent.
a second therapy or therapeutic agent is administered at about the same time as an administration or delivery of a provided agent.
a second therapy or therapeutic agent is administered subsequently to an administration or delivery of a provided agent.
a subject is exposed to both a provided agent and a second therapeutic agent.
a subject is exposed to a therapeutic effect of a provided agent and a therapeutic effect of a second therapeutic agent.
a second therapy is or comprises surgery.
a second therapy is or comprises radiation therapy.
a second therapy is or comprises immunotherapy.
a second therapeutic agent is or comprises a drug.
a second therapeutic agent is or comprises a cancer drug.
a second therapeutic agent is or comprises a chemotherapeutic agent.
a second therapeutic agent is or comprises a hormone therapy agent.
a second therapeutic agent is or comprises a kinase inhibitor.
a second therapeutic agent is or comprises a checkpoint inhibitor (e.g., antibodies against PD-1, PD-L1, CTLA-4, etc.).
a provide agent can be administered with lower unit dose and/or total dose compared to being used alone.
a second agent can be administered with lower unit dose and/or total dose compared to being used alone.
one or more side effects associated with administration of a provided agent and/or a second therapy or therapeutic agent are reduced.
a combination therapy provides improved results, e.g., when compared to each agent utilized individually.
a combination therapy achieves one or more better results, e.g., when compared to each agent utilized individually.
FIG. 1 BRIEF DESCRIPTION OF THE DRAWING [0037] Figure 1. Provided technologies can inhibit beta-catenin driven gene transcription selectively in cells expressing beta-catenin. Stapled peptides inhibited endogenous gene expression in wild HAP1 isogenic cell but not in CTNNB1 knockout (KO) cells.
A beta-catenin levels.
CHIR CHIR99021, which can activate beta-catenin pathway and increase AXIN2 and SP5 expression.
B SP5 expression (24h).
FIG. 1 For each group, from left to right, DMSO (“0” and “0”), Peptide A (1 and 5 uM), I-66 (1 and 5 uM) and I-470 (1 and 5 uM). Expression assessed after 24 hour treatment.
Figure 2. Provided technologies can reduce nuclear beta-catenin levels. Results for total beta- catenin in nuclear fraction (24 h) are shown as examples.
Figure 3. Provided technologies can inhibit cell proliferation, modulate transcription and/or induce cell cycle arrest.
A Provided technologies can reduce cell proliferation.
FIG. 1 Provided technologies can reduce cell proliferation.
FIG. 1 Provided technologies can reduce cell proliferation.
FIG. 1 For each group, from left to right, DMSO (“0” and “0”), Peptide A (1 and 5 uM), I-66 (1 and 5 uM) and I-470 (1 and 5 uM). Expression assessed after 24 hour treatment.
Figure 3. Provided technologies can reduce nuclear beta-catenin levels. Results for total beta- catenin in nuclear fraction (24
FIG. 4 Provided technologies can provide robust, dose-dependent anti-tumor effects in vivo. Both dose levels assessed provided robust reduction of tumor sizes, and the higher dose levels provided greater reductions. COLO320DM cells (colon cancer, mutations: APC, TP53) were utilized for the presented data. Top line is for vehicle treatment, the middle line is for I-66, 30 mg/kg, Q4D, and the bottom line is for I-66, 75 mg/kg, Q4D. [0041] Figure 5. Provided technologies can provide sustained tumor exposure, suitable pharmacokinetic profiles and broad tissue distribution.
the top spectra can provide improved resolution for various peaks.
Figure 11. A region of 1 H NMR of I-66 as an example. Top: 900 MHz 1 H of a preparation of I- 66 in methanol-d4, 298 K, about 10 mg/750 uL. Bottom: 500 MHz 1 H of a preparation of I-66 in methanol- d4, 298 K, about 10 mg/500 uL.
the top spectra can provide improved resolution for various peaks; certain NH peaks at about 7.7ppm that were observed in the 500 MHz data were absent in the 900 MHz data. As those skilled in the art appreciate, NH peaks may change depending on sample environments.
Spectrometer Frequency (900.30, 900.30). Spectra Width: (9014.4, 9009.0). Lowest Frequency: (-5.7, -3.0). Acquired Size: (2048, 1024). Spectral Size: (2048, 1024). Digital Resolution: (4.40, 8.80). [0054] Figure 18. Expansion of 1 H- 1 H NOESY of a preparation of I-66 as an example. Solvent: methanol-d4. About 10 mg/750 uL. Temperature: 298 K. Number of scans: 8. Receiver gain: 57.7. Relaxation Delay: 2.0000. Acquisition Time: 0.2272. Spectrometer Frequency: (900.30, 900.30).
CRC PDX tumor (PIK3CA H1047R).
Administration IP.
protein and mRNA single-dose vehicle (10 mg/mL arginine/6% PEG400 phosphate), I-6660 mg/kg 2 days post-dose, I-6660 mg/kg 7 days post-dose; protein and mRNA 2 doses (Q4D): vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose; protein and mRNA 3 doses (Q4D): vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose.
protein and mRNA single-dose vehicle (10 mg/mL arginine/6% PEG400 phosphate), I-6660 mg/kg 2 days post-dose, I-6660 mg/kg 7 days post-dose; protein and mRNA 2 doses (Q4D): vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose; protein and mRNA 3 doses (Q4D): vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose.
ns not significant; *, **, ***, and **** reflect different significance levels.
CRC PDX tumor (PIK3CA H1047R and mutant APC).
Administration IP.
vehicle (10 mg/mL arginine/6% PEG400 phosphate), I-66 60 mg/kg 2 days post-dose, I-6660 mg/kg 7 days post-dose;
mRNA 2 doses (Q4D): vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose;
mRNA 3 doses (Q4D): vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose.
FIG. 23 Provided technologies can reduce levels of Ki67.
CRC PDX tumor PIK3CA H1047R and mutant APC.
Administration IP. 2 doses (Q4D). From left to right: vehicle, I-47020 mg/kg 2 days post last dose, I-6620 mg/kg 2 days post last dose, I-6660 mg/kg 2 days post last dose, I-6660 mg/kg 7 days post last dose. ns: not significant; *, **, ***, and **** reflect different significance levels.
Figure 24 Provided technologies can reduce tumor growth in various models in vivo. For example, I-66 can reduce tumor volume in various models.
provided technologies can suppress tumor growth and/or lead to tumor stasis and/or regression in various tumors.
Figure 26 Provided technologies can reduce tumor growth in vivo. Among other things, Figure 26 confirms that provided technologies can suppress tumor growth and in some cases lead to tumor regression even with weekly administration. Model 2. Lines from top to bottom: vehicle; I-6620 mg/kg Q&D; I-6660 mg/kg Q7D (squares) and I-6660 mg/kg Q4D (triangles). The two 60 mg/kg largely overlap with each other. #: end of in-life. [0063] Figure 27. Provided technologies can provide rapid regression.
Figure 27 confirms that provided technologies are active in vivo and can provide rapid tumor regression at various dose levels. Arrows indicate I-66 doses. Lines: top line at day 4 is vehicle, the second is I-47020 mg/kg; at day 8, from top going down are I-47020 mg/kg, I-6620 mg/kg and I-60 mg/kg. Model 2. 500mm 3 starting volume. [0064] Figure 28. Provided technologies can modulate expression in vivo. (A) I-66 reduced Axin2 polypeptide levels in Model 2.
Administration typically refers to the administration of a composition to a subject or system.
routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
administration may be ocular, oral, parenteral, topical, etc.
administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
bronchial e.g., by bronchial instillation
buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc
enteral intra-arterial, intradermal, intragastric
administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
Affinity is a measure of the tightness with a particular ligand (e.g., an agent) binds to its partner (e.g., beta-catenin or a portion thereof). Affinities can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay.
binding partner concentration may be fixed to be in excess of ligand concentration so as to mimic physiological conditions.
binding partner concentration and/or ligand concentration may be varied.
affinity may be compared to a reference under comparable conditions (e.g., concentrations).
the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof.
the term may be used to refer to a natural product in that it is found in and/or is obtained from nature.
the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form.
potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them.
the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties.
the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties.
the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.
an agent is a compound.
an agent is a stapled peptide.
Aliphatic means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation (but not aromatic), or combinations thereof.
aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms.
aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms.
Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
Alkenyl As used herein, the term “alkenyl” refers to an aliphatic group, as defined herein, having one or more double bonds.
Alkyl As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C 1 -C 20 for straight chain, C 2 -C 20 for branched chain), and alternatively, about 1-10.
cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure.
an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C 1 -C 4 for straight chain lower alkyls).
Amino acid In its broadest sense, as used herein, refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
an amino acid comprising an amino group and an a carboxylic acid group.
an amino acid has the structure of NH(R a1 ) ⁇ L a1 ⁇ C(R a2 )(R a3 ) ⁇ L a2 ⁇ COOH, wherein each variable is independently as described in the present disclosure.
an amino acid has the general structure NH(R’)–C(R’) 2 –COOH, wherein each R’ is independently as described in the present disclosure.
an amino acid has the general structure H 2 N–C(R’) 2 –COOH, wherein R’ is as described in the present disclosure.
an amino acid has the general structure H 2 N– C(H)(R’)–COOH, wherein R’ is as described in the present disclosure.
an amino acid is a naturally-occurring amino acid.
an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.
Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
an amino acid including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above.
an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, one or more hydrogens, and/or the hydroxyl group) as compared with the general structure.
such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
amino acid may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
Analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways.
an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.
Animal As used herein refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, of either sex and at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development.
the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
an animal may be a transgenic animal, genetically engineered animal, and/or a clone.
the term “approximately” or “about,” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
Aryl The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” “aryloxyalkyl,” etc.
an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members.
an aryl group is a biaryl group.
aryl may be used interchangeably with the term “aryl ring.”
aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents.
aryl is a group in which an aromatic ring is fused to one or more non–aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like, where a radical or point of attachment is on an aryl ring.
two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
a particular entity e.g., nucleic acid (e.g., genomic DNA, transcripts, mRNA, etc.), polypeptide, genetic signature, metabolite, microbe, etc..
a particular entity e.g., nucleic acid (e.g., genomic DNA, transcripts, mRNA, etc.), polypeptide, genetic signature, metabolite, microbe, etc..
binding typically refers to a non- covalent association between or among agents. In many embodiments herein, binding is addressed with respect to particular agents and beta-catenin.
binding is assessed with respect to beta-catenin. In some embodiments, binding is assessed with respect to one or more amino acid residues of beta-catenin. In some embodiments, binding is assessed with respect to one or more amino acid residues corresponding to (e.g., similarly positioned in three dimensional space and/or having certain similar properties and/or functions) those of beta-catenin.
binding site refers to a region of a target polypeptide, formed in three-dimensional space, that includes one or more or all interaction residues of the target polypeptide.
binding site may refer to one or more amino acid residues which comprise or are one or more or all interaction amino acid residues of a target polypeptide.
a binding site may include residues that are adjacent to one another on a linear chain, and/or that are distal to one another on a linear chain but near to one another in three-dimensional space when a target polypeptide is folded.
a binding site may comprise amino acid residues and/or saccharide residues.
carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like.
carriers are or include one or more solid components.
Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable.
sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.
composition may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form – e.g., gas, gel, liquid, solid, etc.
Cycloaliphatic refers to saturated or partially unsaturated aliphatic monocyclic, bicyclic, or polycyclic ring systems having, e.g., from 3 to 30, members, wherein the aliphatic ring system is optionally substituted.
Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl.
the cycloalkyl has 3–6 carbons.
cycloaliphatic may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where a radical or point of attachment is on an aliphatic ring.
a carbocyclic group is bicyclic.
a carbocyclic group is tricyclic.
a carbocyclic group is polycyclic.
cycloaliphatic refers to a monocyclic C 3 -C 10 , or C 3 - C 6 hydrocarbon, or a C 4 -C 10 , or C 8 -C 10 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, or a C 9 -C 16 tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic.
Derivative As used herein, the term “derivative” refers to a structural analogue of a reference substance.
a “derivative” is a substance that shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways.
a derivative is a substance that can be generated from the reference substance by chemical manipulation.
a derivative is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.
Dosage form or unit dosage form may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject.
each such unit contains a predetermined quantity of active agent.
such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
Dosing regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts.
a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen). [0087] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man.
a peptide may be considered to be engineered if its amino acid sequence has been selected by man.
an engineered agent has an amino acid sequence that was selected based on preferences for corresponding amino acids at particular sites of protein- protein interactions.
an engineered sequence has an amino acid sequence that differs from the amino acid sequence of polypeptides included in the NCBI database that binds to a TCF site of beta- catenin.
provided agents are engineered agents.
engineered agents are peptide agents comprising non-natural amino acid residues, non-natural amino acid sequences, and/or peptide staples.
provided agents comprise or are engineered peptide agents which comprise engineered sequences.
Halogen means F, Cl, Br, or I.
Heteroaliphatic The term “heteroaliphatic” is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms are replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).
Heteroalkyl The term “heteroalkyl” is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).
heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.
Heteroaryl The terms “heteroaryl” and “heteroar—,” used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or polycyclic ring systems having, for example, a total of five to thirty, e.g., 5, 6, 9, 10, 14, etc., ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom.
a heteroatom is nitrogen, oxygen or sulfur.
a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms.
a heteroaryl group has 6, 10, or 14 ⁇ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
a heteroaryl is a heterobiaryl group, such as bipyridyl and the like.
heteroaryl and “heteroar—”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where a radical or point of attachment is on a heteroaromatic ring.
Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H–quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3–b]–1,4–oxazin–3(4H)–one.
heteroaryl group may be monocyclic, bicyclic or polycyclic.
heteroaryl may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted.
heteroarylkyl refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.
Heteroatom means an atom that is not carbon and is not hydrogen.
a heteroatom is oxygen, sulfur, nitrogen, phosphorus, boron or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl); etc.).
a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.
a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus.
a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur.
Heterocyclyl As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.
a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur.
a heterocyclyl group is a stable 5– to 7–membered monocyclic or 7– to 10–membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen.
the nitrogen may be N (as in 3,4–dihydro–2H–pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N–substituted pyrrolidinyl).
a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
heterocycle used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H–indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where a radical or point of attachment is on a heteroaliphatic ring.
a heterocyclyl group may be monocyclic, bicyclic or polycyclic.
heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
homology refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.
Typical amino acid categorizations are summarized below (hydrophobicity scale of Kyte and Doolittle, 1982: A simple method for displaying the hydropathic character of a protein. J. Mol. Biol.157:105-132):
the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.
the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position.
the percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
the percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
Interaction residues refers to, with respect to an agent, residues or motifs in an agent that are designed to interact with particular target residues in a target polypeptide, or with respect to a target polypeptide, residues in a target polypeptide that interact with particular motifs (e.g., aromatic groups, amino acid residues, etc.) of an agent.
interaction residues and motifs of various agents are selected and arranged within the agents so that they will be displayed in three dimensional space within a predetermined distance (or volume) of identified target residues (e.g., upon binding, docking or other interaction assays).
interaction residues are direct-binding residues.
an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc).
comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance).
Partially unsaturated refers to a moiety that includes at least one double or triple bond.
the term “partially unsaturated” is intended to encompass groups having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties.
Peptide The term “peptide” as used herein refers to a polypeptide.
a peptide is a polypeptide that is relatively short, for example having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids.
a length is about 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 10-20, 10-19, 10-18, 10- 17, 10-16, 10-15, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
pharmaceutical composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingual
oral administration for example,
compositions or vehicles which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydrox
compositions that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable salts are well known. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other known methods such as ion exchange.
nontoxic acid addition salts which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other known methods such as ion exchange.
pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
pharmaceutically acceptable salts include, but are not limited to, nontoxic base addition salts, such as those formed by acidic groups of provided compounds with bases.
Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like.
pharmaceutically acceptable salts are ammonium salts (e.g., ⁇ N(R)3 + ).
pharmaceutically acceptable salts are sodium salts.
pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
Polypeptide As used herein refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature.
a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
a polypeptide may comprise or consist of natural amino acids, non- natural amino acids, or both.
a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids.
a polypeptide may comprise D-amino acids, L-amino acids, or both.
a polypeptide may comprise only D-amino acids.
a polypeptide may comprise only L-amino acids.
a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof.
such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
a polypeptide may be cyclic, and/or may comprise a cyclic portion.
a polypeptide is not cyclic and/or does not comprise any cyclic portion.
a polypeptide is linear.
a polypeptide may be or comprise a stapled polypeptide.
the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
a common sequence motif e.g., a characteristic sequence element
shares a common activity in some embodiments at a comparable level or within a designated range
a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
a conserved region that may in some embodiments be or comprise a characteristic sequence element
Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
Prevent or prevention refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
Protecting group The term “protecting group,” as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.
Suitable amino–protecting groups include methyl carbamate, ethyl carbamante, 9–fluorenylmethyl carbamate (Fmoc), 9–(2–sulfo)fluorenylmethyl carbamate, 9–(2,7– dibromo)fluoroenylmethyl carbamate, 2,7–di–t–butyl–[9–(10,10–dioxo–10,10,10,10– tetrahydrothioxanthyl)]methyl carbamate (DBD–Tmoc), 4–methoxyphenacyl carbamate (Phenoc), 2,2,2– trichloroethyl carbamate (Troc), 2–trimethylsilylethyl carbamate (Teoc), 2–phenylethyl carbamate (hZ), 1– (1–adamantyl)–1–methylethyl carbamate (Adpoc), 1,1–dimethyl–2–haloethy
suitable mono-protected amines include, but are not limited to, aralkylamines, carbamates, allyl amines, amides, and the like.
suitable mono-protected amino moieties include t-butyloxycarbonylamino (—NHBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (—NHAlloc), benzyloxocarbonylamino (–NHCBZ), allylamino, benzylamino (–NHBn), fluorenylmethylcarbonyl (–NHFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, t
suitable di-protected amines include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides, such as phthalimide, maleimide, succinimide, and the like.
suitable di-protected amines include pyrroles and the like, 2,2,5,5-tetramethyl- [1,2,5]azadisilolidine and the like, and azide.
Suitably protected carboxylic acids further include, but are not limited to, silyl–, alkyl–, alkenyl–, aryl–, and arylalkyl–protected carboxylic acids.
Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t–butyldimethylsilyl, t–butyldiphenylsilyl, triisopropylsilyl, and the like.
Examples of suitable alkyl groups include methyl, benzyl, p–methoxybenzyl, 3,4–dimethoxybenzyl, trityl, t–butyl, tetrahydropyran–2–yl.
Examples of suitable alkenyl groups include allyl.
Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl.
Suitable arylalkyl groups include optionally substituted benzyl (e.g., p–methoxybenzyl (MPM), 3,4–dimethoxybenzyl, O–nitrobenzyl, p– nitrobenzyl, p–halobenzyl, 2,6–dichlorobenzyl, p–cyanobenzyl), and 2– and 4–picolyl.
suitable protected carboxylic acids include, but are not limited to, optionally substituted C 1–6 aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, and the like.
ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, wherein each group is optionally substituted.
Additional suitable protected carboxylic acids include oxazolines and ortho esters.
Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t–butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p– methoxybenzyloxymethyl (PMBM), (4–methoxyphenoxy)methyl (p–AOM), guaiacolmethyl (GUM), t– butoxymethyl, 4–pentenyloxymethyl (POM), siloxymethyl, 2–methoxyethoxymethyl (MEM), 2,2,2– trichloroethoxymethyl, bis(2–chloroethoxy)methyl, 2–(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3–bromotetrahydropyranyl, tetrahydrothiopyranyl, 1–methoxycyclohexyl, 4– methoxytetrahydropyr
the protecting groups include methylene acetal, ethylidene acetal, 1–t– butylethylidene ketal, 1–phenylethylidene ketal, (4–methoxyphenyl)ethylidene acetal, 2,2,2– trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p–methoxybenzylidene acetal, 2,4–dimethoxybenzylidene ketal, 3,4– dimethoxybenzylidene acetal, 2–nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1–methoxyethy
a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl, 1 -ethoxyethyl, 1 -(2-chloroethoxy)ethyl, 2- trimethylsilylethyl, p- chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6- dichlorobenzyl, diphenylmethyl, p- nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trich
each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t- butyldimethylsilyl, t- butyldiphenylsilyl and 4,4'-dimethoxytrityl.
the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl group.
a phosphorous linkage protecting group is a group attached to the phosphorous linkage (e.g., an internucleotidic linkage) throughout oligonucleotide synthesis.
a protecting group is attached to a sulfur atom of an phosphorothioate group. In some embodiments, a protecting group is attached to an oxygen atom of an internucleotide phosphorothioate linkage. In some embodiments, a protecting group is attached to an oxygen atom of the internucleotide phosphate linkage.
a protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o- nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4- oxopentyl, 4-methylthio-l-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2- trifluoroacetyl)amino]butyl.
Protected thiols are well known in the art and include those described in detail in Greene (1999). Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioester, to name but a few.
Reference As used herein describes a standard or control relative to which a comparison is performed.
an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
a reference or control is a historical reference or control, optionally embodied in a tangible medium.
a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
Specificity is a measure of the ability of a particular ligand (e.g., an agent) to distinguish its binding partner (e.g., beta-catenin) from other potential binding partners (e.g., another protein, another portion (e.g., domain) of beta-catenin).
binding partner e.g., beta-catenin
substitution As described herein, compounds of the disclosure may contain optionally substituted and/or substituted moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, example substituents are described below.
Suitable monovalent substituents on R° are independently halogen, —(CH 2 ) 0–2 R ⁇ , –(haloR ⁇ ), –(CH 2 ) 0–2 OH, –(CH 2 ) 0–2 OR ⁇ , –(CH 2 ) 0–2 CH(OR ⁇ ) 2 ; –O(haloR ⁇ ), –CN, –N 3 , –(CH 2 ) 0–2 C(O)R ⁇ , –(CH 2 ) 0– 2 C(O)OH, –(CH 2 ) 0–2 C(O)OR ⁇ , –(CH 2 ) 0–2 SR ⁇ , –(CH 2 ) 0–2 SH, –(CH 2 ) 0–2 NH 2 , –(CH 2 ) 0
R * is selected from hydrogen, C 1–6 aliphatic which may be substituted as defined below, or an unsubstituted
Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: – O(CR * 2 ) 2–3 O–, wherein each independent occurrence of R * is selected from hydrogen, C 1–6 aliphatic which may be substituted as defined below, or an unsubstituted 3–6 (e.g., 3-5, 5-6, etc.)–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
Suitable substituents on the aliphatic group of R * are halogen, –R ⁇ , -(haloR ⁇ ), –OH, ⁇ OR ⁇ , – O(haloR ⁇ ), –CN, –C(O)OH, –C(O)OR ⁇ , –NH 2 , –NHR ⁇ , –NR ⁇ 2 , or –NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1–4 aliphatic, –CH 2 Ph, –O(CH 2 ) 0–1 Ph, or a 3–6 (e.g., 3-5, 5-6, etc.)–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
halogen e.g., 3-5, 5-6, etc.
suitable substituents on a substitutable nitrogen are –R ⁇ , ⁇ NR ⁇ 2, –C(O)R ⁇ , –C(O)OR ⁇ , –C(O)C(O)R ⁇ , –C(O)CH 2 C(O)R ⁇ , –S(O) 2 R ⁇ , –S(O) 2 NR ⁇ 2, ⁇ C(S)NR ⁇ 2, –C(NH)NR ⁇ 2, or – N(R ⁇ )S(O) 2 R ⁇ ; wherein each R ⁇ is independently hydrogen, C 1–6 aliphatic which may be substituted as defined below, unsubstituted –OPh, or an unsubstituted 3–6 (e.g., 3-5, 5-6, etc.)–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding
Suitable substituents on the aliphatic group of R ⁇ are independently halogen, ⁇ R ⁇ , -(haloR ⁇ ), – OH, –OR ⁇ , –O(haloR ⁇ ), –CN, –C(O)OH, –C(O)OR ⁇ , –NH 2 , –NHR ⁇ , –NR ⁇ 2 , or –NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1–4 aliphatic, –CH 2 Ph, –O(CH 2 ) 0–1 Ph, or a 3–6 (e.g., 3-5, 5-6, etc.)–membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
Subject refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition. In some embodiments, a subject is a human.
Susceptible to An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
Target polypeptide A “target polypeptide”, as that term is used herein, is a polypeptide with which an agent interacts.
a target polypeptide is a beta-catenin polypeptide.
a target polypeptide comprises, consists essentially of, or is a binding site of beta-catenin polypeptide.
Target residue is a residue within a target polypeptide with which an agent is designed to interact.
an agent may be characterized by particular interaction motifs (e.g., aromatic groups as described herein) and/or residues (e.g., amino acid residues comprising aromatic groups as described herein) selected and arranged (by virtue of being presented on the selected scaffold) to be within a certain predetermined distance (or volume) of a target residue.
a target residue is or comprises an amino acid residue.
Therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
Therapeutic regimen A “therapeutic regimen”, as that term is used herein, refers to a dosing regimen whose administration across a relevant population may be correlated with a desired or beneficial therapeutic outcome.
therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
Treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
Unit dose refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition.
a unit dose contains a predetermined quantity of an active agent.
a unit dose contains an entire single dose of the agent.
more than one unit dose is administered to achieve a total single dose.
administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect.
a unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described infra.
acceptable carriers e.g., pharmaceutically acceptable carriers
diluents e.g., diluents, stabilizers, buffers, preservatives, etc.
a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment.
the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.
Unsaturated means that a moiety has one or more units of unsaturation.
salts such as pharmaceutically acceptable acid or base addition salts, stereoisomeric forms, and tautomeric forms, of provided compound are included.
the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional/second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.
a provided agent is or comprises a peptide.
a provided agent is a peptide.
a peptide is a stapled peptide.
a provided agent is a stapled peptide.
a peptide is a stitched peptide.
a provided agent is a stitched peptide.
a stitched peptide comprises two or more staples, wherein two staples are bonded to the same peptide backbone atom.
Stapled peptides as described herein are typically peptides in which two or more amino acids of a peptide chain are linked through connection of two peptide backbone atoms of the amino acid residues and, as is understood by those skilled in the art, the connection is not through the peptide backbone between the linked amino acid residues.
a staple as described herein is a linker that link one amino acid residue to another amino acid residue, e.g., through bonding to a peptide backbone atom of each of the amino acid residues and, as is understood by those skilled in the art, the connection through a staple is not through the peptide backbone between the linked amino acid residues.
a staple bonds to the peptide backbone by replacing one or more hydrogen and/or substituents (e.g., side chains, O, S, etc.) on peptide backbone atoms (e.g., C, N, etc.).
substituents e.g., side chains, O, S, etc.
side chains form portions of staples.
a staple is bonded to two carbon backbone atoms, e.g., two alpha carbon atoms.
a staple comprises C(R’) 2 or N(R’), either individually or as part of a large moiety, wherein R’ is R and is taken together with another group attached to a backbone atom which can be R (e.g., R a3 ) and their intervening atoms to form a ring as described herein (e.g., when PyrS2 is stapled in various peptides).
a stapled peptide comprises one or more staples.
a stapled peptide comprises two or more staples.
a stapled peptide comprises three or more staples.
a stapled peptide comprises four or more staples.
a peptide e.g., a stapled peptide
a peptide is or comprise a helical structure.
a peptide is a stapled peptide.
a stapled peptide is as described in WO 2022/261257 which is incorporated herein by reference.
an peptide is as described in WO 2022/261257.
an agent is as described in WO 2022/261257.
a staple is a hydrocarbon staple.
a staple as described herein is a non-hydrocarbon staple.
a non-hydrocarbon staple comprises one or more chain heteroatoms wherein a chain of a staple is the shortest covalent connection within the staple from one end of the staple to the other end of the staple.
a staple comprises one or more amino groups, e.g., ⁇ N(R’) ⁇ , wherein each R’ is independently as described herein.
⁇ N(R’) ⁇ bonds to two carbon atoms. In some embodiments, ⁇ N(R’) ⁇ bonds to two carbon atoms, wherein neither of the two carbon atoms are bond to any heteroatoms through a double bond. In some embodiments, ⁇ N(R’) ⁇ bonds to two sp3 carbon atoms.
a staple comprises one or more ⁇ C(O) ⁇ N(R’) ⁇ groups, wherein each R’ is independently as described herein. In some embodiments, a staple comprises one or more carbamate groups, e.g., one or more ⁇ (O) ⁇ C(O) ⁇ N(R’) ⁇ , wherein each R’ is independently as described herein.
R’ is ⁇ H. In some embodiments, R’ is optionally substituted C 1-6 aliphatic. In some embodiments, R’ is optionally substituted C 1-6 alkyl. In some embodiments, R’ is C 1-6 aliphatic. In some embodiments, R’ is C 1-6 alkyl. In some embodiments, R’ is methyl.
peptides, e.g., staple peptides, of the present disclosure is or comprises a helix structure. As those skilled in the art will appreciate, helixes can have various lengths. In some embodiments, lengths of helixes range from 5 to 30 amino acid residues.
a length of a helix is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more, amino acid residues.
Amino acids stapled together can have various number of amino acid residues in between, e.g., 1- 20, 1-15, 1-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.
a staple is (i, i+4) which means there are three amino acid residues between the two amino acids (at positions i and i+4, respectively) that bond to the staple (at positions i+1, i+2, i+3, respectively).
a staple is (i, i+2).
a staple is (i, i+3).
a staple is (i, i+5). In some embodiments, a staple is (i, i+6). In some embodiments, a staple is (i, i+7). In some embodiments, a staple is (i, i+8). In some embodiments, a stapled peptide comprises two staples, one is (i, i+2) and the other is (i, i+7). In some embodiments, a stapled peptide comprises two staples, one is (i, i+3) and the other is (i, i+7). In some embodiments, a stapled peptide comprises two staples, one is (i, i+3) and the other is (i, i+4).
a stapled peptide comprises two staples, one is (i, i+4) and the other is (i, i+7). In some embodiments, a stapled peptide comprises two staples, one is (i, i+3) and the other is (i, i+3). In some embodiments, a stapled peptide comprises two staples, one is (i, i+4) and the other is (i, i+4). In some embodiments, a stapled peptide comprises two staples, one is (i, i+7) and the other is (i, i+7). In some embodiments, the two staples are bonded to a common backbone atom, e.g., an alpha carbon atom of an amino acid residue.
a stapled peptide further comprises a third staple.
a third staple is (i, i+3).
a third staple is (i, i+4).
a third staple is (i, i+7).
a stapled peptide further comprises a fourth staple.
a fourth staple is (i, i+3).
a fourth staple is (i, i+4).
a fourth staple is (i, i+7).
a staple can be of various lengths, in some embodiments, as represent by the number of chain atoms of a staple.
a chain of a staple is the shortest covalent connection in the staple from a first end (connection point with a peptide backbone) of a staple to a second end of the staple, wherein the first end and the second end are connected to two different peptide backbone atoms.
a staple comprises 5-30 chain atoms, e.g., 5-20, 5-15, 5, 6, 7, 8, 9, or 10 to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chain atoms.
a staple comprises 5 chain atoms.
a staple comprises 6 chain atoms.
a staple comprises 7 chain atoms.
a staple comprises 8 chain atoms.
a staple comprises 9 chain atoms. In some embodiments, a staple comprises 10 chain atoms. In some embodiments, a staple comprises 11 chain atoms. In some embodiments, a staple comprises 12 chain atoms. In some embodiments, a staple comprises 13 chain atoms. In some embodiments, a staple comprises 14 chain atoms. In some embodiments, a staple comprises 15 chain atoms. In some embodiments, a staple comprises 16 chain atoms. In some embodiments, a staple comprises 17 chain atoms. In some embodiments, a staple comprises 18 chain atoms. In some embodiments, a staple comprises 19 chain atoms. In some embodiments, a staple comprises 20 chain atoms.
a staple has a length of 5 chain atoms. In some embodiments, a staple has a length of 6 chain atoms. In some embodiments, a staple has a length of 7 chain atoms. In some embodiments, a staple has a length of 8 chain atoms. In some embodiments, a staple has a length of 9 chain atoms. In some embodiments, a staple has a length of 10 chain atoms. In some embodiments, a staple has a length of 11 chain atoms. In some embodiments, a staple has a length of 12 chain atoms. In some embodiments, a staple has a length of 13 chain atoms. In some embodiments, a staple has a length of 14 chain atoms.
a staple has a length of 15 chain atoms. In some embodiments, a staple has a length of 16 chain atoms. In some embodiments, a staple has a length of 17 chain atoms. In some embodiments, a staple has a length of 18 chain atoms. In some embodiments, a staple has a length of 19 chain atoms. In some embodiments, a staple has a length of 20 chain atoms. In some embodiments, a staple has a length of 8-15 chain atoms. In some embodiments, a staple has 8-12 chain atoms. In some embodiments, a staple has 9-12 chain atoms. In some embodiments, a staple has 9-10 chain atoms.
a staple has 8-10 chain atoms.
length of a staple can be adjusted according to the distance of the amino acid residues it connects, for example, a longer staple may be utilized for a (i, i+7) staple than a (i, i+4) or (i, i+3) staple.
a (i, i+2) staple has about 5-10, 5-8, e.g., about 5, 6, 7, 8, 9 or 10 chain atoms.
a (i, i+2) staple has 5 chain atoms.
a (i, i+2) staple has 6 chain atoms.
a (i, i+2) staple has 7 chain atoms.
a (i, i+2) staple has 8 chain atoms. In some embodiments, a (i, i+2) staple has 9 chain atoms. In some embodiments, a (i, i+2) staple has 10 chain atoms. In some embodiments, a (i, i+3) staple has about 5-10, 5-8, e.g., about 5, 6, 7, 8, 9 or 10 chain atoms. In some embodiments, a (i, i+3) staple has 5 chain atoms. In some embodiments, a (i, i+3) staple has 6 chain atoms. In some embodiments, a (i, i+3) staple has 7 chain atoms.
a (i, i+3) staple has 8 chain atoms. In some embodiments, a (i, i+3) staple has 9 chain atoms. In some embodiments, a (i, i+3) staple has 10 chain atoms. In some embodiments, a (i, i+4) staple has about 5-12, 5- 10, 7-12, 5-8, e.g., about 5, 6, 7, 8, 9, 10, 11 or 12 chain atoms. In some embodiments, a (i, i+4) staple has 5 chain atoms. In some embodiments, a (i, i+4) staple has 6 chain atoms. In some embodiments, a (i, i+4) staple has 7 chain atoms.
a (i, i+4) staple has 8 chain atoms. In some embodiments, a (i, i+4) staple has 9 chain atoms. In some embodiments, a (i, i+4) staple has 10 chain atoms. In some embodiments, a (i, i+4) staple has 11 chain atoms. In some embodiments, a (i, i+4) staple has 12 chain atoms. In some embodiments, a (i, i+7) staple has about 8-25, 10-25, 10-16, 12-15, e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 chain atoms. In some embodiments, a (i, i+7) staple has 8 chain atoms.
a (i, i+7) staple has 9 chain atoms. In some embodiments, a (i, i+7) staple has 10 chain atoms. In some embodiments, a (i, i+7) staple has 11 chain atoms. In some embodiments, a (i, i+7) staple has 12 chain atoms. In some embodiments, a (i, i+7) staple has 13 chain atoms. In some embodiments, a (i, i+7) staple has 14 chain atoms. In some embodiments, a (i, i+7) staple has 15 chain atoms. In some embodiments, a (i, i+7) staple has 16 chain atoms.
a (i, i+7) staple has 17 chain atoms. In some embodiments, a (i, i+7) staple has 18 chain atoms. In some embodiments, a (i, i+7) staple has 19 chain atoms. In some embodiments, a (i, i+7) staple has 20 chain atoms. In some embodiments, a (i, i+7) staple has 21 chain atoms. In some embodiments, a (i, i+7) staple has 22 chain atoms. In some embodiments, a stapled peptide comprises three or more staples, each of which is independently such a (I, i+2), (i, i+3), (i, i+4) or (i, i+7) staple.
a stapled peptide comprises such a (i, i+2) staple, such a (i, i+4) staple and such a (i, i+7) staple.
a stapled peptide comprises such a (i, i+3) staple, such a (i, i+4) staple and such a (i, i+7) staple.
a stapled peptide comprises such a (i, i+3) staple, such a (i, i+7) staple and such a (i, i+7) staple.
staple lengths may be described as the total number of chain atoms and non-chain ring atoms, where a non-chain ring atom is an atom of the staple which forms a ring with one or more chain atoms but is not a chain atom in that it is not within the shortest covalent connection from a first end of the staple to a second end of the staple.
staples formed using Monomer A which comprises an azetidine moiety
Monomer B which comprises a pyrrolidine moiety
Monomer C which comprises a pyrrolidine moiety
a staple has no heteroatoms in its chain. In some embodiments, a staple comprises at least one heteroatom in its chain. In some embodiments, a staple comprises at least one nitrogen atom in its chain. [0144] In some embodiments, a staple is L s , wherein L s is an optionally substituted, bivalent C 8-14 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S s(O) 2 ⁇ , ⁇ S
a staple is L s , wherein L s is an optionally substituted, bivalent C9-13 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
a staple is L s , wherein L s is an optionally substituted, bivalent C 10-15 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
a staple is L s , wherein L s is an optionally substituted, bivalent C 11-14 aliphatic group wherein one or more methylene units of the aliphatic group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
a staple is a (i, i+2) staple in that not including the two amino acid residues that are directly connected to the staple, there are one amino acid residue between the two amino acid residues that are directly connected to the staple.
a staple is a (i, i+3) staple in that not including the two amino acid residues that are directly connected to the staple, there are two amino acid residues between the two amino acid residues that are directly connected to the staple.
a staple is a (i, i+4) staple in that not including the two amino acid residues that are directly connected to the staple, there are three amino acid residues between the two amino acid residues that are directly connected to the staple.
a staple is a (i, i+7) staple in that not including the two amino acid residues that are directly connected to the staple, there are six amino acid residues between the two amino acid residues that are directly connected to the staple.
a staple comprises a double bond.
a staple comprises a double bond may be formed by olefin metathesis of two olefins.
staples are formed by metathesis reactions, e.g., involving one or more double bonds in amino acid residues as described herein.
an olefin e.g., in a staple, is converted into ⁇ CHR’ ⁇ CHR’ ⁇ , wherein each R’ is independently as described herein.
R’ is R as described herein.
R’ is ⁇ H.
each R’ is ⁇ H. In some embodiments, R’ is ⁇ OR, wherein R is as described herein. In some embodiments, R’ is ⁇ OH. In some embodiments, R’ is ⁇ N(R) 2 wherein each R is independently as described herein. In some embodiments, R’ is ⁇ SR wherein R is as described herein. In some embodiments, R’ is R wherein R is optionally substituted aliphatic, e.g., C 1-10 aliphatic. In some embodiments, R’ is R wherein R is optionally substituted aliphatic, e.g., C 1-10 alkenyl.
R’ is R wherein R is optionally substituted aliphatic, e.g., C 1-10 alkynyl.
⁇ CHR’ ⁇ CHR’ ⁇ is ⁇ CH 2 ⁇ CH 2 ⁇ .
each of the two olefins is independently of a side chain of an amino acid residue.
each olefin is independently a terminal olefin.
each olefin is independently a mono-substituted olefin.
amino acids that are useful for stapling, or are stapled are described in WO 2022/261257, e.g., those of formula A-II, A-II-b, A-III, A-IV, A-V, or A-VI, or salts thereof, which are incorporated herein by reference.
an amino acid is selected from Tables A-I, A-II, A-III and A-IV (may be presented as Fmoc-protected).
Fmoc-protected amino groups and carboxyl groups may independently form amide connections with other amino acid residues (or N- or C-terminus capping groups, or exist as N- or C- terminus amino or carboxyl groups).
Olefins including those in Alloc groups, may be utilized to form staples through olefin metathesis.
Staples comprising olefins may be further modified, e.g., through hydrogenation to convert olefin double bonds into single bonds, and/or through CO 2 extrusion to convert carbamate moieties (e.g., ⁇ O ⁇ (CO) ⁇ N(R’) ⁇ ) into amine moieties (e.g., ⁇ N(R’) ⁇ ).
an agent is or comprises a stapled peptide (e.g., a stapled peptide described according to Table E2 or Table E3) or a salt thereof, in which stapled peptide each double bond is converted into a single bond.
a conversion is achieved through hydrogenation which adds a ⁇ H to each olefin carbon atom.
an olefin double bond is replaced with ⁇ CHR’ ⁇ CHR’ ⁇ , wherein each R’ is independently as described herein.
R’ is R as described herein.
R’ is ⁇ H.
each R’ is ⁇ H.
R’ is ⁇ OR, wherein R is as described herein.
R’ is ⁇ OH.
R’ is ⁇ N(R) 2 wherein each R is independently as described herein.
R’ is ⁇ SR wherein R is as described herein.
R’ is R wherein R is optionally substituted aliphatic, e.g., C 1-10 aliphatic. In some embodiments, R’ is R wherein R is optionally substituted aliphatic, e.g., C 1-10 alkenyl. In some embodiments, R’ is R wherein R is optionally substituted aliphatic, e.g., C 1-10 alkynyl. In some embodiments, ⁇ CHR’ ⁇ CHR’ ⁇ is ⁇ CH 2 ⁇ CH 2 ⁇ . [0148] Table A-I. Exemplary amino acids (Fmoc-Protected). [0149] Table A-II. Exemplary amino acids (Fmoc-Protected). [0150] Table A-III.
an amino acid is an alpha-amino acid.
an amino acid is an L-amino acid.
an amino acid is a D-amino acid.
the alpha-carbon of an amino acid is achiral.
an amino acid is a beta-amino acid.
an amino acid is a gamma-amino acid.
an olefin in a staple is a Z-olefin. In some embodiments, an olefin in a staple in an E-olefin.
a provided composition comprises stapled peptides comprising a staple that contains a Z-olefin and stapled peptides comprising a staple that contains an E-olefin. In some embodiments, a provided composition comprises stapled peptides comprising a staple that contains a Z- olefin. In some embodiments, a provided composition comprises stapled peptides comprising a staple that contains an E-olefin. In some embodiments, otherwise identical stapled peptides that differ only in the E/Z configuration of staple olefin demonstrate different properties and/or activities as demonstrated herein.
stapled peptides with E-olefin in a staple may provide certain desirable properties and/or activities given the context.
stapled peptides with Z-olefin in a staple may provide certain desirable properties and/or activities given the context.
the present disclosure provides compositions comprising stapled peptides.
a composition comprises one and only one stereoisomer of a stapled peptide (e.g., E or Z isomer, and/or a single diastereomer/enantiomer with respect to a chiral center, etc.).
a composition comprises two or more stereoisomers (e.g., both E and Z isomers of one or more double bonds, and/or one or more diastereomers/enantiomers with respect to a chiral center, etc.).
a composition corresponds to a single peak in a chromatographic separation, e.g., HPLC.
a peak comprises one and only one stereoisomers.
a peak comprises two or more stereoisomers.
two staples may be bonded to the same atom of the peptide backbone, forming a stitched peptide.
a staple is pro-lock wherein one end of the staple is bonded to the alpha-carbon of a proline residue.
a staple is a staple illustrated in Tables S-1, S-2, S-3, S-4, S-5 or S-6 of WO 2022/261257.
the double bond in a (i, i+3) staple is Z.
the double bond in a (i, i+4) staple is Z.
the double bond in a (i, i+7) staple is Z.
the double bond in a (i, i+3) staple is E.
the double bond in a (i, i+4) staple is E.
the double bond in a (i, i+7) staple is E.
a staple comprises ⁇ S ⁇ .
a staple is formed from cysteine stapling. Certain such staples are described in, e.g., WO 2022/261257.
Certain useful agents (peptides prior to stapling and stapled peptides post stapling) and compositions thereof are presented in Table E2 or Table E3 as examples, which includes various amino acid residues and N- and C-terminus capping groups for various positions as examples; also illustrated are various stapling patterns, e.g., X 1 ⁇ X 4 ⁇ X 11 , X 1 ⁇ X 3 , X 3 ⁇ X 7 , X 3 ⁇ X 10 , X 4 ⁇ X 11 , X 7 ⁇ X 10 , X 7 ⁇ X 14 , X 10 ⁇ X 14 , etc.
provided technologies can deliver improved useful properties and/or activities.
a provided agent, a peptide, or a stapled peptide is a compound as described herein.
a provided agent has a structure selected from Table E2 or Table E3, or a salt thereof.
a provided agent is a stereoisomer of a structure selected from Table E2 or Table E3, or a salt thereof.
a provided agent is a stereoisomer, with respect to a chiral center bonded to two staples (e.g., in B4, B5, etc.), of a structure selected from Table E2 or Table E3, or a salt thereof.
a provided agent is a stereoisomer, with respect to olefin double bond(s) in staple(s), of a structure selected from Table E2 or Table E3, or a salt thereof.
a provided agent is a stereoisomer, with respect to olefin double bond(s) in staple(s) and/or a chiral center bonded to two staples (e.g., in B4, B5, etc.), of a structure selected from Table E2 or Table E3, or a salt thereof.
a provided composition is a composition described in Table E2 or Table E3.
a compound has the structure of or a salt thereof.
a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of
a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of or a salt thereof.
a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of or a salt thereof. In some
a compound has the structure of ) or a salt thereof. In some embodiments, a compound has the structure of or a salt thereof. In some embodiments, a
a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of or a salt thereof. In some embodiments, a
a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of or a salt thereof. In some embodiments, a compound has the structure of
a compound has the structure of or a salt thereof.
a double bond of a (i, i+2) staple is E.
a double bond of a (i, i+2) staple is Z.
a double bond of a (i, i+3) staple is E.
a double bond of a (i, i+3) staple is Z.
a double bond of a (i, i+7) staple is E.
a double bond of a (i, i+7) staple is Z.
both double bonds are E.
both double bonds are Z.
a (i, i+3) staple is E, and the other is Z.
a (i, i+3) staple is Z, and the other is E.
a (i, i+4) staple is E, and the other is Z.
a (i, i+4) staple is Z, and the other is E.
a double bond of a (i, i+7) staple is Z, and a double bond of a second staple (e.g., (i, i+2), (i, i+3), (i, i+4), etc.) is E.
a double bond of a (i, i+7) staple is Z, and a double bond of a second staple (e.g., (i, i+2), (i, i+3), (i, i+4), etc.) is Z.
a double bond of a (i, i+7) staple is E, and a double bond of a second staple (e.g., (i, i+2), (i, i+3), (i, i+4), etc.) is E.
a double bond of a (i, i+7) staple is E
a double bond of a second staple e.g., (i, i+2), (i, i+3), (i, i+4), etc.
two staples are bonded to a chiral center (e.g., a carbon atom in B5), and the chiral center is R.
two staples are bonded to a chiral center (e.g., a carbon atom in B5), and the chiral center is S.
a compound has the structure selected from below or a salt thereof:
an agent is SP-1-1 or a salt thereof. In some embodiments, an agent is SP-1-2 or a salt thereof. In some embodiments, an agent is SP-1-3 or a salt thereof. In some embodiments, an agent is SP-1- 4 or a salt thereof. In some embodiments, an agent is SP-1-5 or a salt thereof. In some embodiments, an agent is SP-1-6 or a salt thereof. In some embodiments, an agent is SP-1-7 or a salt thereof. In some embodiments, an agent is SP-1-8 or a salt thereof. In some embodiments, an agent is SP-2-1 or a salt thereof. In some embodiments, an agent is SP-2-2 or a salt thereof. In some embodiments, an agent is SP-2-3 or a salt thereof.
an agent is SP-2-4 or a salt thereof. In some embodiments, an agent is SP-2- 5 or a salt thereof. In some embodiments, an agent is SP-2-6 or a salt thereof. In some embodiments, an agent is SP-2-7 or a salt thereof. In some embodiments, an agent is SP-2-8 or a salt thereof. In some embodiments, an agent is SP-3-1 or a salt thereof. In some embodiments, an agent is SP-3-2 or a salt thereof. In some embodiments, an agent is SP-4-1 or a salt thereof. In some embodiments, an agent is SP-4-2 or a salt thereof. In some embodiments, an agent is SP-4-3 or a salt thereof. In some embodiments, an agent is SP-4- 4 or a salt thereof.
an agent is SP-4-5 or a salt thereof. In some embodiments, an agent is SP-4-6 or a salt thereof. In some embodiments, an agent is SP-4-7 or a salt thereof. In some embodiments, an agent is SP-4-8 or a salt thereof. In some embodiments, an agent is SP-5-1 or a salt thereof. In some embodiments, an agent is SP-5-2 or a salt thereof. In some embodiments, an agent is SP-5-3 or a salt thereof. In some embodiments, an agent is SP-5-4 or a salt thereof. In some embodiments, an agent is SP-5- 5 or a salt thereof. In some embodiments, an agent is SP-5-6 or a salt thereof. In some embodiments, an agent is SP-5-7 or a salt thereof.
an agent is SP-5-8 or a salt thereof. In some embodiments, an agent is SP-6 or a salt thereof. In some embodiments, an agent is SP-7-1 or a salt thereof. In some embodiments, an agent is SP-7-2 or a salt thereof. In some embodiments, an agent is SP-7-3 or a salt thereof. In some embodiments, an agent is SP-7-4 or a salt thereof. In some embodiments, an agent is SP-7- 5 or a salt thereof. In some embodiments, an agent is SP-7-6 or a salt thereof. In some embodiments, an agent is SP-7-7 or a salt thereof. In some embodiments, an agent is SP-7-8 or a salt thereof. In some embodiments, an agent is SP-8-1 or a salt thereof.
an agent is SP-8-2 or a salt thereof. In some embodiments, an agent is SP-8-3 or a salt thereof. In some embodiments, an agent is SP-8-4 or a salt thereof. In some embodiments, an agent is SP-8-5 or a salt thereof. In some embodiments, an agent is SP-8- 6 or a salt thereof. In some embodiments, an agent is SP-8-7 or a salt thereof. In some embodiments, an agent is SP-8-8 or a salt thereof. In some embodiments, an agent is SP-9-1 or a salt thereof. In some embodiments, an agent is SP-9-2 or a salt thereof. In some embodiments, an agent is SP-9-3 or a salt thereof. In some embodiments, an agent is SP-9-4 or a salt thereof.
an agent is SP-9-5 or a salt thereof. In some embodiments, an agent is SP-9-6 or a salt thereof. In some embodiments, an agent is SP-9- 7 or a salt thereof. In some embodiments, an agent is SP-9-8 or a salt thereof. In some embodiments, an agent is SP-10-1 or a salt thereof. In some embodiments, an agent is SP-10-2 or a salt thereof. In some embodiments, an agent is SP-10-3 or a salt thereof. In some embodiments, an agent is SP-10-4 or a salt thereof. In some embodiments, an agent is SP-10-5 or a salt thereof. In some embodiments, an agent is SP- 10-6 or a salt thereof. In some embodiments, an agent is SP-10-7 or a salt thereof.
an agent is SP-10-8 or a salt thereof.
an agent is SP-11-1 or a salt thereof.
an agent is SP-11-2 or a salt thereof.
an agent is SP-11-3 or a salt thereof.
an agent is SP-11-4 or a salt thereof.
an agent is SP- 11-5 or a salt thereof.
an agent is SP-11-6 or a salt thereof.
an agent is SP-11-7 or a salt thereof.
an agent is SP-11-8 or a salt thereof.
an agent is SP-12-1 or a salt thereof.
an agent is SP-12-2 or a salt thereof.
an agent is SP-12-3 or a salt thereof.
an agent is SP-
an agent is SP-12-5 or a salt thereof. In some embodiments, an agent is SP-12-6 or a salt thereof. In some embodiments, an agent is SP-12-7 or a salt thereof. In some embodiments, an agent is SP-12-8 or a salt thereof. In some embodiments, an agent is SP-13-1 or a salt thereof. In some embodiments, an agent is SP-13-2 or a salt thereof. In some embodiments, an agent is SP-
an agent is SP-13-4 or a salt thereof. In some embodiments, an agent is SP-13-5 or a salt thereof. In some embodiments, an agent is SP-13-6 or a salt thereof. In some embodiments, an agent is SP-13-7 or a salt thereof. In some embodiments, an agent is SP-13-8 or a salt thereof. In some embodiments, an agent is SP-14-1 or a salt thereof. In some embodiments, an agent is SP-
an agent is SP-14-3 or a salt thereof. In some embodiments, an agent is SP-14-4 or a salt thereof. In some embodiments, an agent is SP-14-5 or a salt thereof. In some embodiments, an agent is SP-14-6 or a salt thereof. In some embodiments, an agent is SP-14-7 or a salt thereof. In some embodiments, an agent is SP-14-8 or a salt thereof. In some embodiments, an agent is SP-
an agent is SP-15-2 or a salt thereof. In some embodiments, an agent is SP-15-3 or a salt thereof. In some embodiments, an agent is SP-15-4 or a salt thereof. In some embodiments, an agent is SP-15-5 or a salt thereof. In some embodiments, an agent is SP-15-6 or a salt thereof. In some embodiments, an agent is SP-15-7 or a salt thereof. In some embodiments, an agent is SP- 15-8 or a salt thereof.
an agent is selected from:
Agents e.g., peptides including stapled peptides, can contain various numbers of amino acid residues.
a length of a peptide agent is about 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 10- 20, 10-19, 10-18, 10-17, 10-16, 10-15, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.
a length is about 10 amino acid residues. In some embodiments, a length is about 11 amino acid residues. In some embodiments, a length is about 12 amino acid residues. In some embodiments, a length is about 13 amino acid residues. In some embodiments, a length is about 14 amino acid residues. In some embodiments, a length is about 15 amino acid residues. In some embodiments, a length is about 16 amino acid residues. In some embodiments, a length is about 17 amino acid residues. In some embodiments, a length is about 18 amino acid residues. In some embodiments, a length is about 19 amino acid residues. In some embodiments, a length is about 20 amino acid residues.
one or more staples independently comprise an olefin double bond (e.g., formed through olefin metathesis). In some embodiments, one or more staples independently comprise an amide group (e.g., formed through amidation). In some embodiments, at least one staple does not contain an olefin double bond. In some embodiments, there is at least one staple whose formation does not comprise reactions of olefins such as olefin metathesis and/or modification of olefin double bonds (e.g., hydrogenation, epoxidation, etc.).
a residue of a staple (e.g., B5) is so positioned that if its position is P (e.g., X 4 ), a first acidic amino acid residue is at position P-2 (e.g., X 2 ), a second acidic amino acid residue is positioned at P+1 (e.g., X 5 ), a third acidic amino acid residue is positioned at P+2 (e.g., X 6 ), a hydrophobic amino acid residue is positioned at P+4 (e.g., X 8 ), a first aromatic amino acid residue is positioned at P+5 (e.g., X 9 ), a second aromatic amino acid residue is positioned at P+8 (e.g., X 12 ), and/or a third aromatic amino acid residue is positioned at P+9 (e.g., X 13 ).
a staple is a (i, i+7) staple, and the other residue of the staple is positioned at P+7 (e.g., X 11 ).
a first acidic amino acid residue is at position P-2 (e.g., X 2 ).
a second acidic amino acid residue is positioned at P+1 (e.g., X 5 ).
a third acidic amino acid residue is positioned at P+2 (e.g., X 6 ).
a hydrophobic amino acid residue is positioned at P+4 (e.g., X 8 ).
a first aromatic amino acid residue is positioned at P+5 (e.g., X 9 ).
a second aromatic amino acid residue is positioned at P+8 (e.g., X 12 ).
a third aromatic amino acid residue is positioned at P+9 (e.g., X 13 ).
a first acidic amino acid residue is at position P-2 (e.g., X 2 ), a second acidic amino acid residue is positioned at P+1 (e.g., X 5 ), a first aromatic amino acid residue is positioned at P+5 (e.g., X 9 ), a second aromatic amino acid residue is positioned at P+8 (e.g., X 12 ), and a third aromatic amino acid residue is positioned at P+9 (e.g., X 13 ).
a first acidic amino acid residue is at position P-2 (e.g., X 2 ), a second acidic amino acid residue is positioned at P+1 (e.g., X 5 ), a third acidic amino acid residue is positioned at P+2 (e.g., X 6 ), a hydrophobic amino acid residue is positioned at P+4 (e.g., X 8 ), a first aromatic amino acid residue is positioned at P+5 (e.g., X 9 ), a second aromatic amino acid residue is positioned at P+8 (e.g., X 12 ), and a third aromatic amino acid residue is positioned at P+9 (e.g., X 13 ).
a stapled peptide agent comprises acidic amino acid residues at positions P-2 and P+1, and aromatic amino acid residues at positions P+5, P+8 and P+9. In some embodiments, a stapled peptide agent comprises acidic amino acid residues at positions P-2, P+1 and P+2, and aromatic amino acid residues at positions P+5, P+8 and P+9. In some embodiments, a stapled peptide agent comprises acidic amino acid residues at positions P-2 and P+1, a hydrophobic amino acid residue at position P+4, and aromatic amino acid residues at positions P+5, P+8 and P+9.
a stapled peptide agent comprises acidic amino acid residues at positions P-2, P+1 and P+2, a hydrophobic amino acid residue at position P+4, and aromatic amino acid residues at positions P+5, P+8 and P+9.
P is 3.
P is 4.
P is 5.
P is 6.
P is 7.
an amino acid residue at position P comprises two groups for stapling, e.g., B4, B5, B6, etc. In some embodiments, it is B4. In some embodiments, it is B5. In some embodiments, it is B6.
an agent comprises a staple and a first additional staple, e.g., a (i, i+3) or (i, i+4) staple.
a staple and a first additional staple are bonded to the same residue (e.g., B5, B6, etc.).
the other residue of a first additional residue is at position P-2 (e.g., when a moiety for stapling like a terminal olefin is in a P-terminal group which is considered a portion of X 1 ), P-3 or P-4.
an agent comprises a second additional staple, e.g., a (i, i+4) staple (e.g., stapling residues at positions P+6 (e.g., X 10 ) and P+10 (e.g., X 14 ), a (i, i+3) staple (e.g., stapling residues at positions P+3 (e.g., X 7 ) and P+6 (e.g., X 10 ), a (i, i+7) staple (e.g., stapling residues at positions P+3 (e.g., X 7 ) and P+10 (e.g., X 14 ), etc.).
a (i, i+4) staple e.g., stapling residues at positions P+6 (e.g., X 10 ) and P+10 (e.g., X 14
a (i, i+4) staple e.g., stapling residues
an agent comprises a second additional staple which is a (i, i+4) staple stapling residues at positions P+6 (e.g., X 10 ) and P+10 (e.g., X 14 ).
an agent comprises a third additional staple, e.g., a (i, i+4) staple stapling residues at positions P-1 (e.g., X 3 ) and P+3 (e.g., X 7 ).
stapled agents comprising so positioned staples and residues can provide various desired properties and activities.
positioning of one or more staples may be shifted relevant to various acidic, hydrophobic and/or aromatic amino acid residues described herein, e.g., in some embodiments, stapled peptide agents comprise stapled residues at position P and P+7 (and optionally P-3 or P-4), acidic amino acid residues are at positions P-1, and P+2, and aromatic amino acid residues at positions P+6, P+9 and P+10, and optionally an acid amino acid residue at P+3 and/or a hydrophobic amino acid residue at position P+5.
Beta-catenin [0166] Among other things, the present disclosure provides technologies for modulating one or more beta-catenin functions. In some embodiments, the present disclosure provides useful technologies for inhibiting one or more beta-catenin functions that are associated with cancer or hyperplasia.
a condition, disorder or disease is cancer.
Beta-catenin is reported to have various functions. For example, it can regulate and coordinate transcription of various genes. It is reported that high beta-catenin activity and/or expression levels may contribute to the development various conditions, disorders or diseases including cancer. Mutations and overexpression of beta-catenin are reported to be associated with conditions, disorders or diseases including many cancers including colorectal cancer, lung cancer, and breast cancer.
Dysregulation of the Wnt/ ⁇ - catenin signaling pathway has reportedly been linked to a number of conditions, disorders or diseases, including neurodegenerative diseases, psychiatric diseases, cancers, asthma, and even wound healing.
An abundance of published research, both clinical and preclinical, has indicated that hyperactivated Wnt/beta- catenin activity drives tumorigenesis and is required for tumor maintenance in various cancers.
Many Wnt inhibitors largely modulate this pathway at the extracellular ligand/receptor level, e.g., by preventing Wnt ligand secretion or by blocking Wnt ligand interaction with its receptors at the plasma membrane.
Wnt pathway- activating mutations are found in APC and/or CTNNB1, which are downstream of membrane-proximal events.
the present disclosure encompasses the recognition that many agents at the extracellular ligand/receptor level are insufficient to treat many relevant patients, e.g., those with downstream mutations/abnormalities.
Wnt pathway- activating mutations converge on beta-catenin/TCF node.
the present disclosure targets beta-catenin/TCF interaction, e.g., as a therapeutic approach.
Agents that can modulate beta-catenin functions are useful for various purposes including preventing and/or treating various conditions, disorders or diseases associated with beta-catenin.
beta-catenin is in a mutant form which comprises one or more mutations. In some embodiments, a mutant form is associated with a condition, disorder or disease.
Binding Sites may interact with various agents at various binding sites each independently comprising a set of amino acid residues that interact with binding agents. For example, certain binding sites are utilized for beta-catenin interactions with Axin, APC, C-cadherin, E-cadherin, TCF3, and Bcl9. For interactions with TCF3, it has been reported that two or more binding sites may be utilized simultaneously to interact with different portions of TCF3. See, e.g., Graham et al. Cell, Vol.103, 885–896, 2000.
provided agents bind to beta-catenin at a unique binding site.
provided agents interact with beta-catenin at a set of amino acid residues that are different from previously reported binding sites, e.g., those for Axin, APC, C-cadherin, E-cadherin, TCF3 or Bcl9.
provided agents interact with one or more or all (e.g., about 1-23, 1-20, 1-15, 1-10, 1-5, 5-23, 5-20, 5-15, 5-10, 6-23, 6-20, 6-15, 6-10, 7-23, 7-20, 7-15, 7-10, 8-23, 8-20, 8-15, 8-10, 9-23, 9-20, 9-15, 9-10, 10-23, 10-20, 10-15, 11-23, 11-20, 11-15, 12-23, 12-20, 12-15, 13-23, 13- 20, 13-15, 13-23, 14-20, 15-23, 15-20, 16-23, 16-20, 17-23, 17-20, 18-23, or 18-20, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, etc.) of a set of amino acid residues that are or correspond to amino acid residues in SEQ ID NO: 1, e.g., in some embodiments, the following amino acid residues of SEQ ID NO:
a set of amino acid residues are or correspond to amino acid residues A305, Y306, G307, N308, Q309, K312, R342, K345, V346, V349, Q375, Q379, N380, L382, W383, R386, N387, D413, N415, V416, T418, and C419 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues A305, Y306, G307, N308, Q309, K312, K345, V346, V349, Q379, N380, L382, W383, R386, N387, D413, N415, V416, T418, and C419 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues G307, K312, K345, W383, N387, D413, and N415 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues G307, K312, K345, Q379, L382, W383, N387, N415 and V416 of SEQ ID NO: 1. In some embodiments, a set of amino acid residues are or correspond to amino acid residues Y306, G307, K312, K345, Q379, L382, W383, N387, N415 and V416 of SEQ ID NO: 1. In some embodiments, a set of amino acid residues are or correspond to amino acid residues G307, K312, K345, Q379, L382, W383, R386, N387, N415 and V416 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues Y306, G307, K312, K345, Q379, L382, W383, R386, N387, N415 and V416 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues Y306, G307, K312, K345, V349, Q379, L382, W383, N387, N415 and V416 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues Y306, G307, K312, K345, V349, Q379, L382, W383, R386, N387, N415 and V416 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues G307, K312, K345, W383, and N387 of SEQ ID NO: 1.
a set of amino acid residues are or correspond to amino acid residues Y306, G307, K312, R386 and N387 of SEQ ID NO: 1.
provided agents interact with Y306 or an amino acid residue corresponding thereto.
provided agents interact with G307 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with K312 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with K345 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with V349 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with Q379 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with L382 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with W383 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with R386 or an amino acid residue corresponding thereto.
provided agents interact with N387 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with N415 or an amino acid residue corresponding thereto. In some embodiments, provided agents interact with V416 or an amino acid residue corresponding thereto. [0172] In some embodiments, a present agent interacts with a polypeptide whose sequence corresponds to aa 146-aa665 of human beta-catenin.
a present agent interacts with a polypeptide whose sequence comprises or is SEQ ID NO: 2: SVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIIL ASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCL WTLRNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEAL VRT (SEQ ID NO: 2). [0173] In some embodiments, all amino acid residues that interact with a provided agent is with SEQ ID NO: 2.
amino acid residues that interact with a provided agent interacts with an agent through hydrogen bonding, hydrophobic interactions or salt bridge.
a provided agent e.g., one or more amino acid residues in an agent
they are typically within a certain range of distances when, e.g., assessed using crystallography, NMR, etc.
certain amino acid residues reported to interact with one or more polypeptides are not significantly involved in interactions between provided and beta-catenin.
provided agents do not interact with an Axin binding site.
provided agents do not interact with a Bcl9 binding site.
provided agents do not interact with one or more or all of amino acid residues that are or correspond to N426, C429, K435, R469, H470, S473, R474, K508 and N516 of SEQ ID NO: 1. In some embodiments, provided agents do not interact with N426 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with C429 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with K435 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with R469 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with H470 or an amino acid residue corresponding thereto.
provided agents do not interact with S473 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with R474 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with K508 or an amino acid residue corresponding thereto. In some embodiments, provided agents do not interact with N516 or an amino acid residue corresponding thereto. [0175] In some embodiments, mutation of one or more amino acid residues outside of SEQ ID NO: 2 in beta-catenin does not significantly (e.g., not exceeding 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more) reduce interactions of beta-catenin with a provided agent.
mutation of one or more or all of amino acid residues that are or correspond to N426, C429, K435, R469, H470, S473, R474, K508 and N516 of SEQ ID NO: 1 does not significantly reduce interactions of beta-catenin with a provided agent.
mutation of N426 or an amino acid residue corresponding thereto does not significantly reduce interaction of beta-catenin with an agent.
mutation of Q379 or an amino acid residue corresponding thereto (e.g., to Ala, Glu, Phe, Trp, etc.) does not significantly reduce interaction of beta-catenin with an agent.
an agent binds to a TCF site of beta-catenin.
an agent interacts with one or more but not all amino acid residues that interact with TCF.
an agent interacts with one or more but not all amino acid residues that interact with an extended region of XTcf3-CBD.
an agent does not interact with beta-catenin amino acid residues that interact with a beta-hairpin module of XTcf3-CBD.
an agent does not interact with beta-catenin amino acid residues that interact with a helix module of XTcf3-CBD.
an agent competes with TCF for beta-catenin binding.
an agent competes with an extended region of TCF (e.g., Ala14-Glu24, or Asp16-Glu24, as described in Graham et al. Cell, Vol.103, 885–896, 2000) for beta-catenin binding.
an agent compared to an extended region of TCF, an agent does not compete, or competes at a less degree, with Axin for beta-catenin binding.
an agent does not compete, or competes at a less degree, with Bcl9 for beta-catenin binding. In some embodiments, compared to an extended region of TCF, an agent does not compete, or competes at a less degree, with a beta-hairpin module of XTcf3-CBD for beta-catenin binding. In some embodiments, compared to an extended region of TCF, an agent does not compete, or competes at a less degree, with a helix module of XTcf3-CBD for beta- catenin binding. In some embodiments, an agent competes with E-cadherin for beta-catenin binding.
the present disclosure provides complexes of peptides (e.g., polypeptides whose sequences are or comprises SEQ ID NO: 1 or 2) and provided agents.
polypeptides and provided agents interact with one or more or all amino acid residues as described herein, and optionally do not interact with one or more or all amino acid residues as described herein.
the present disclosure provides complexes comprising a provided agent and a beta-catenin polypeptide or a portion thereof. In some embodiments, a portion thereof comprises one or more or all of the interacting residues as described herein.
an agent and a beta- catenin polypeptide or a portion thereof interact with other at one or more or all of the interacting residues.
Certain Agents [0180]
the present disclosure provides an agent having the structure of formula I: R N ⁇ L P1 ⁇ L AA1 ⁇ L P2 ⁇ L AA2 ⁇ L P3 ⁇ L AA3 ⁇ L P4 ⁇ L AA4 ⁇ L P5 ⁇ L AA5 ⁇ L P6 ⁇ L AA6 ⁇ L P7 ⁇ R C , I or a salt thereof, wherein: R N is a peptide, an amino protecting group or R’ ⁇ L RN ⁇ ; each of L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 is independently L, wherein L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 comprise:
the present disclosure provides an agent having the structure of formula I: R N ⁇ L P1 ⁇ L AA1 ⁇ L P2 ⁇ L AA2 ⁇ L P3 ⁇ L AA3 ⁇ L P4 ⁇ L AA4 ⁇ L P5 ⁇ L AA5 ⁇ L P6 ⁇ L AA6 ⁇ L P7 ⁇ R C , I or a salt thereof, wherein: R N is a peptide, an amino protecting group or R’ ⁇ L RN ⁇ ; each of L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 is independently L, wherein L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 comprise: a first R’ group and a second R’ group which are taken together to form ⁇ L s ⁇ which is bonded to the atom to which a first R’ group is
a compound of formula I is a stapled peptide as described herein.
Various agents including embodiments for various variables, e.g., R’, R, R N , L P1 , L AA1 , L P2 , L AA2 , L P3 , L AA3 , L P4 , L AA4 , L P5 , L AA5 , L P6 , L AA6 , L P7 , R C , L AS , etc., are described in WO 2022/261257 and incorporated herein by reference.
L P1 is or comprises ⁇ [X] p ⁇ X 1 ⁇ , wherein each of p, X and X 1 is independently as described herein, and X 1 is bonded to L AA1 .
L P1 is or comprises ⁇ X 1 ⁇ .
such a carbon atom is an alpha carbon of an amino acid residue.
L AA1 is X 2 as described herein.
L P2 is or comprises ⁇ [X]pX 4 [X]p’ ⁇ , wherein each of p, p’, X and X 4 is independently as described herein.
L P2 is or comprises ⁇ [X]pX 3 X 4 [X]p’ ⁇ , wherein each X, X 3 and X 4 is independently an amino acid residue, and each of p and p’ is independently 0-10.
L P2 is or comprises ⁇ X 3 X 4 ⁇ , wherein each X 3 and X 4 is independently as described herein, and X 4 is bonded to L AA2 .
L AA2 is X 5 as described herein.
L P3 is or comprises ⁇ [X]pX 6 X 7 [X]p’ ⁇ , wherein each X, X 6 and X 7 is independently an amino acid residue, and each of p and p’ is independently 0-10.
L P3 is or comprises ⁇ X 6 X 7 ⁇ , wherein each X 6 and X 7 is independently an amino acid residue.
L AA3 is X 8 as described herein.
L P4 is or comprises ⁇ [X]pX 7 X 8 [X]p’ ⁇ , wherein each X, X 7 and X 8 is independently an amino acid residue, and each of p and p’ is independently 0- 10.
L P4 is or comprises ⁇ X 7 X 8 ⁇ , wherein each X 7 and X 8 is independently as described herein, and X 8 is bonded to L AA4 .
L AA4 is X 9 as described herein.
L P5 is or comprises ⁇ [X]pX 11 [X]p’ ⁇ , wherein each variable is independently as described herein.
L P5 is or comprises ⁇ X 10 X 11 ⁇ , wherein each X 10 and X 11 is independently as described herein, and X 11 is bonded to L AA5 .
L AA5 is or comprises amino acid residue.
L AA5 is or comprises an amino acid residue that comprises a side chain comprising an aromatic group. In some embodiments, L AA5 is X 12 as described herein. In some embodiments, L P6 is or comprises an amino acid residue. In some embodiments, L P6 is or comprises a peptide. In some embodiments, L AA6 is X 13 as described herein. In some embodiments, L P7 is or comprises ⁇ X 14 ⁇ [X]p’ ⁇ , wherein p’ is 0-10. In some embodiments, X 14 is bonded to L AA6 . [0185] In some embodiments, an agent of formula I is a stapled peptide as described herein.
an agent of formula I is an agent selected from Table E2 or a pharmaceutically acceptable salt thereof. In some embodiments, an agent of formula I is an agent selected from Table E3 or a pharmaceutically acceptable salt thereof. [0186] Among other things, the present disclosure provides agents, e.g. peptides, that can bind to beta- catenin.
an agent is or comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 wherein each of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 and X 14 is independently an amino acid residue.
an agent is or comprises [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein each of p0, p15, p16 and p17 is independently 0 or 1, and each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue.
each of X 2 and X 5 is independently an acidic residue as described herein.
each of X 2 , X 5 and X 6 is independently an acidic residue as described herein.
each of X 9 , X 12 and X 13 are independently an amino acid residue comprising a side chain that comprises an aromatic group.
X 2 is an acidic residue.
X 2 comprises a side chain that comprises ⁇ COOH or a derivative thereof. In some embodiments, X 2 comprises a side chain that comprises ⁇ COOH. In some embodiments, X 2 is Asp. Various other amino acid residues for X 2 are described else in the present disclosure.
X 5 is an acidic residue. In some embodiments, X 5 comprises a side chain that comprises ⁇ COOH or a derivative thereof. In some embodiments, X 5 comprises a side chain that comprises ⁇ COOH. In some embodiments, X 5 is Asp. Various other amino acid residues for X 5 are described else in the present disclosure. [0191] In some embodiments, X 6 is an acidic residue.
X 6 comprises a side chain that comprises ⁇ COOH or a derivative thereof. In some embodiments, X 6 comprises a side chain that comprises ⁇ COOH. In some embodiments, X 6 is Asp. Various other amino acid residues for X 6 are described else in the present disclosure. [0192] In some embodiments, X 9 comprises a side chain that comprises an aromatic group. In some embodiments, X 9 comprises a side chain that comprises ⁇ R, wherein R is an optionally substituted group selected from phenyl, 10-membered bicyclic aryl, 5-membered heteroaryl having 1-3 hetereoatoms, and 9-10 membered bicyclic heteroaryl having 1-5 heteroatoms.
each heteroatom is independently sleeved from nitrogen, oxygen and sulfur.
X 9 is Phe.
Various other amino acid residues for X 9 are described else in the present disclosure.
X 12 comprises a side chain that comprises an aromatic group.
X 12 comprises a side chain that comprises ⁇ R, wherein R is an optionally substituted group selected from phenyl, 10-membered bicyclic aryl, 5-membered heteroaryl having 1-3 hetereoatoms, and 9-10 membered bicyclic heteroaryl having 1-5 heteroatoms.
each heteroatom is independently sleeved from nitrogen, oxygen and sulfur.
X 12 is 3Thi. In some embodiments, X 12 is 2F3MeF. In some embodiments, X 12 is Phe. Various other amino acid residues for X 12 are described else in the present disclosure. [0194] In some embodiments, X 13 comprises a side chain that comprises an aromatic group. In some embodiments, X 13 comprises a side chain that comprises ⁇ R, wherein R is an optionally substituted group selected from phenyl, 10-membered bicyclic aryl, 5-membered heteroaryl having 1-3 hetereoatoms, and 9-10 membered bicyclic heteroaryl having 1-5 heteroatoms. In some embodiments, each heteroatom is independently sleeved from nitrogen, oxygen and sulfur.
X 13 is BtzA. In some embodiments, X 13 is 34ClF. In some embodiments, X 13 is 2NapA. Various other amino acid residues for X 13 are described else in the present disclosure.
a peptide is a stapled peptide.
an agent is or comprises a peptide, wherein a peptide is a stapled peptide. In some embodiments, a peptide is a stitched peptide. In some embodiments, a peptide comprises three or more staples as described herein.
a peptide comprises three or more staples within a region having a length of, e.g., 11-15, such as 11, 14, etc., amino acid residues as described herein.
such a peptide provides improved rigidity, activity, delivery, solubility, and/or other desired properties comprising a reference peptide that is not stapled or that comprises fewer staples.
the present disclosure provides an agent, e.g., a peptide, comprising X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 , wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , and X 14 are each independently an amino acid residue and comprises two or more pairs of amino acid residues, wherein each pair of amino acid residues are independently two amino acid residues suitable for stapling or stapled.
an agent e.g., a peptide, comprising X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 , wherein X 1 , X 2 , X
the present disclosure provides an agent, e.g., a peptide, comprising X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 , wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , and X 14 are each independently an amino acid residue and comprises two or more pairs of amino acid residues, wherein each pair of amino acid residues are independently three amino acid residues suitable for stapling or stapled.
an agent e.g., a peptide, comprising X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 , wherein X 1 , X 2 , X
the present disclosure provides an agent, e.g., a peptide, comprising [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein each of p0, p15, p16 and p17 is independently 0 or 1, and X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 are each independently an amino acid residue and comprises two or more pairs of amino acid residues, wherein each pair of amino acid residues are independently two amino
the present disclosure provides an agent, e.g., a peptide, comprising [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein each of p0, p15, p16 and p17 is independently 0 or 1, and X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 are each independently an amino acid residue and comprises three or more pairs of amino acid residues, wherein each pair of amino acid residues are independently two amino acid residues suitable
each amino acid residue in such pairs of amino acid residues are independently selected from X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , and X 14 .
each pair is independently not stapled.
one or more pairs are independently stapled.
two or more pairs are independently stapled.
a pair is X 1 and X 4 . In some embodiments, a pair is X 4 and X 11 . In some embodiments, a pair is X 1 and X 3 . In some embodiments, a pair is X 4 and X 11 . In some embodiments, a pair is X 10 and X 14 . In some embodiments, a pair is X 7 and X 10 .
a pair is X 7 and X 14 . In some embodiments, a pair is X 3 and X 7 . [0200] In some embodiments, a pair is X 1 and X 14 and a pair is X 4 and X 11 . In some embodiments, a pair is X 1 and X 14 , a pair is X 4 and X 11 and a pair is X 10 and X 14 . In some embodiments, a pair is X 1 and X 14 , a pair is X 4 and X 11 and a pair is X 7 and X 10 .
a pair is X 1 and X 14 , a pair is X 4 and X 11 and a pair is X 7 and X 14 .
a pair is X 1 and X 14 , a pair is X 4 and X 11 , a pair is X 3 and X 7 , and a pair is X 7 and X 14 .
each pair is independently a pair of amino acid residues suitable for stapling.
each pair is independently stapled.
a pair is X 1 and X 3 , a pair is X 4 and X 11 , and a pair is X 10 and X 14 .
each pair is independently a pair of amino acid residues suitable for stapling. In some embodiments, each pair is independently stapled.
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16
X 2 comprises a side chain comprising an acidic or a polar group. In some embodiments, X 2 comprises a side chain comprising an acidic group. In some embodiments, X 2 comprises a side chain comprising a polar group. In some embodiments, X 5 comprises a side chain comprising an acidic or a polar group. In some embodiments, X 5 comprises a side chain comprising an acidic group. In some embodiments, X 5 comprises a side chain comprising a polar group. In some embodiments, X 13 comprises a side chain comprising an optionally substituted aromatic group.
two or more of X 1 , X 3 , X 4 , X 7 , X 10 , X 11 and X 14 are each independently an amino acid residue suitable for stapling, or are each independently stapled.
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11
three or more of X 1 , X 3 , X 4 , X 7 , X 10 , X 11 and X 14 are each independently an amino acid residue suitable for stapling, or are each independently stapled. In some embodiments, four or more of X 1 , X 3 , X 4 , X 7 , X 10 , X 11 and X 14 are each independently an amino acid residue suitable for stapling, or are each independently stapled. In some embodiments, five of X 1 , X 3 , X 4 , X 7 , X 10 , X 11 and X 14 are each independently an amino acid residue suitable for stapling, or are each independently stapled.
X 1 and X 4 are each independently an amino acid residue suitable for stapling.
X 1 and X 3 are each independently an amino acid residue suitable for stapling.
X 4 and X 11 are each independently an amino acid suitable for stapling.
X 1 , X 4 , and X 11 are each independently an amino acid residue suitable for stapling.
X 10 and X 14 are each independently an amino acid residue suitable for stapling.
X 7 and X 10 are each independently an amino acid residue suitable for stapling.
X 7 and X 14 are each independently an amino acid residue suitable for stapling.
X 3 and X 7 are each independently an amino acid residue suitable for stapling.
X 1 and X 4 are connected by a staple.
X 1 and X 3 are connected by a staple.
X 4 and X 11 are connected by a staple.
X 1 and X 4 connected by a staple, and X 4 and X 11 are connected by a staple.
X 10 and X 14 are connected by a staple.
X 7 and X 10 are connected by a staple.
X 7 and X 14 are connected by a staple.
X 3 and X 7 are connected by a staple.
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group;
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group;
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group;
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group;
the present disclosure provides an agent, which is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group;
X 2 comprises a side chain comprising an acidic (e.g., ⁇ COOH) or a polar group. In some embodiments, X 2 comprises a side chain comprising an acid group. In some embodiments, X 5 comprises a side chain comprising an acidic or a polar group. In some embodiments, X 5 comprises a side chain comprising an acid group. In some embodiments, X 6 comprises a side chain comprising an acidic or a polar group. In some embodiments, X 6 comprises a side chain comprising an acid group. In some embodiments, X 9 comprises a side chain comprising an optionally substituted aromatic group.
an acidic e.g., ⁇ COOH
X 2 comprises a side chain comprising an acid group.
X 5 comprises a side chain comprising an acidic or a polar group. In some embodiments, X 5 comprises a side chain comprising an acid group. In some embodiments, X 6 comprises a side chain comprising an acidic or a
X 12 comprises a side chain comprising an optionally substituted aromatic group.
X 13 comprises a side chain comprising an optionally substituted aromatic group.
X 2 and X 5 each independently comprise a side chain comprising an acidic or a polar group.
X 2 and X 6 each independently comprise a side chain comprising an acidic or a polar group.
X 5 and X 6 each independently comprise a side chain comprising an acidic or a polar group.
X 2 and X 5 each independently comprise a side chain comprising an acidic group.
X 2 and X 6 each independently comprise a side chain comprising an acidic group.
X 5 and X 6 each independently comprise a side chain comprising an acidic group.
X 2 , X 5 and X 6 each independently comprise a side chain comprising an acidic or a polar group.
X 2 , X 5 and X 6 each independently comprise a side chain comprising an acidic group.
each of X 9 and X 12 independently comprises a side chain comprising an optionally substituted aromatic group.
each of X 9 and X 13 independently comprises a side chain comprising an optionally substituted aromatic group.
each of X 9 , X 12 and X 13 independently comprises a side chain comprising an optionally substituted aromatic group.
each of X 2 and X 5 independently comprises a side chain comprising an acidic group (e.g., ⁇ COOH), and each of X 9 , X 12 and X 13 independently comprises a side chain comprising an optionally substituted aromatic group.
each of X 2 , X 5 and X 6 independently comprises a side chain comprising an acidic group (e.g., ⁇ COOH), and each of X 9 , X 12 and X 13 independently comprises a side chain comprising an optionally substituted aromatic group.
amino acid residues e.g., those of amino acids having the structure of formula A-I, A-II, A-III, A-IV, A-V, A-VI, etc. as described herein or in WO 2022/261257 and incorporated herein by reference
Certain examples are described herein for X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , etc.
X 1 is an amino acid reside suitable for stapling.
X 1 is stapled.
X 1 is PL3.
⁇ Cy ⁇ is optionally substituted phenylene.
X 2 is a residue of amino acid (e.g., of formula A-I, A-II, A-III, A-IV, A-V, A-VI, etc. or a salt thereof) that comprises an acidic or polar group.
X 2 is a residue of amino acid whose side chain comprises an acidic group (in some embodiments, may be referred to as an “acidic amino acid residue”).
X 2 is Asp.
X 2 is Glu.
X 3 is a hydrophobic amino acid residue.
X 3 is a residue of an amino acid whose side chain is C 1-10 aliphatic optionally substituted with one or more non-polar and non-charged groups. In some embodiments, it is a residue of an amino acid whose side chain is C 1-10 aliphatic optionally substituted with one or more hydrophobic substituents. In some embodiments, it is a residue of an amino acid whose side chain is C 1-10 aliphatic. In some embodiments, it is a residue of an amino acid whose side chain is C 1-10 alkyl.
X 3 is a residue of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Npg, Leu, Cha, Val, nLeu, Ile, CypA, CyLeu, Chg, DiethA, Ala, Aib, OctG, or Cba.
X 3 is a residue of Npg, Leu, or Cha.
X 3 is a residue of Npg.
X 3 is a residue of Leu.
X 3 is a residue of Cha.
X 3 is a residue of Val.
X 3 is a residue of nLeu.
X 3 is a residue of Ile. In some embodiments, X 3 is a residue of CypA. In some embodiments, X 3 is a residue of CyLeu. In some embodiments, X 3 is a residue of Chg. In some embodiments, X 3 is a residue of DiethA. In some embodiments, X 3 is a residue of Ala. In some embodiments, X 3 is a residue of Aib. In some embodiments, X 3 is a residue of OctG. In some embodiments, X 3 is a residue of Cba. In some embodiments, X 4 is residue of an amino acid suitable for stapling.
X 4 is a residue of an amino acid which comprises two functional groups suitable for stapling. In some embodiments, X 4 is a residue of an amino acid which comprises two olefins, e.g., two terminal olefins. In some embodiments, X 4 is stapled. In some embodiments, X 4 is stapled with X 1 and/or X 11 . In some embodiments, X 4 is B5. In some embodiments, X 4 is R5. In some embodiments, X 4 is R4. In some embodiments, X 4 is R6. In some embodiments, X 4 is S5. In some embodiments, X 6 is a residue of amino acid that comprises an acidic or polar group.
X 6 is a residue of amino acid whose side chain comprises an acidic group, e.g., a ⁇ COOH group or a salt form thereof.
X 6 is a residue of 3COOHF, TfeGA, Asp, [CH2CMe2CO2H]TriAzDap, Glu, 2OH3COOHF, 4OH3COOHF, 4COOHF, 2COOHF, 5F3Me2COOHF, 4F3Me2COOHF, 5F3Me3COOHF, 4F3Me3COOHF, 3F2COOHF, or dGlu.
X 6 is a residue of 3COOHF, TfeGA, Asp, or [CH2CMe2CO2H]TriAzDap. In some embodiments, X 6 is a residue of 3COOHF. In some embodiments, X 6 is a residue of TfeGA. In some embodiments, X 6 is a residue of Asp. In some embodiments, X 6 is a residue of Glu. In some embodiments, X 7 is a hydrophobic amino acid residue, e.g., those described for X 3 . In some embodiments, X 7 is a residue of an amino acid whose side chain is C 1-10 aliphatic optionally substituted with one or more non-polar and non-charged groups.
X 7 is a residue of Ala. In some embodiments, X 7 is a residue of Aib. In some embodiments, X 7 is a residue of nLeu. In some embodiments, X 7 is a residue of Cha. In some embodiments, X 7 is a residue of Npg. In some embodiments, X 7 is a residue of sAla. In some embodiments, X 7 is a residue of Val. In some embodiments, X 7 is a residue of CyLeu.. In some embodiments, X 7 is a residue of Leu. In some embodiments, X 7 is a residue of Cpg. In some embodiments, X 7 is a residue of Cbg.
X 7 is a residue of aMeL. In some embodiments, X 7 is a residue of DaMeL. In some embodiments, X 7 is a residue of aMeV. In some embodiments, X 8 is a hydrophobic amino acid residue, e.g., those described for X 3 . In some embodiments, X 8 is a residue of Ala. In some embodiments, X 8 is a residue of Aib. In some embodiments, X 8 is a residue of Cpg. In some embodiments, X 8 is a residue of Val. In some embodiments, X 8 is a residue of Leu. In some embodiments, X 8 is a residue of nLeu.
X 8 is a residue of Cba.
X 9 comprises a side chain comprising an optionally substituted aromatic group.
X 9 is an aromatic amino acid residue as described herein.
X 9 is Phe.
X 9 is 3COOHF.
X 9 is 2NapA.
X 9 is Tyr.
X 9 is 3Thi.
X 9 is 4FF.
X 9 is 4ClF.
X 9 is 4BrF.
X 9 is 3FF.
X 9 is 3ClF.
X 9 is 3BrF. In some embodiments, X 9 is 2FF. In some embodiments, X 9 is 3OMeF. In some embodiments, X 9 is 4CNF. In some embodiments, X 9 is 3CNF. In some embodiments, X 9 is 4MeF. In some embodiments, X 9 is 3MeF. In some embodiments, X 10 is a residue of an amino acid whose side chain comprises a polar group. In some embodiments, X 10 is Asn, Gln, Lys, etc. In some embodiments, X 10 is stapled. In some embodiments, X 10 is not stapled. In some embodiments, X 10 is stapled with X 14 .
a staple comprises ⁇ C(O)NH ⁇ .
X 12 is a residue of an amino acid selected from Phe, 1NapA, 2cmbF, 2F3MeF, 2ClF, 2FurA, 2OMeF, 2MeF, 2BrF, 2CNF, 2NO2F, 2PyrA, 2Thi, 3PyrA, 3Thi, or 4PyrA.
X 12 is a residue of Phe.
X 12 is a residue of 2F3MeF.
X 12 is a residue of 2ClF.
X 12 is a residue of 2FurA.
X 12 is a residue of 2OMeF.
X 12 is a residue of 2MeF. In some embodiments, X 12 is a residue of 2BrF. In some embodiments, X 12 is a residue of 2CNF. In some embodiments, X 12 is a residue of 2NO2F. In some embodiments, X 12 is a residue of 2PyraA. In some embodiments, X 12 is a residue of 3PyrA. In some embodiments, X 12 is a residue of 3Thi. In some embodiments, X 12 is a residue of 4PyrA. In some embodiments, X 12 is a residue of His. In some embodiments, X 12 is a residue of 1NapA.
X 12 is a residue of 2Thi. In some embodiments, X 12 is a residue of 2cmbF. In some embodiments, X 13 comprises a side chain which is or comprises an optionally substituted aromatic group. In some embodiments, X 13 is an aromatic amino acid residue as described herein.
X 13 is a residue of BztA, 34ClF, 3Thi, Phe, 34MeF, 3BrF, 7FBztA, 2BrF, 3F3MeF, 4F3MeF, RbMe2NapA, RbMeBzta, SbMeBzta, 5IndA, 7ClBztA, 7MeBztA, 2ClF, 3ClF, 4BrF, 4ClF, or 3MeF.
X 13 is BztA.
X 13 is 34ClF.
X 13 is 3Thi.
X 13 is Phe.
X 14 is an amino acid residue suitable for stapling.
X 14 is stapled.
X 14 is stapled with X 10 as described herein.
X 14 is stapled with X 7 as described herein.
X 14 is GlnR, Lys, sAla, Gln, Cys, TriAzLys, AsnR, hGlnR, 4PipA, sAbu, Orn, dGlnR, [4mampiperidine]GlnR, [39N2spiroundecane]GlnR, [29N2spiroundecane]GlnR, iPrLys, sCH2S, [diaminobutane]GlnR, or [4aminopiperidine]GlnR.
X 14 is GlnR.
X 14 is Lys.
X 14 is sAla. In some embodiments, X 14 is Gln. In some embodiments, X 14 is Cys. In some embodiments, X 14 is TriAzLys. In some embodiments, X 14 is AsnR. In some embodiments, X 14 is hGlnR. In some embodiments, X 14 is 4PipA. In some embodiments, X 14 is sAbu. In some embodiments, X 14 is Orn. In some embodiments, X 14 is dGlnR. In some embodiments, X 14 is [4mampiperidine]GlnR. In some embodiments, X 14 is [39N2spiroundecane]GlnR.
X 14 is [29N2spiroundecane]GlnR. In some embodiments, X 14 is iPrLys. In some embodiments, X 14 is sCH2S. In some embodiments, X 14 is [diaminobutane]GlnR. In some embodiments, X 14 is [4aminopiperidine]GlnR. In some embodiments, X 14 is a C-terminus amino acid residue. In some embodiments, p15 is 1. In some embodiments, p15 is 0.
X 15 is a residue of Ala, Leu, Val, Aib, MorphNva, Thr, dAla, dLeu, [BiotinPEG8]Lys, Glu, or AzLys.
p16 is 1. In some embodiments, p16 is 0. In some embodiments, p17 is 1. In some embodiments, p17 is 0. In some embodiments, p18 is 1. In some embodiments, p18 is 0. In some embodiments, p19 is 1. In some embodiments, p19 is 0. In some embodiments, p20 is 1. In some embodiments, p20 is 0. In some embodiments, p21 is 1. In some embodiments, p21 is 0.
an agent is or comprises a peptide having the structure of: R N ⁇ [X] p ⁇ [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 ⁇ [X] p’ ⁇ R C , or a salt thereof, wherein: each X is independently an amino acid residue; each p and p’ is independently 0-10; R N is independently a peptide, an amino protecting group or R’ ⁇ L RN ⁇ ; R C is independently a peptide, a carboxyl protecting group, ⁇ L RC ⁇ R’, ⁇ O ⁇ L RC
p is 0. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, p is 9. In some embodiments, p is 10. [0216] In some embodiments, p’ is 0. In some embodiments, p’ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p’ is 1. In some embodiments, p’ is 2. In some embodiments, p’ is 3.
R N is an N-terminus capping group. In some embodiments, R N is ⁇ C(O)R, wherein R is as described herein. In some embodiments, R is ⁇ H. In some embodiments, R is optionally substituted C 1-6 aliphatic. In some embodiments, R is optionally substituted C 1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R N is Ac.
R N is a group suitable for stapling, or is stapled. In some embodiments, R N is 4pentenyl. In some embodiments, R N is 5hexenyl. In some embodiments, R N is BzAm2OAllyl.
R N is Ac, NPyroR3, 5hexenyl, 4pentenyl, Bua, C3a, Cpc, Cbc, CypCO, Bnc, CF3CO, 2PyCypCO, 4THPCO, Isobutyryl, Ts, 15PyraPy, 2PyBu, 4PymCO, 4PyPrpc, 3IAPAc, 4MePipzPrpC, MePipAc, MeImid4SO2, BzAm2OAllyl, Hex, 2PyzCO, 3Phc3, MeOPr, lithocholate, 2FPhc, PhC, MeSO2, Isovaleryl, EtHNCO, TzPyr, 8IAP, 3PydCO, 2PymCO, 5PymCO, 1Imidac, 2F2PyAc, 2IAPAc, 124TriPr, 6QuiAc, 3PyA
R C is a C-terminus capping group. In some embodiments, R C comprises a PEG moiety. In some embodiments, R C is ⁇ O ⁇ L RC ⁇ R’ wherein each of L RC and R’ is independently as described herein. In some embodiments, R C is ⁇ OR’, wherein R’ is optionally substituted C 1 -C 10 aliphatic. In some embodiments, R C is ⁇ OR’, wherein R’ is optionally substituted C 1 -C 10 alkyl. In some embodiments, R C is ⁇ OR’, wherein R’ is optionally substituted C 1-30 heteroaliphatic having 1-10 heteroatoms.
R C is ⁇ L RC ⁇ R’, wherein one or more methylene unit of L RC are replaced with ⁇ O ⁇ and R’ is as described herein.
R C is ⁇ O ⁇ L RC ⁇ R’, wherein L RC is optionally substituted C 1–6 alkylene and R’ is as described herein.
R C is ⁇ O ⁇ L RC ⁇ R’, wherein L RC is optionally substituted C 1–6 alkylene and R’ is an optionally substituted group selected from C 6-30 aryl and 5-30 membered heteroaryl having 1-10 heteroatoms.
R C is ⁇ OR’ wherein R’ is optionally substituted C 6-30 arylaliphatic. In some embodiments, R C is ⁇ N(R’) 2 wherein each R’ is independently as described herein. In some embodiments, R C is ⁇ NHR’ wherein R’ is as described herein. In some embodiments, R C is ⁇ N(R) 2 wherein each R is independently as described herein. In some embodiments, R C is ⁇ NHR wherein R is as described herein. In some embodiments, R is ⁇ H. In some embodiments, R is optionally substituted C 1-6 aliphatic. In some embodiments, R is optionally substituted C 1-6 alkyl. In some embodiments, R is methyl.
R is ethyl. In some embodiments, R C is ⁇ NH 2 . In some embodiments, R C is ⁇ NHEt. [0219] In some embodiments, R C is ⁇ NHC(CH 3 )CH 2 OH. In some embodiments, R C is ⁇ (S) ⁇ NHC(CH 3 )CH 2 OH. In some embodiments, R C is ⁇ (R) ⁇ NHC(CH 3 )CH 2 OH. In some embodiments, R C is C In some embodiments, R is . In some embodiments, R C is .
R C is C In some embodiments, R is [0220] In some embodiments, R C is ⁇ Alaol, wherein the amino group of ⁇ Alaol is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is C In some embodiments, R is ⁇ dAlaol, wherein the amino group of ⁇ dAlaol is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is In some embodiments, R C is ⁇ Prool, wherein the amino group of ⁇ Prool is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is In some embodiments, R C is ⁇ Throl, wherein the amino group of ⁇ Throl is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is C In some embodiments, R is ⁇ Serol, wherein the amino group of ⁇ Serol is bonded to the last ⁇ C(O) ⁇ of
an agent is or comprises a peptide analog. In some embodiments, an agent is or comprises an analog of a peptide described herein or a portion thereof. In some embodiments, an agent is or comprises an analog of a peptide described herein. In some embodiments, in a peptide analog, one or more of amino (e.g., ⁇ NH ⁇ ), carbonyl or amide group (e.g., ⁇ C(O)NH ⁇ ) are independently replaced with L R each of which is independently L as described herein. In some embodiments, in an analog of a peptide, one or more of backbone amino, carbonyl or amide group are independently replaced with L R as described herein.
L R is a bivalent moiety with a length of 0, 1, 2, 3, 4, or 5 atoms between its two covalent bonds (e.g., for ⁇ CH 2 ⁇ , one atom (one carbon atom between the two covalent bonds); for ⁇ CH 2 CH 2 ⁇ , two atoms (two carbon atoms between the two covalent bonds), etc.).
a length is 1 atom.
a length is 2 atoms.
a length is 3 atoms.
a length is 4 atoms.
a length is 5 atoms.
the length of L R (or L, or any moiety that can be L), measured by the number of atoms between its two covalent bonds, are the same as, or no more than about 1 or 2 atoms longer or shorter than, a group it replaces. In some embodiments, the length of L R (or L, or any moiety that can be L), measured by the number of atoms between its two covalent bonds, are the same as a group it replaces. In some embodiments, L R is a covalent bond. In some embodiments, L R is optionally substituted ⁇ CH 2 ⁇ . In some embodiments, L R is optionally substituted ⁇ CH 2 ⁇ CH2 ⁇ .
a carbonyl group is replaced with optionally substituted ⁇ CH 2 ⁇ .
an amide group is replaced with a covalent bond.
an amide group is replaced with optionally substituted ⁇ CH 2 ⁇ .
an amide group is replaced with optionally substituted ⁇ CH 2 ⁇ CH 2 ⁇ .
an amide group is replaced with ⁇ C(R’) 2 ⁇ C(R’) 2 ⁇ .
an amide group is replaced with optionally substituted ⁇ CHR’ ⁇ CHR’ ⁇ .
Amino Acids [0223] As appreciated by those skilled in the art, various amino acids may be utilized in accordance with the present disclosure. For example, both naturally occurring and non-naturally occurring amino acids can be utilized in accordance with the present disclosure.
an amino acid is a compound comprising an amino group that can form an amide group with a carboxyl group and a carboxyl group.
an amino acid is an alpha amino acid.
an amino acid is a beta- amino acid.
an amino acid is a D-amino acid.
an amino acid is a L-amino acid.
an amino acid is an naturally encoded amino acid, e.g., in mammalian cells.
an amino acid is a compound having the structure of formula A-I: N(R a1 ) 2 ⁇ L a1 ⁇ C(R a2 )(R a3 ) ⁇ L a2 ⁇ COOH, A-I or a salt thereof, wherein: each of R a1 , R a2 , R a3 is independently ⁇ L a ⁇ R’; each of L a , L a1 and L a2 is independently L; each L is independently a covalent bond, or an optionally substituted, bivalent C 1 -C 25 aliphatic or heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇
a compound having the structure of formula A-I or a salt thereof has the structure of NH(R a1 ) ⁇ L a1 ⁇ C(R a2 )(R a3 ) ⁇ L a2 ⁇ COOH or a salt thereof.
a ring moiety of, e.g., ⁇ Cy ⁇ , R (including those formed by R groups taken together), etc. is monocyclic. In some embodiments, a ring moiety is bicyclic or polycyclic.
a monocyclic ring is an optionally substituted 3-10 (3, 4, 5, 6, 7, 8, 9, or 10, 3-8, 3-7, 4-7, 4-6, 5-6, etc.) membered, saturated, partially unsaturated or aromatic ring having 0-5 heteroatoms.
each monocyclic ring unit of a bicyclic or polycyclic ring moiety is independently an optionally substituted 3-10 (3, 4, 5, 6, 7, 8, 9, or 10, 3-8, 3-7, 4-7, 4-6, 5-6, etc.) membered, saturated, partially unsaturated or aromatic ring having 0-5 heteroatoms.
each heteroatom is independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
each heteroatom is independently selected from oxygen, nitrogen, and sulfur.
L a1 is a covalent bond.
a compound of formula A- 1 is of the structure NH(R a1 ) ⁇ C(R a2 )(R a3 ) ⁇ L a2 ⁇ COOH.
L a2 is a covalent bond.
a compound of formula A- 1 is of the structure NH(R a1 ) ⁇ C(R a2 )(R a3 ) ⁇ L a2 ⁇ COOH.
L a1 is a covalent bond and L a2 is a covalent bond.
a compound of formula A-1 is of the structure NH(R a1 ) ⁇ C(R a2 )(R a3 ) ⁇ COOH.
an amino acid is suitable for stapling.
an amino acid comprises a terminal olefin. Certain such amino acids are exemplified herein (e.g., those described in or utilized in peptides of various Tables).
an agent comprises a detectable moiety, which can either be detected directly or indirectly.
a detectable moiety is or comprises a fluorescent group.
a detectable moiety is or comprises a biotin moiety.
a detectable moiety is connected to the rest of an agent at an amino acid residue, e.g., through a side chain, optionally through a linker (e.g., L as described herein).
a detectable moiety is ⁇ N 3 , which may be detected after a click chemistry reaction with a labeled agent comprising an alkyne.
a compound e.g., an amino acid or a protected and/or activated form thereof or a salt thereof comprises 1) a first group which is an optionally protected amino group, 2) a second group which is an optionally protected and/or activated carboxyl group, and 3) a side chain (typically bonded to an atom between the first and second groups (“a side chain attachment atom”)) which comprises an optionally protected and/or activated carboxyl group and a) an optionally substituted ring (which ring is typically between the optionally protected and/or activated carboxyl group of the side chain and a side chain attachment atom) or b) an amino group (which amino group is typically between the optionally protected and/or activated carboxyl group of the side chain and a side chain attachment atom).
a provided compound is an optionally protected and/or activated amino acid or a salt thereof, wherein the side chain of the amino acid comprises an optionally protected and/or activated carboxyl group, and an optionally substituted ring or an amino group, wherein the optionally substituted ring or an amino group is between the optionally protected and/or activated carboxyl group and a backbone atom to which a side chain is attached (e.g., an atom between an amino and carboxyl group, both of which can be optionally and independently protected and/or activated (e.g., an alpha carbon atom in an amino acid)).
the side chain of the amino acid comprises an optionally protected and/or activated carboxyl group, and an optionally substituted ring or an amino group, wherein the optionally substituted ring or an amino group is between the optionally protected and/or activated carboxyl group and a backbone atom to which a side chain is attached (e.g., an atom between an amino and carboxyl group, both of which
the present disclosure provides compounds having the structure of formula PA: N(R PA )(R a1 ) ⁇ L a1 ⁇ C(R a2 )(R a3 ) ⁇ L a2 ⁇ C(O)R PC , PA or a salt thereof, wherein: R PA is ⁇ H or an amino protecting group; each of R a1 and R a3 is independently ⁇ L a ⁇ R’; R a2 is ⁇ L aa ⁇ C(O)R PS ; each of L a , L a1 and L a2 is independently L; ⁇ C(O)R PS is optionally protected or activated ⁇ COOH; ⁇ C(O)R PC is optionally protected or activated ⁇ COOH; each L is independently a covalent bond, or an optionally substituted, bivalent C 1 -C 25 aliphatic or heteroaliphatic group having 1-10 heteroatoms wherein one or more methylene units of the group are optionally and independently
each L is independently a covalent bond, or an optionally substituted, bivalent C 1 -C 25 aliphatic or heteroaliphatic group having 1-10 heteroatoms wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O) 2 ⁇ , ⁇ C(O) 2 N(R’) ⁇ , ⁇ C(O) 2 ⁇ , ⁇ C(O) 2 N(R’) ⁇ ,
L is a covalent bond.
L (or L a , L aa , L a1 , L a2 , L s1 , L s2 , L s3 , or another variable or moiety that can be L, or a linker moiety) is an optionally substituted, bivalent C 1 -C 25 , C 1 -C 20 , C 1 -C 15 , C 1 -C 10 , C 1 -C 9 , C 1 -C 8 , C 1 -C 7 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , or C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 ,
L, L a , L aa , L a1 , L a2 , L s1 , L s2 , L s3 , L”, or another variable or moiety that can be L, or a linker moiety is an optionally substituted, bivalent C 1 -C 25 , C 1 -C 20 , C 1 -C 15 , C 1 -C 10 , C 1 -C 9 , C 1 -C 8 , C 1 -C 7 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , or C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 ,
it is an optionally substituted, bivalent C 1 -C 10 , C 1 -C 9 , C 1 -C 8 , C 1 -C 7 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , or C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , or C 10 , aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C
it is an optionally substituted, bivalent C 2 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
it is an optionally substituted, bivalent C 3 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
it is an optionally substituted, bivalent C 4 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
it is an optionally substituted, bivalent C 5 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
it is an optionally substituted, bivalent C 6 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
the bivalent aliphatic is saturated. In some embodiments, the bivalent aliphatic is linear. In some embodiments, the bivalent aliphatic is branched. In some embodiments, it is an optionally substituted, bivalent linear saturated C 6 aliphatic wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
each replacement if any is independently with ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
each replacement if any is independently with ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ .
each replacement if any is independently with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) 2 ⁇ , ⁇ S(O) 2 N(R’) ⁇ , ⁇ C(O)S ⁇ , or ⁇ C(O)O ⁇ . In some embodiments, each replacement if any is independently with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
L, L a , L aa , L a1 , L a2 , L s1 , L s2 , L s3 , L is an optionally substituted, bivalent C 1 -C 6 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is an optionally substituted, bivalent C 1 -C 5 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is an optionally substituted, bivalent C 1 -C 4 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is an optionally substituted, bivalent C 1 -C 3 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is an optionally substituted, bivalent C 1 -C 2 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is a bivalent C 1 -C 6 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ . In some embodiments, it is a bivalent C 1 -C 5 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is a bivalent C 1 -C 4 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is a bivalent C 1 -C 3 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
it is a bivalent C 1 -C 2 linear saturated aliphatic wherein one or more methylene units is optionally and independently replaced with ⁇ O ⁇ , ⁇ S ⁇ , ⁇ N(R’) ⁇ , or ⁇ C(O) ⁇ .
R’ in each moiety that is utilized to replace a methylene unit (e.g., ⁇ N(R’) ⁇ ) as described herein is hydrogen or optionally substituted C 1–6 aliphatic or phenyl.
R’ is each such moiety is hydrogen or optionally substituted C 1–6 alkyl. In some embodiments, R’ is each such moiety is hydrogen or C 1–6 alkyl. In some embodiments, each ⁇ Cy ⁇ is optionally substituted bivalent ring selected from 3-10, 3-9, 3-8, 3-7, 5-10, 5-9, 5-8, 5-7, 5-6, or 3, 4, 5, 6, 7, 8, 9, or 10 membered cycloaliphatic and heterocyclylene having 1-3 heteroatoms, phenylene, and 5-6 membered heteroarylene having 1-3 heteroatoms.
⁇ Cy ⁇ is optionally substituted bivalent 3-10, 3-9, 3-8, 3-7, 5-10, 5-9, 5- 8, 5-7, 5-6, or 3, 4, 5, 6, 7, 8, 9, or 10 membered cycloaliphatic. In some embodiments, ⁇ Cy ⁇ is optionally substituted 3-10, 3-9, 3-8, 3-7, 5-10, 5-9, 5-8, 5-7, 5-6, or 3, 4, 5, 6, 7, 8, 9, or 10 membered heterocyclylene having 1-3 heteroatoms.
⁇ Cy ⁇ is optionally substituted 3-10, 3-9, 3-8, 3-7, 5-10, 5-9, 5-8, 5-7, 5-6, or 3, 4, 5, 6, 7, 8, 9, or 10 membered heterocyclylene having 1 heteroatom.
⁇ Cy ⁇ is optionally substituted phenylene.
⁇ Cy ⁇ is phenylene.
⁇ Cy ⁇ is optionally substituted 5-6 membered heteroarylene having 1-3 heteroatoms.
⁇ Cy ⁇ is optionally substituted 5-6 membered heteroarylene having 1 heteroatom.
a heteroatom is nitrogen.
a heteroatom is oxygen.
a heteroatom is sulfur.
L, L a , L aa , L aa , L a1 , L a2 , L s1 , L s2 , L s3 , L”, or another variable or moiety that can be L, or a linker moiety is optionally substituted ⁇ (CH 2 )n ⁇ . In some embodiments, it is ⁇ (CH 2 )n ⁇ .
n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8.
n is 9. In some embodiments, n is 10. [0241]
L, L a , L aa , L a1 , L a2 , L s1 , L s2 , L s3 , L”, or another variable or moiety that can be L, or a linker moiety is an optionally substituted, bivalent heteroaliphatic group having 1-10 heteroatoms wherein one or more methylene units of the group are optionally and independently replaced with ⁇ C(R’) 2 ⁇ , ⁇ Cy ⁇ , ⁇ O ⁇ , ⁇ S ⁇ , ⁇ S ⁇ S ⁇ , ⁇ N(R’) ⁇ , ⁇ C(O) ⁇ , ⁇ C(S) ⁇ , ⁇ C(NR’) ⁇ , ⁇ C(O)N(R’) ⁇ , ⁇ N(R’)C(O)N(R’) ⁇ , ⁇ N(R’)C(O)O ⁇ , ⁇ S(O) ⁇ , ⁇ S(O) ⁇ , ⁇ S(
each R’ is independently ⁇ R, ⁇ C(O)R, ⁇ CO 2 R, or ⁇ SO 2 R.
R’ is ⁇ L a ⁇ R. In some embodiments, R’ is R.
R’ is ⁇ C(O)R. In some embodiments, R’ is ⁇ CO 2 R. In some embodiments, R’ is ⁇ SO 2 R. In some embodiments, R’ is ⁇ H.
each R is independently ⁇ H, or an optionally substituted group selected from C 1-30 aliphatic, C 1-30 heteroaliphatic having 1-10 heteroatoms, C 6-30 aryl, C 6-30 arylaliphatic, C 6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms;
R is ⁇ H. In some embodiments, R is not ⁇ H. In some embodiments, R is optionally substituted C 1-10 aliphatic. In some embodiments, R is optionally substituted C 1-10 alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is isopropyl. In some embodiments, R is ⁇ CF 3 . In some embodiments, R is ⁇ CH 2 CF 3 . In some embodiments, R is butyl. In some embodiments, R is t-butyl. In some embodiments, R is optionally substituted C 3-10 cycloaliphatic.
R is optionally substituted C 3-10 cycloalkyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is optionally substituted 5-membered heteroaryl having 1-3 heteroatoms. In some embodiments, R is optionally substituted 5-membered heteroaryl having 1 heteroatom.
R is optionally substituted 6-membered heteroaryl having 1-3 heteroatoms. In some embodiments, R is optionally substituted 6-membered heteroaryl having 1 heteroatom. In some embodiments, R is optionally substituted bicyclic 8-10 membered aromatic ring having 0-5 heteroatoms. In some embodiments, R is optionally substituted bicyclic 9-membered aromatic ring having 1- 5 heteroatoms. In some embodiments, R is optionally substituted bicyclic 10-membered aromatic ring having 1-5 heteroatoms. In some embodiments, R is optionally substituted bicyclic 9-membered aromatic ring having 1 heteroatom. In some embodiments, R is optionally substituted bicyclic 10-membered aromatic ring having 1 heteroatom.
R is optionally substituted bicyclic 10-membered aromatic ring having no heteroatom. In some embodiments, R is optionally substituted 3-10 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is optionally substituted 5-14 membered bicyclic heterocyclyl having 1-5 heteroatoms.
two R groups (or two groups that can be R, e.g., two groups each independently selected from R’, R a1 , R a2 , R a3 , R a5 , R RN , etc.) are taken together with their intervening atom(s) to form an optionally substituted 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms.
a formed ring is substituted.
a formed ring is unsubstituted.
a formed ring is 3-30, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3- 6, 4-10, 4-9, 4-8, 4-7, 4-6, 5-10, 5-9, 5-8, 5-7, 5-6, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 membered.
a formed ring is 3-10 membered.
a formed ring is 3-7 membered.
a formed ring is 4-10 membered.
a formed ring is 4-7 membered.
a formed ring is 5-10 membered.
a formed ring is 5-7 membered. In some embodiments, a formed ring is 3-membered. In some embodiments, a formed ring is 4-membered. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6-membered. In some embodiments, a formed ring is 7-membered. In some embodiments, a formed ring is 8-membered. In some embodiments, a formed ring is 9-membered. In some embodiments, a formed ring is 10-membered. In some embodiments, a formed ring is monocyclic. In some embodiments, a formed ring is bicyclic.
a formed ring is polycyclic. In some embodiments, a formed ring has no heteroatoms in addition to the intervening atom(s). In some embodiments, a formed ring has 1-10, e.g., 1-5, 1-3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 heteroatoms in addition to the intervening atom(s). In some embodiments, a formed ring is saturated. In some embodiments, a formed ring is partially unsaturated. In some embodiments, a formed ring comprises one or more aromatic ring. In some embodiments, a formed ring is bicyclic or polycyclic, and each monocyclic unit is independently 3-10 membered, saturated, partially unsaturated or aromatic and having 0-5 heteroatoms.
each heteroatom is independently selected from nitrogen, oxygen and sulfur.
a group that can be R e.g., R’, R a1 , R a2 , R a3 , R a5 , R RN , etc., is R as described herein. Those skilled in the art appreciate that embodiments described for one group that can be R may also be utilized for another group that can be R to the extent that such embodiments fall within the definition of R.
each heteroatom is independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
each heteroatom is independently selected from oxygen, nitrogen and sulfur.
each heteroatom of heteroaryl, heterocyclyl, heteroaliphatic, ring e.g., ⁇ Cy ⁇ , ring formed by groups taken together (e.g., two R groups taken together) with their intervening atom(s) (if any), etc.), etc., is independently selected from nitrogen, oxygen and sulfur.
each heteroatom is independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus.
rings e.g., cycloaliphatic, aryl, heteroaryl, heterocyclyl, etc.
rings in variables (e.g., R, R’ etc.), formulae, etc.) contain 1-30 (e.g., 2-30, 1-20, 2-20, 1-15, 2-15, 1-10, 2-10, 1-6, 2-6, etc.) ring carbon atoms and 0-15 (e.g., 0, 1-15, 0-10, 0-5, 1-10, 1-5, etc.) ring heteroatoms.
rings contain 1-20 ring carbon atoms and 0-10 ring heteroatoms.
rings contain 2-20 ring carbon atoms and 0-10 ring heteroatoms.
rings contain 1-10 ring carbon atoms and 0-5 ring heteroatoms.
rings contain 2-10 ring carbon atoms and 0-5 ring heteroatoms. In some embodiments, rings contain 2-10 ring carbon atoms and 0-5 ring heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments, rings formed by groups taken together (e.g., two R groups taken together) contain, in addition to the intervening atom(s) of such groups, 1-20 (e.g., 1-15, 1-10, 1-5, etc.) additional ring carbon atoms and 0-10 (e.g., 0, 1-10, 0-5, 1-5, etc.) additional ring heteroatoms. In some embodiments, such rings contain 1-15 additional ring carbon atoms and 0-5 ring heteroatoms.
such rings contain 1-10 additional ring carbon atoms and 0-5 ring heteroatoms.
each ring heteroatom is independently selected from nitrogen, oxygen and sulfur.
various groups may be optionally substituted. Substituents are routinely utilized in chemistry including in development of various therapeutics. Many substituents can be utilized in accordance with the present disclosure.
a substituent is a hydrocarbon group.
a substituent comprises a heteroatom.
a substituent comprises multiple heteroatoms.
each atom in a substituent is independently selected from hydrogen, carbon, halogen, nitrogen, oxygen, sulfur, phosphorus and silicon.
each atom in a substituent is independently selected from hydrogen, carbon, halogen, nitrogen, oxygen, and sulfur. In some embodiments, each atom in a substituent is independently selected from hydrogen, carbon, fluorine, chlorine, bromine, iodine, nitrogen, oxygen, and sulfur.
the total number of carbon and non-halogen heteroatom(s) in a substituent is about or no more than about 1; in some embodiments, it is no more than about 2; in some embodiments, it is no more than about 3; in some embodiments, it is no more than about 4; in some embodiments, it is no more than about 5; in some embodiments, it is no more than about 6; in some embodiments, it is no more than about 7; in some embodiments, it is no more than about 8; in some embodiments, it is no more than about 9; in some embodiments, it is no more than about 10; in some embodiments, it is no more than about 11; in some embodiments, it is no more than about 12; in some embodiments, it is no more than about 13; in some embodiments, it is no more than about 14; in some embodiments, it is no more than about 15; in some embodiments, it is no more than about 20.
the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 20. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 15. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 10. In some embodiments, the total number of carbon and non-halogen heteroatom(s) in each substituent is independently no more than about 6.
amino acids and structure moieties are described in WO 2022/020651, WO 2022/020652, and WO 2022/261257, the amino acids and structure moieties of each of which are independently incorporated herein by reference, and can be utilized in accordance with the present disclosure.
an amino acid, or a structure moiety, of an amino acid or an agent e.g., a peptide
a N-terminal cap is connected via R 1 to the amino group (R 1 ) of the first amino acid (AA1).
a N-Term cap may be properly considered as part of AA1.
each carboxylate (R 2 ) of that amino acid is connected to the amino group (R 1 ) of the subsequent amino acid, until the carboxylate (R 2 ) of the final amino acid is connected to R 1 of a C-terminal group.
R 1 of the monomer in brackets is attached to R 3 of the amino acid.
R 3 and R4 if two branches are indicated, the R 1 of the first branch is connected to R 3 , and R 1 of the second branch connected to R4; in some embodiments, only one branch is indicated and R4 is ⁇ H.
the R 3 groups of each of those amino acids are linked to each other.
the R 3 groups of those amino acids are linked to each other.
R 1 is attached to the branching amino acid adjacent to it in the sequence, and the R 2 group of the branching monomer is attached to R 3 of the amino acid with no branching monomer designated.
amino acid residues e.g., D-amino acid residues, homologated amino acid residues, alkyl (e.g., methyl) amino acid residues, etc.
amino acid residues e.g., alkyl amino acid residues, hydrophobic amino acid residues, etc.
moieties useful as, e.g., amino acid residues e.g., polar amino acid residues, basic amino acid residues, etc.
Certain moieties useful as, e.g., amino acid residues e.g., acidic amino acid residues, non-aromatic amino acid residues, etc.
Certain moieties e.g., moieties utilized in [] in various agents
moieties e.g., moieties utilized in [] in various agents, amino acid residues, etc.
R 1 of the first is connected to R 1 of the latter.
R 1 of Ac is connected to R 1 of dPEG2.
R 2 of dPEG2 can be connected to other moieties, e.g., in [Ac-dPEG2]-Lys, R 3 of Lys.
the present disclosure provides an agent, e.g., a peptide agent (in various embodiments, a stapled peptide agent), comprising a moiety selected from the table above.
a residue is stapled, e.g., forming a staple with another moiety.
an agent comprises a staple formed between two moieties each independently selected from the table above.
a staple comprises a double bond.
a staple comprises an E double bond.
a staple comprises a Z double bond.
a double bond is converted into another moiety, e.g., to a saturated bond through hydrogenation, an epoxide through epoxidation, etc.
a moiety, e.g., an amino acid residue comprises two groups that can be utilized for stapling.
an amino acid residue comprises two groups for stapling, e.g., B3, B4, B5, B6, Dap7Gly, Dap7Pent, DapAc7EDA, DapAc7PDA, Dap7Abu, etc.
a N-terminal group e.g., 4pentenyl, 5hexenyl, etc., may be considered as part of the first amino acid residue for stapling.
amino acid residues with N-terminal groups comprise two groups, e.g., two double bonds, for stapling.
a group for stapling is a double bond.
each group for stapling is independently a double bond.
a group for stapling is a double bond and the other is not (e.g., amino group, or a group which is or comprises R 3 ).
an agent comprise one and no more than one residue comprising two or more residues for stapling.
agents comprising one and no more than one amino acid residue that is bonded to two staples.
agents comprise staples having different types of structures and/or formed by different types of transformations.
an agent comprises a staple whose formation does not comprises an olefin metathesis transformation and/or modification of a carbon-carbon double bond (e.g., hydrogenation).
such agents may provide improved properties, activities, design flexibility, manufacturing efficiency, etc.
a compound has a structure selected from the table above, wherein R 1 is ⁇ OH. In some embodiments, a compound has a structure selected from the table above, wherein R 1 is ⁇ H.
a compound is a compound has the structure selected from the table above, wherein R 1 is ⁇ H or amino protecting group (e.g., Fmoc, tBoc, etc.) and R 2 is ⁇ OH, a carboxyl protecting or activating group, or a salt thereof.
R 1 is ⁇ H or amino protecting group
R 2 is ⁇ OH, or a salt thereof.
a compound is a compound has the structure selected from the table above, wherein R 1 is ⁇ H and R 2 is ⁇ OH, or a salt thereof.
a compound is a compound has the structure selected from the table above, wherein R 1 is ⁇ H, R 2 is ⁇ OH and R 3 is ⁇ H, or a salt thereof. In some embodiments, R 3 is ⁇ H or a protecting group. In some embodiments, R 3 is ⁇ H. In some embodiments, a compound has a structure selected from the table above, wherein R 1 is an amino protection group, e.g., Fmoc, tBoc, etc.
a compound has a structure selected from the table above, wherein R 1 is an amino protecting group, e.g., Fmoc, tBoc, etc., and R 2 is ⁇ OH, or ⁇ COR 2 is an optionally substituted, protected or activated carboxyl group. In some embodiments, R 2 is ⁇ OH.
an amino acid residue has a structure selected from the table above, wherein each of R 1 and R 2 independently represents a connection site (e.g., for structure , the residue is of the structure
an agent, a peptide or a stapled peptide comprises such an amino acid residue.
the present disclosure provides peptides, including stapled peptides, comprising residues of amino acids described herein.
the present disclosure provides various methods comprising utilizing amino acids, optionally protected and/or activated, as described herein.
the present disclosure provides methods for preparing peptides, comprising utilizing amino acids, typically protected and/or activated, as described herein.
various amino groups are Fmoc protected for peptide synthesis (particularly for forming backbone peptide bonds).
various side chain carboxylic acid groups are t-Bu protected ( ⁇ C(O) ⁇ O ⁇ tBu).
an agent e.g., a peptide, a stapled peptide, a stitched peptide, etc.
an agent is less than about 5000 Daltons in mass.
an agent is greater than or equal to about 900 Daltons and less than about 5000 Daltons in mass.
an agent is greater than or equal to about 1500 Daltons and less than about 5000 Daltons in mass.
an agent is greater than or equal to about 2000 Daltons and less than about 5000 Daltons in mass.
an agent is greater than or equal to about 2500 Daltons and less than about 5000 Daltons in mass.
an agent is greater than or equal to about 1000 Daltons and less than about 3000 Daltons in mass. In some embodiments, an agent is greater than or equal to about 1500 Daltons and less than about 3000 Daltons in mass. In some embodiments, an agent is greater than or equal to about 1500 Daltons and less than about 2500 Daltons in mass. In some embodiments, an agent is greater than or equal to about 1600 Daltons and less than about 2200 Daltons in mass. In some embodiments, the agent is no more than about 900 Daltons in mass. In some embodiments, an agent is no more than about 500 Daltons in mass. In some embodiments, an agent is no more than about 300 Daltons in mass. In some embodiments, an agent is no more than about 200 Daltons in mass.
agents e.g., peptides
binding characteristics e.g., with respect to a particular target of interest (e.g., beta-catenin or a portion thereof), stability characteristics, for example in solution or in dried form, cell permeability characteristics, solubility, lipophilicity, etc.
a binding characteristic may be or comprise specificity, affinity, on-rate, off-rate, etc, optionally under (or over a range of) specified conditions such as, for example, concentration, temperature, pH, cell type, presence or level of a particular competitor, etc.
assessments of characteristics as described herein may involve comparison with an appropriate reference (e.g., a positive or negative control) which may, in some embodiments, be a contemporaneous reference or, in some embodiments, a historical reference.
an appropriate reference e.g., a positive or negative control
desirable characteristics may be, for example: binding to a desired target (e.g., a dissociation constant (K D ) of at least less than about 1 ⁇ M, and preferably a K D of less than about 50 nM); cell penetration (e.g., as measured by fluorescence-based assays or mass spectrometry of cellular fractions, etc.); solubility (e.g., soluble at less than about 1000 uM agent, or soluble at less than about 500 uM agent, or soluble at less than about 100 uM agent, or less than about 50 uM, or less than about 35 uM); activity (e.g., modulating one or more functions of a target, which may be assessed in a cellular reporter assay (e.g., with an IC50 of less than a concentration, e.g., less than about 1 ⁇ M, less than about 500 nM, less than about 50 nM, less than about 10 nM, etc.),
K D dissociation constant
an agent of the invention comprises an affinity of less than about 10 nM, for example, an IC50 of 7 nM).
provided agents can bind to targets, e.g., beta-catenin, with an EC 50 of no more than about 2000 nM.
an EC50 is no more than about 1500 nM.
an EC50 is no more than about 1000 nM.
an EC50 is no more than about 500 nM.
an EC50 is no more than about 300 nM.
an EC50 is no more than about 200 nM.
an EC50 is no more than about 100 nM. In some embodiments, an EC50 is no more than about 75 nM. In some embodiments, an EC50 is no more than about 50 nM. In some embodiments, an EC50 is no more than about 25 nM. In some embodiments, an EC50 is no more than about 10 nM. In some embodiments, an EC50 is no more than about 5 nM. In some embodiments, an EC50 is measured by fluorescence polarization as described in the Examples. [0262] In some embodiments, the present disclosure provides agents, e.g., stapled peptides, with suitable solubility for various purposes.
solubility of provided agents is about or at least about 5-100 uM (e.g., about or at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 uM). In some embodiments, solubility is about or at least about 25 uM. In some embodiments, solubility is about or at least about 30 uM. In some embodiments, solubility is about or at least about 40 uM. In some embodiments, solubility is about or at least about 50 uM.
provided agents are protein bound in serum; in some embodiments, they are at least about 85%, 90%, or 95% protein bound in serum. In some embodiments, provided agents are over 95% protein bound in serum.
provided agents can traverse a cell membrane of an animal cell. In some embodiments, provided agents can traverse a cell membrane of a human cell.
provided agents can bind to motifs, residues, or polypeptides. In some embodiments, provided agents bind to beta-catenin. In some embodiments, a dissociation constant (K D ) is about 1 nM to about 1 uM.
a K D is no more than about 1 uM. In some embodiments, a K D is no more than about 500 nM. In some embodiments, a K D is no more than about 250 nM. In some embodiments, a K D is no more than about 100 nM. In some embodiments, a K D is no more than about 50 nM. In some embodiments, a K D is no more than about 25 nM. In some embodiments, a K D is no more than about 10 nM. In some embodiments, a K D is no more than about 5 nM. In some embodiments, a K D is no more than about 1 nM.
K D is measured by Surface Plasmon Resonance (SPR) as illustrated herein.
SPR Surface Plasmon Resonance
provided agents binds to a polypeptide whose sequence is or comprising SEQ ID NO: 2, or a fragment thereof: SVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIIL ASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCL WTLRNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEAL VRT (SEQ ID NO: 2).
provided agents have one or more or all of the following interactions with beta-catenin: Direct interactions (), water mediated [], non-polar contacts ⁇ LQIL ⁇ AY ⁇ (G) ⁇ NQ ⁇ ES(K)LIILA (residue 301-317 of Uniprot P35222 sequence) (SEQ ID NO: 3) SRVL ⁇ (K)V ⁇ LS ⁇ V ⁇ CSSN (residue 341-353 of Uniprot P35222 sequence) (SEQ ID NO: 4) RLV ⁇ QN ⁇ C ⁇ L ⁇ (W)TL ⁇ R ⁇ (N)LSDA (residue 376-391 of Uniprot P35222 sequence) (SEQ ID NO: 5) LGSD[D]I(N) ⁇ V ⁇ V ⁇ TC ⁇ AAGI (residue 409-423 of Uniprot P35222 sequence) (SEQ ID NO: 6) [0267] In some embodiments, an agent, e.g., a peptide, binds to beta-catenin
an agent e.g., a peptide, binds to beta-catenin and interacts with one or more residues that are or correspond to at least two, or at least three, or at least four, or at least five, or at least six, or seven of the following amino acid residues in SEQ ID NO: 1 at the indicated positions: G307, K312, K345, W383, R386, N387, D413, and N415.
an agent binds to beta-catenin and interacts with one or more residues that are or correspond to at least two, or at least three, or at least four, or at least five, or at least six, or seven of the following amino acid residues in SEQ ID NO: 1 at the indicated positions: G307, K312, K345, W383, N387, D413, and N415.
provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) of G307, K312, K345, Q379, L382, W383, N387, N415 and V416.
provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of Y306, G307, K312, K345, Q379, L382, W383, N387, N415 and V416. In some embodiments, provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of G307, K312, K345, Q379, L382, W383, R386, N387, N415 and V416.
provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) of Y306, G307, K312, K345, Q379, L382, W383, R386, N387, N415 and V416.
provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) of Y306, G307, K312, K345, V349, Q379, L382, W383, R386, N387, N415 and V416.
provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of G307, K312, K345, W383, R386, N387, D413 and N415. In some embodiments, provided agents interact with beta-catenin at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of G307, K312, K345, W383, N387, D413 and N415. In some embodiments, provided agents interact with beta- catenin at one or both of K312 and R386. In some embodiments, provided agents interact with G307. In some embodiments, provided agents interact with K312.
provided agents interact with beta-catenin at one or more of K345, W383, D413 and N415. In some embodiments, provided agents interact with beta-catenin at one or more of K345 and W383. In some embodiments, provided agents interact with beta-catenin at one or more of D413 and N415. In some embodiments, provided agents interact with Y306. In some embodiments, provided agents interact with G307. In some embodiments, provided agents interact with K312. In some embodiments, provided agents interact with K345. In some embodiments, provided agents interact with V349. In some embodiments, provided agents interact with Q379. In some embodiments, provided agents interact with L382. In some embodiments, provided agents interact with W383.
provided agents interact with R386. In some embodiments, provided agents interact with N387. In some embodiments, provided agents interact with D413. In some embodiments, provided agents interact with N415. In some embodiments, provided agents interact with V416. [0269] In some embodiments, provided agents interact with one or more of amino acid residues that are or correspond to K312, R386, K345 and W383 of SEQ ID NO: 1. In some embodiments, provided agents interact with one or more of amino acid residues that are or correspond to K312 and R386 of SEQ ID NO: 1. In some embodiments, interaction with an amino acid residue can be assessed through mutation of such an amino acid residue (e.g., mutation of K, R, etc. to D, E, etc.).
interactions with beta-catenin may be assessed by contacting an agent with either a full-length or a portion of beta-catenin.
a portion of beta-catenin comprises the interacting residues above.
a portion of beta-catenin is or comprises SEQ ID NO: 2.
a portion of beta-catenin is expressed with a tag (e.g., for purification, detection, etc.).
a tag is a fluorescent tag.
a tag is for detection.
a tag is for purification and detection.
a tag is a purification tag.
a tag is or comprises biotin. Many other types of tags are available in the art and can be utilized in accordance with the present disclosure.
Various technologies can be utilized for characterizing and/or assessing provided technologies (e.g., agents (e.g., various peptides), compositions, methods, etc.) in accordance with the present disclosure.
a useful technology is or comprises fluorescence polarization.
a useful technology assesses LogP or LogD.
a useful technology is or comprises a CHI LogD assay.
a useful technology assesses solubility.
a useful technology is or comprises NanoBRET.
a useful technology is or comprises a reporter assay (e.g., DLD1 reporter assay).
a useful technology is or comprises alphascreen.
Certain useful protocols are described in the Examples. Those skilled in the art appreciate that suitable adjustments may be made to such protocols, e.g., according to specific conditions, agents, purposes, etc.
tumor models are utilized to assess technologies.
a tumor model is a patient-derived tumor model.
a number of tumor models are utilized.
a number of patient-derived tumor models are utilized.
tumor models carry various mutations one or more of which may be shared by two or more models. Certain mutations are described in the Examples.
tumor models can have different expression profiles.
tumor models carrying APC and/or beta-catenin mutations may have different expression profiles with respect to genes downstream of active (and hyper-active) beta-catenin/TCFs (e.g., Axin2, SP5, etc.).
results observed from tumor models are different from those observed in in vitro models.
dependence of cancer cells on the Wnt pathway/beta-catenin is not preserved in an in vitro culture.
Wnt pathway/beta-catenin dependence is more dramatic in organoid and in vivo contexts.
breadth and degree of efficacy observed in in vivo tumor models are greater than that observed using in vitro models.
multiple tumor models some of which are responsive to technologies herein (e.g., a stapled peptide agent) and some of which are not, are utilized to identify useful biomarkers, either individually or in combination, for selecting patients for treatment by technologies herein.
the present disclosure provides biomarkers, individually and in combination, to demonstrate pharmacodynamic activity in patients and/or to predict drug responses in vivo.
a subject is identified for treatment based on pathway mutation (e.g., APC, beta-catenin), expression of certain genes (e.g., AXIN2), beta-catenin levels and/or localization, and/or presence or absence of additional oncogenic drivers.
pathway mutation e.g., APC, beta-catenin
AXIN2 genes that promote beta-catenin levels and/or localization
beta-catenin levels and/or localization e.g., beta-catenin levels and/or localization
additional oncogenic drivers e.g., I-66
I-66 can provide tumor regression in multiple APC mutant CRC PDX models.
Some of these responsive models contain TP53 mutation, some contain Ras mutation (K/N), some contain mutations in both, and some contain no mutations in TP53 or Ras.
a level of expression, transcript, polypeptide, etc. is relative to that of relevant normal cells, diseased cells (e.g., cancer cells) of one or more other subjects, etc. In some embodiments, a level of expression, transcript, polypeptide, etc., is relative to that of absence of administration of an agent and/or administration of a reference (e.g., in some embodiments, I-470 as a reference agent for I-66).
a reference e.g., in some embodiments, I-470 as a reference agent for I-66.
Stapling may be performed during and/or after peptide chain synthesis.
the present disclosure provides an unstapled peptide agent whose sequence is one described in Table E2 or Table E3.
amino acid residues are optionally protected for peptide synthesis (e.g., peptide synthesis using Fmoc-protected amino acids wherein certain side chains may be protected).
one or more stapling are achieved through olefin metathesis.
two or more stapling are formed through one olefin metathesis process.
the present disclosure provides a stapled peptide agent described in Table E2 or Table E3 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof).
the present disclosure provides a stereoisomer of a stapled peptide agent described in Table E2 or Table E3 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof).
the present disclosure provides a E/Z stereoisomer of a stapled peptide agent described in Table E2 or Table E3 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof).
an olefin double bond in the first staple that comprising such a bond is Z
an olefin double in the second staple that comprising such a bond is E (Z-E); in some embodiments, it is (Z-Z); in some embodiments, it is (E-Z); in some embodiments, it is (E-E).
an olefin double bond in the first (i, i+2), (i, i+3) or (i, i+4) staple that comprising such a bond is Z
an olefin double in the first (i, i+7) staple that comprising such a bond is E (Z-E); in some embodiments, it is (Z-Z); in some embodiments, it is (E-Z); in some embodiments, it is (E-E).
an agent comprises an olefin double bond in a third staple, and it is E; in some embodiments, it is Z.
an agent comprises an olefin double bond in a fourth staple, and it is E; in some embodiments, it is Z.
one or more or all staples are formed after chain extension. In some embodiments, one or more or all staples are formed during chain extension. In some embodiments, one or more or all staples by metathesis are formed after chain extension. In some embodiments, one or more or all staples by metathesis are formed during chain extension. In some embodiments, two or more staples each independently comprising an olefin are formed in one step.
an agent e.g., I-66 or I-67 or an isomer thereof
metathesis is performed on an optionally protected agent (e.g., ⁇ COOH protected as esters such as t-butyl ester) comprising amino acid residues comprising olefin moieties, e.g., PL3, B5 and PyrS2 (which agent is optionally on a solid support).
an optionally protected agent e.g., ⁇ COOH protected as esters such as t-butyl ester
amino acid residues comprising olefin moieties, e.g., PL3, B5 and PyrS2 (which agent is optionally on a solid support).
metathesis is performed on an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3- PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof, wherein each PG is independently a suitable protecting group for ⁇ COOH.
an agent is Ac-PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)- Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt thereof.
metathesis is performed in the presence of a metal complex, e.g., Grubbs M102 complex.
PG is a protecting group stable to peptide synthesis.
PG is a protecting group stable to Fmoc-based peptide synthesis.
PG is a protecting group orthogonal to Fmoc.
PG is a protecting group that can be removed under an acidic condition.
PG is t-butyl.
t-butyl ester protection e.g., after metathesis
a method comprises releasing a peptide from a solid support, e.g., through TFA treatment.
the present disclosure provides a method, comprising a) preparing a first compound comprising two moieties each of which independently comprises an olefin double bond; b) providing a second compound by stapling the two moieties by olefin metathesis of an olefin double bond of one moiety with an olefin double bond of the other to form a first-formed staple; c) add one or more additional moieties to the second compound to provide a third compound which comprising two moieties each of which independently comprises an olefin double bond; and d) providing a fourth compound by stapling the two moieties in the third compound by olefin metathesis of an olefin double bond of one moiety with an olefin double bond of the other to form a second- formed staple.
a moiety is an amino acid residue. In some embodiments, each moiety is independently an amino acid residue. In some embodiments, each moiety is independently an amino acid residue comprising a terminal olefin as described herein. In some embodiments, there are two olefin double bonds in one moiety, e.g., of the first compound. For example, in some embodiments, such a moiety is B5. In some embodiments, two moieties of a first compound is independently X 4 and X 11 . In some embodiments, a first-formed staple is a (i, i+7) staple.
a first compound comprises ⁇ X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 ⁇ . In some embodiments, a first compound comprises ⁇ X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 ⁇ . In some embodiments, a first compound comprises a staple. In some embodiments, a staple is a (i, i+4) staple. In some embodiments, a staple is between X 10 and X 14 . In some embodiments, an olefin double bond in a third compound is present in the first compound (e.g., an unstapled olefin double bond of B5).
one and only one amino acid residue comprises an olefin double bond is added to the second compound.
ae third compound is or comprises ⁇ X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 ⁇ .
a third compound is or comprises ⁇ X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 ⁇ .
a first- and second-formed staples are bonded to the same amino acid residue.
a first- and second-formed staples are bonded to the same atom.
a second-formed staple is a (i, i+2), (i, i+3) or (i, i+4) staple.
two moieties in the third compound is independently X 1 and X 4 .
a first-formed staple is formed with E selectivity as described herein (e.g. about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, or more).
a second-formed staple is formed with Z selectivity as described herein (e.g., about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, or more).
synthesis may be performed on a solid support (e.g., solid phase peptide synthesis), and a compound or an agent may be on a solid support.
stapling during chain extension, or individually performed stapling for one or more staples can provide advantages, e.g., increased selectivity, yield, purity, etc.
two or more staples are formed in a metathesis reaction.
all staples formed by metathesis are formed in a metathesis reaction. In some embodiments, each of such staples are formed through olefin metathesis of terminal olefins. In some embodiments, multiple staples are formed after full lengths of peptides have been achieved. In some embodiments, one or more staples comprising double bonds are formed after full lengths of peptides have been achieved. In some embodiments, all staples comprising double bonds are formed after full lengths of peptides have been achieved. In some embodiments, one or more staples formed through metathesis are formed after full lengths of peptides have been achieved. In some embodiments, all staples formed through metathesis are formed after full lengths of peptides have been achieved.
stepwise stapling in which two or more staples are formed in two or more steps, were performed.
stepwise stapling provides improved levels of selectivity to form a desired product (e.g., I-66) over other compounds, e.g., stereoisomers (e.g., for I-66, I-67).
a desired product e.g., I-66
stereoisomers e.g., for I-66, I-67
an improvement is about or at least about 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 fold.
an improvement is assessed by comparing percentage of a desired product among all related stereoisomers.
an improvement is assessed by ratios of a desired product versus a stereoisomer (e.g., I-66 versus I-67).
two staples comprising olefin double bonds are formed in two separate steps.
two staples formed by metathesis are formed in two separate steps.
two staples bonded to the same amino acid residue are formed in two separate steps.
two staples bonded to the same atom are formed in two separate steps.
two staples bonded to the same carbon atom are formed in two separate steps.
two staples formed from B5 are formed in two separate steps.
a provided technologies comprise a third step forming a third staple.
each staple is formed in a separate step.
the present disclosure provides a method for preparing a stapled peptide, comprising: 1) reacting a first reactive group with a second reactive group to form a first staple, wherein the first and second reactive groups are in two different amino acid residues; and 2) reacting a third reactive group with a fourth reactive group to form a second staple, wherein the third and fourth reactive groups are in two different amino acid residues.
a method comprises reacting a fifth reactive group with a sixth reactive group to form a third staple, wherein the fifth and sixth reactive groups are in two different amino acid residues.
a third staple is formed before a first and second staples.
a first staple is formed through a metathesis reaction.
each of the first and second reactive groups independently is or comprises a double bond.
each of the first and second reactive groups is independently a terminal olefin.
a first staple is formed through olefin metathesis.
a first staple is an (i, i+7) staple.
a metathesis reaction is performed in the presence of a catalyst.
a catalyst is Hoveyda-Grubbs M720 catalyst (CAS 301224-40-8).
a first staple is between X 4 and X 11 .
a second staple is formed through a metathesis reaction.
each of the third and fourth reactive groups independently is or comprises a double bond.
each of the third and fourth reactive groups is independently a terminal olefin.
a second staple is formed through olefin metathesis.
a second staple is an (i, i+3) staple.
Various metathesis technologies may be utilized in accordance with the present disclosure to form a second staple.
a metathesis reaction is performed in the presence of a catalyst.
a catalyst is Grubbs M102 catalyst (CAS 172222-30-9).
a second staple is between X 1 and X 4 .
one of the first and second reactive groups, and one of the third and fourth reactive groups are in the same amino acid residues. In some embodiments, they are independently in a side chain and the two side chains are bonded to the same atom. In some embodiments, the two side chains are bonded to the same carbon atom, e.g., as in B5. In some embodiments, the first and second staples are bonded to the same amino acid residue. In some embodiments, they are bonded to same atom.
an agent e.g., I-66 or I-67 or an isomer thereof
metathesis is performed on an optionally protected agent (e.g., ⁇ COOH protected as esters such as t-butyl ester) comprising two amino acid residues comprising olefin moieties, e.g., PyrS2 and B5 (which agent is optionally on a solid support).
metathesis is performed on an agent comprising -B5- Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof, wherein each PG is independently a suitable protecting group for ⁇ COOH.
PG is t-butyl.
metathesis is performed on an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- 3Thi-BztA-GlnR*3-Ala- or a salt form thereof.
metathesis is performed on an agent comprising -Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof.
metathesis is performed on an agent comprising -Asp-Npg-B5- Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof.
an agent is Fmoc-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi- BztA-GlnR*3-Ala-solid support or a salt thereof.
metathesis is performed in the presence of a metal complex, e.g., Hoveyda-Grubbs M720 complex.
a product agent is a corresponding agent wherein B5 and Pyrs are stapled.
additional amino acid residue(s) is added.
an additional amino acid residue comprising an olefin in its side chain e.g., PL3, is added.
an N-terminus is capped, e.g., by Ac.
an amino acid residue comprising an olefin in its side chain, e.g., PL3, forms a staple with another amino acid residue comprising an olefin in its side chain (e.g., with an olefin in B5, which may have optionally formed a staple through one side chain) through a metathesis reaction.
a metathesis reaction is performed in the presence of a metal complex, e.g., Grubbs M102 complex.
an agent comprises -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)- Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof, wherein there is a staple between B5 and PyrS2.
an agent comprises -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3- PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof, wherein there is a staple between B5 and PyrS2.
an agent comprises -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe- Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt form thereof, wherein there is a staple between B5 and PyrS2.
an agent is Ac-PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib- Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt thereof, wherein there is a staple between B5 and PyrS2.
a third staple comprises an amide group, e.g., ⁇ C(O)N(R’) ⁇ wherein R’ is as described herein.
a third staple comprises ⁇ C(O)NH ⁇ .
a third staple is a (i, i+4) staple.
one of the fifth and the sixth reactive groups is or comprises an amino group or an activated form thereof, and the other is or comprises an acid group, e.g., a carboxyl group, or an activated form thereof.
a third staple is formed through an amidation reaction.
a third staple is not formed by a metathesis reaction.
a third staple does not comprise an olefin double bond.
Various amidation technologies are available and may be utilized herein. As described herein, other types of staples may be utilized and constructed as well. See, for example, preparation of I-66, I-335, etc. in the Examples.
a third staple is between X 10 and X 14 .
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt form thereof, wherein each of PGL and PGG is independently a suitable protecting group is deprotected, e.g., contacted with a Pd agent (e.g., tetrakis(triphenylphosphine)palladium(0)) when PGL is Alloc and PGG is Oallyl, to form an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt form thereof.
a Pd agent e.g., tetrakis(triphenylphosphine)palladium(0)
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala- or a salt form thereof is contacted with a Pd agent (e.g., tetrakis(triphenylphosphine)palladium(0)) to form an agent comprising -Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof.
a Pd agent e.g., tetrakis(triphenylphosphine)palladium(0)
⁇ NH 2 in the side chain of Lys (a salt form thereof) is coupled with ⁇ COOH (a salt form thereof) in the side chain of Glu to provide an agent comprising -Lys*3-PyrS2-3Thi-BztA-GlnR*3- a salt form thereof.
⁇ NH 2 in the side chain of Lys (a salt form thereof) is coupled with ⁇ COOH (a salt form thereof) in the side chain of Glu to provide an agent comprising -Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- a salt form thereof.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala- or a salt form thereof comprises -Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala- or a salt form thereof; in some embodiments, it comprises -Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala-solid support or a salt form thereof; in some embodiments, it is Fmoc-Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala- solid support or a salt thereof; in some embodiments, it comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe- Lys(
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt form thereof comprises -Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof; in some embodiments, it comprises -Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof; in some embodiments, it comprises -Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt form thereof; in some embodiments, it is Fmoc-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu- Ala-solid support or a salt thereof; in some embodiments, it comprises -Asp(PG)-3COOHF(PG)-Aib-Ala- Phe
an agent comprising -Lys*3-PyrS2-3Thi-BztA-Glu*3-Ala- or a salt form thereof comprises -Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-Glu*3-Ala- or a salt form thereof; in some embodiments, it comprises -Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-Glu*3-Ala-solid support or a salt form thereof; in some embodiments, it is Fmoc-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-Glu*3-Ala-solid support or a salt thereof; in some embodiments, it comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA
PGL and PGG can be deprotected under the same conditions.
PGL and PGG are orthogonal to peptide synthesis, e.g., Fmoc-based peptide synthesis.
PG is stable under de-protection conditions of PGL and PGG.
PG, PGL and PGG are stable under peptide synthesis, e.g., Fmoc-based peptide synthesis, conditions.
PGL and PGG comprises allyl.
PGL is Alloc.
PGG is Oallyl. Certain examples are described in Exemplification.
one or more stapling steps are independently performed before full lengths are achieved.
a third staple is formed before the two amino acid residues comprising the first and second reactive groups are both installed.
a first staple is formed before the two amino acid residues comprising the third and fourth reactive groups are both installed.
a third staple is formed after an amino acid residue comprising one of the first and second reactive group is installed but before an amino acid residue comprising the other of the first and second reactive group is installed.
a first staple is formed after an amino acid residue comprising one of the third and fourth reactive group is installed but before an amino acid residue comprising the other of the third and fourth reactive group is installed.
two or more stapling steps are performed based on the positions of the related staples and the directions of peptide synthesis, and one or more staples closer to the starting termini are formed before one or more staples further away from the starting termini.
peptide synthesis is performed from C-terminus to N-terminus.
the one that first has both related residues installed is formed first.
a staple between X 4 and X 11 is formed before a staple between X 1 and X 4 .
a metal complex is a Grubbs catalyst.
a metal complex is a Hoveyda-Grubbs catalyst.
it is Grubbs I M102.
a catalyst provides product E/Z selectivity.
catalysts can be utilized at various suitable levels, e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 40%, 50% mol or more.
the present disclosure provides technologies for controlling ratio of E/Z isomers of one or more or each olefin double bond formed during olefin metathesis.
one or more or each olefin double bond is formed with a isomer ratio of about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, or more.
one or more or each olefin double bond has an isomer ratio of about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, or more. In some embodiments, it is independently about 1.5:1 or more. In some embodiments, it is independently about 2:1 or more. In some embodiments, it is independently about 3:1 or more. In some embodiments, it is independently about 4:1 or more. In some embodiments, it is independently about 5:1 or more. In some embodiments, it is independently about 6:1 or more. In some embodiments, it is independently about 7:1 or more. In some embodiments, it is independently about 8:1 or more.
stapling creates one or more chiral centers. For example, in some embodiments, when B5 forms two staples with two other amino acid residues, a chiral center may form. In some embodiments, a formed chiral center is R in an agent.
a formed chiral center is S in an agent.
a composition comprises both agents being R and S at a chiral center.
a chiral center is formed with stereoselectivity (e.g., in some embodiments, diastereoselectivity when other chiral elements are present in the same molecule).
the selectivity is about or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%(when selectivity is 98%, 98% of all product molecules share the same stereochemistry at the chiral center.).
a composition described herein e.g., a pharmaceutical composition
it is about or at least about 70%.
it is about or at least about 75%.
it is about or at least about 80%.
it is about or at least about 85%.
an olefin double bond in a staple may be further modified.
an olefin double bond in a staple is hydrogenated thus converting it into a single bond.
a modification is epoxidation.
a modification is halogenation.
crude product compositions are purified, e.g., through chromatography technologies such as liquid chromatography.
one or more product compositions are collected based on separated portions, e.g., HPLC peaks, with the correct observed mass.
each product composition independently corresponds to a different peak (e.g., in some embodiments, by UV detection at a suitable wavelength, e.g., 220 nm) with the correct observed mass.
a peak area of one or more or each product composition is independently about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more of the total peak area of all peak(s) with the correct mass.
a product composition comprises one isomer. In some embodiments, a product composition comprises two or more isomers (e.g., those that cannot be sufficiently separated).
each product composition independently has a purity and/or stereopurity as described herein, e.g., in some embodiments, for one or more (e.g., 1, 2, 3, 4, 5 or more) or each olefin double bond in a staple, the ratio of the two stereoisomers is independently about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1 or more. In some embodiments, ratios may be assessed by NMR, HPLC, etc.
certain stapled peptides may be provided in and/or produced by a biological system and reacting with a provided reagent, e.g., one having the structure of R x ⁇ L s2 ⁇ R x , or a salt thereof, wherein R x can react with ⁇ SH groups under suitable conditions.
a provided reagent e.g., one having the structure of R x ⁇ L s2 ⁇ R x , or a salt thereof, wherein R x can react with ⁇ SH groups under suitable conditions.
each R x is a suitable leaving group.
each R x is independently ⁇ Br.
amide formation e.g., peptide synthesis, formation of amide-containing staples, etc.
peptides are prepared on solid phase on a synthesizer using, typically, Fmoc chemistry.
the present disclosure provides protected and/or activated amino acids for synthesis.
a useful solid support is Rink Amide MBHA resin.
reagents such as OxymaPure, carbodiimide (e.g., N,N'-Diisopropylcarbodiimide), etc. are utilized for amide coupling.
reagents such as PyAOP are utilzed for amidation. Certain examples are described in the Examples. [0295]
staples are formed by olefin metathesis.
a product double bond of metathesis is reduced/hydrogenated.
CO 2 are extruded from a carbamate moiety of a staple.
provided stapled peptides are further modified, and/or conjugated to other entities. Conditions and/or reagents of these reactions are widely known in the art and can be performed in accordance with the present disclosure to provide stapled peptides. [0296] Properties and/or activities of provided stapled peptides can be readily assessed in accordance with the present disclosure, for example, through use of one or more methods described in the examples.
technologies for preparing and/or assessing stapled peptides include those described in US 9617309, US 2015-0225471, US 2016-0024153, US 2016-0215036, US2016-0244494, WO 2017/062518, etc.
technologies for preparing and/or assessing stapled peptides are described in WO 2022/261257 and are incorporated herein by reference.
the present disclosure provides products manufactured and/or characterized by processes and/or technologies described herein.
Provided compounds can be provided in high purity.
a provided compound is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.
provided compounds e.g., amino acids optionally protected/activated, are essentially free of impurities, including stereoisomers.
an agent may have one or more stereoisomers which may independently co-exist in a composition or preparation.
a provided agent has a stereopurity of about or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, it is about or at least about 80%.
stereoisomers are essentially free from a preparation or composition (e.g., cannot be reliably observed in NMR or HPLC).
an agent comprises one or more staples independently comprising one or more olefin double bond.
stereopurity is with respect to E/Z stereoisomers.
the ratio of the two stereoisomers is independently about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1 or more. In some embodiments, it is independently about 1.5:1 or more. In some embodiments, it is independently about 2:1 or more. In some embodiments, it is independently about 3:1 or more. In some embodiments, it is independently about 4:1 or more. In some embodiments, it is independently about 5:1 or more.
it is independently about 6:1 or more. In some embodiments, it is independently about 7:1 or more. In some embodiments, it is independently about 8:1 or more. In some embodiments, it is independently about 9:1 or more. In some embodiments, it is independently about 10:1 or more. In some embodiments, it is independently about 20:1 or more. In some embodiments, it is independently about 30:1 or more. In some embodiments, it is independently about 40:1 or more. In some embodiments, it is independently about 50:1 or more. In some embodiments, it is independently about 60:1 or more. In some embodiments, it is independently about 70:1 or more. In some embodiments, it is independently about 80:1 or more.
the present disclosure provides compounds, agents, preparations, compositions, etc., that have low metal levels. In some embodiments, the present disclosure provides compounds, agents, preparations, compositions, etc., that have low ruthenium levels. In some embodiments, the present disclosure provides compounds, agents, preparations, compositions, etc., that have low palladium levels.
a level is no more than about 1000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 ppm (weight/weight). In some embodiments, a level is no more than about 1000 ppm. In some embodiments, a level is no more than about 500 ppm. In some embodiments, a level is no more than about 100 ppm. In some embodiments, a level is no more than about 50 ppm. In some embodiments, a level is no more than about 20 ppm. In some embodiments, a level is no more than about 10 ppm. As appreciated by those skilled in the art, various technologies can be utilized to assess metal levels.
compositions that comprise or otherwise relate to provided agents, e.g., small molecule agents, peptide agents (e.g., stapled peptides), as described herein.
provided compositions are or comprise an assay system for characterizing (and optionally including) a stapled peptide as described herein.
provided compositions are pharmaceutical compositions e.g., that comprise or deliver one or more provided agents.
an agent is in a pharmaceutically acceptable salt form.
an agent is a peptide.
an agent is a stapled peptide.
an agent comprises a detectable moiety, e.g., fluorescent moiety, radioactive moiety, biotin, etc.
a detectable moiety is directly detectable.
a detectable antibody is detected indirectly, e.g., utilizing an antibody, an agent that can reacting with a detectable moiety to form a detectable product, etc.
a pharmaceutical composition comprises a provided agent and a pharmaceutically acceptable excipient (e.g., carrier).
a peptide composition may include or deliver a particular form (e.g., a particular optical isomer, diastereomer, salt form, covalent conjugate form [e.g., covalently attached to a carrier moiety], etc., or combination thereof) of an agent as described herein).
an agent included or delivered by a pharmaceutical composition is described herein is not covalently linked to a carrier moiety.
multiple stereoisomers exist for an agent that contains chiral centers and/or double bonds.
level of a particular agent in a composition is enriched relative to one or more or all of its stereoisomers.
a particularly configuration of a double bond (E/Z) is enrich.
a configuration is independently enriched.
a chiral element e.g., a chiral center
one configuration is enriched.
a chiral center bonded to two staples one configuration is enriched.
a configuration is independently enriched.
one or more or all stereochemical element e.g., double bonds, chiral element, etc.
one configuration is independently enriched. In some embodiments, for each double bond in each staple, one configuration is independently enriched, and for a chiral center bonded to two staples, one configuration is enriched. In some embodiments, enrichment for each double bond is independently E or Z. In some embodiments, enrichment for each chiral element is independently R or S.
enrichment for each stereochemical element is about or at least about a certain level, e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (percentage of an agent).
a level is about or at least about 60%. In some embodiments, it is about or at least about 65%. In some embodiments, it is about or at least about 70%. In some embodiments, it is about or at least about 75%. In some embodiments, it is about or at least about 80%. In some embodiments, it is about or at least about 85%.
a provided therapeutic composition may comprise one or more additional therapeutic agents and/or one or more stabilizing agents and/or one or more agents that alters (e.g., extends or limits to a particular tissue, location or site) rate or extent of delivery over time.
a composition is a pharmaceutical composition which comprises or delivers a provided agent (e.g., a stapled peptide) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
a composition comprises one and only stereoisomer of an agent (e.g., a stapled peptide) and/or one or more salts thereof.
a composition comprises two or more stereoisomers of an agent (e.g., a stapled peptide) and/or one or more salts thereof.
the two or more stereoisomers of an agent e.g., a stapled peptide
an agent e.g., a stapled peptide
salts thereof elute as a single peak (e.g., UV and/or MS detection) in a chromatography, e.g., HPLC.
a chromatography e.g., HPLC.
Provided agents and compositions can be utilized for various purposes. For example, certain compounds may be utilized as amino acids, either directly or for preparation of other compounds such as peptides. Certain agents, e.g., peptides, may be utilized to prepare stapled peptides.
agents that are or comprise peptides, particularly stapled peptides, and compositions thereof, are biologically active and can be utilized for various purposes, e.g., as therapeutics toward various conditions, disorders or diseases, as tools for modulating biological functions, etc.
the present disclosure provides agents and compositions thereof for modulating beta-catenin functions.
beta-catenin is reported to have multiple cellular functions including regulation and coordination of cell-cell adhesion and gene transcription.
agents described herein may inhibit beta-catenin activity and/or level and may, for example, inhibit neoplastic growth.
agents described herein may activate and/or increase level of beta-catenin and may, for example, be used to treat male pattern baldness or alopecia.
beta-catenin can interact with members of the TCF/LEF family at a TCF site on beta-catenin.
provided technologies can decrease, suppress or block one or more of such interactions.
the present disclosure provides methods for modulating an interaction between beta-catenin and its binding partner (e.g., a TCF/LEF family member) comprising contacting beta-catenin with a provided agent.
binding of provided agents to beta-catenin competes or inhibits binding of another agent.
binding of provided agents to beta-catenin competes or inhibits binding of another agent. In some embodiments, binding of provided agents to beta-catenin competes or inhibits binding of TCF or a fragment thereof. [0315] In some embodiments, provided agents compete with TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, APC, CDH1, or CDH2, or a fragment thereof, for beta-catenin binding. [0316] In some embodiments, provided agents interfere with interactions of TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, APC, CDH1, or CDH2, or a fragment thereof, with beta-catenin.
provided technologies can reduce or block beta-catenin’s interactions with all TCF family members, E-cadherin and APC, but did not significantly affect its interactions with ICAT, AXIN and BCL9.
provided technologies can interrupt beta-catenin/TCF interaction at both physical interaction level (e.g., as confirmed by NanoBRET, co-IP, etc.) and transcriptional level (e.g., as confirmed by reporter cell line, endogenous gene expression, etc.).
provided technologies show no effect on beta-catenin stability.
the present disclosure provides methods for modulating interactions of beta-catenin with a partner, e.g., TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, APC, CDH1, or CDH2, or a fragment thereof, comprising contacting beta-catenin with a provided agent or a composition that comprises or delivers a provided agent.
a partner e.g., TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, APC, CDH1, or CDH2, or a fragment thereof.
the present disclosure provides methods for modulating interactions of beta-catenin with a partner, e.g., TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, APC, CDH1, or CDH2, or a fragment thereof, comprising administering or delivering to a system comprising beta-catenin and the partner a provided agent or a composition that comprises or delivers a provided agent.
a system is an in vitro system.
a system is an in vivo system.
a system is or comprises a cell, tissue or organ.
a system is a subject.
the present disclosure provides method for inhibiting cell growth, comprising administering or delivering to a population of cells an effective amount of a provided agent or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure provides method for killing cells associated with a condition, disorder or disease (e.g., cancer), comprising administering or delivering to a population of such cells an effective amount of a provided agent or a pharmaceutically acceptable salt thereof.
a condition, disorder or disease e.g., cancer
the present disclosure provides technologies for modulating levels and/or activities of transcripts and/or products thereof.
levels and/or activities of transcripts and levels and/or activities of products thereof may be modulated independently, or may be increased, decreased or maintained at the same or comparable levels independently.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for modulating levels and/or activities of beta-catenin transcripts and/or products (e.g., polypeptides) thereof.
the present disclosure provides a method for decreasing beta-catenin polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides a method for increasing CTNNB1 mRNA level in a system, comprising administering or delivering to the system an agent or composition.
provided technologies can reduce levels of total beta-catenin polypeptide while increasing CTNNB1 mRNA levels.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for modulating levels and/or activities of c-Myc and/or N-Myc transcripts and/or products (e.g., polypeptides) thereof.
c-Myc transcript levels are reduced.
N-Myc transcript levels are reduced.
c-Myc polypeptide levels are reduced. In some embodiments, N-Myc polypeptide levels are reduced. In some embodiments, c-Myc polypeptide and mRNA levels are reduced. In some embodiments, N-Myc polypeptide and mRNA levels are reduced. In some embodiments, c-Myc and N-Myc polypeptide and mRNA levels are reduced. In some embodiments, provided technologies, e.g., I-66, provide reduction of levels of c-Myc and/or N-Myc transcripts and/or polypeptides 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days post a dose (in some embodiments, last dose of a multi-dose regimen).
the present disclosure provides a method for decreasing c-Myc transcript level in a system, comprising administering or delivering to the system an agent or composition. In some embodiments, the present disclosure provides a method for decreasing c-Myc polypeptide level in a system, comprising administering or delivering to the system an agent or composition. In some embodiments, the present disclosure provides a method for decreasing N-Myc transcript level in a system, comprising administering or delivering to the system an agent or composition. In some embodiments, the present disclosure provides a method for decreasing N-Myc polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for reducing levels and/or activities of Axin2 transcripts and/or products (e.g., polypeptides) thereof.
the present disclosure provides a method for decreasing Axin2 transcript level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides a method for decreasing Axin2 polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for modulating levels and/or activities of Notum transcripts and/or products (e.g., polypeptides) thereof.
the present disclosure provides a method for decreasing Notum polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides a method for increasing Notum mRNA level in a system, comprising administering or delivering to the system an agent or composition.
provided technologies can reduce levels of Notum polypeptide while increasing Notum mRNA levels.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for modulating levels and/or activities of DKK4 transcripts and/or products (e.g., polypeptides) thereof.
the present disclosure provides a method for decreasing DKK4 polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides a method for increasing DKK4 mRNA level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for modulating levels and/or activities of FGF20 transcripts and/or products (e.g., polypeptides) thereof.
the present disclosure provides a method for decreasing FGF20 polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides a method for increasing FGF20 mRNA level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides technologies (e.g., agents, compositions, methods, etc.) for modulating levels and/or activities of Ki67 transcripts and/or products (e.g., polypeptides) thereof.
the present disclosure provides a method for decreasing Ki67 polypeptide level in a system, comprising administering or delivering to the system an agent or composition.
the present disclosure provides a method for decreasing percentage of Ki67 polypeptide- positive cells in a system, comprising administering or delivering to the system an agent or composition.
a reduction is about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
an increase is about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
the present disclosure provide increases and/or decreases at least several days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days or more after administration is completed.
the present disclosure provide increases and/or decreases at least several days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days or more after the first dose. In some embodiments, the present disclosure provide increases and/or decreases at least several days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days or more after two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses. In some embodiments, it is after a first dose. In some embodiments, it is after a second dose. In some embodiments, it is after multiple doses. In some embodiments, it is after the last dose.
a system, cell, tissue, organ or subject comprises or expresses a mutant beta-catenin. In some embodiments, a system, cell, tissue, organ or subject comprises beta-catenin hyperactivity. In some embodiments, a system, cell, tissue, organ or subject comprises upregulated beta- catenin transcript level. In some embodiments, a system, cell, tissue, organ or subject comprises upregulated beta-catenin polypeptide level. In some embodiments, a system, cell, tissue, organ or subject comprises or expresses a mutant c-Myc.
a system, cell, tissue, organ or subject comprises c-Myc hyperactivity. In some embodiments, a system, cell, tissue, organ or subject comprises upregulated c-Myc transcript level. In some embodiments, a system, cell, tissue, organ or subject comprises upregulated c-Myc polypeptide level. In some embodiments, a system, cell, tissue, organ or subject comprises or expresses a mutant N-Myc. In some embodiments, a system, cell, tissue, organ or subject comprises N-Myc hyperactivity. In some embodiments, a system, cell, tissue, organ or subject comprises upregulated N-Myc transcript level. In some embodiments, a system, cell, tissue, organ or subject comprises upregulated N-Myc polypeptide level.
the present disclosure provides methods for preventing a condition, disorder or disease associated with beta-catenin (e.g., a cancer, a neurodegenerative disease, etc.), comprising administering or delivering to a subject susceptible thereto an effective amount of a provided agent or a pharmaceutically acceptable salt thereof.
the present disclosure provides methods for treating a condition, disorder or disease associated with beta-catenin (e.g., aberrant beta-catenin activity and/or expression level), comprising administering or delivering to a subject suffering therefrom an effective amount of a provided agent or a pharmaceutically acceptable salt thereof.
a provided agent is administered as a pharmaceutical composition that comprises or delivers an effective amount of a provided agent or a pharmaceutically acceptable salt thereof.
a condition, disorder or disease is associated with beta-catenin interaction with a partner, e.g., TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, APC, CDH1, and/or CDH2.
a condition, disorder or disease is associated with beta-catenin with TCF.
a condition, disorder or disease is cancer.
provided agents may be administered in combination with another therapy, e.g., immunotherapy.
a condition, disorder, or disease is selected from cancer, cardiac disease, dilated cardiomyopathy, fetal alcohol syndrome, depression, and diabetes.
a condition, disorder, or disease is a heart condition, disorder, or disease.
a condition, disorder, or disease is cancer.
a cancer is selected from: colon cancer, colorectal cancer, rectal cancer, prostate cancer familial adenomatous polyposis (FAP), Wilms Tumor, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, primary hetpatocellular carcinoma, ovarial carcinoma, breast cancer, lung cancer, glioblastoma, pliomatrixoma, medulloblastoma, thyroid tumors, and ovarian neoplasms.
FAP familial adenomatous polyposis
a condition, disorder or disease is a cancer, e.g., colorectal cancer, hepatocellular cancer, melanoma, gastric cancer, bladder cancer, and endometrial cancer.
a cancer is colorectal cancer.
a cancer is hepatocellular cancer.
a cancer is prostate cancer.
a cancer is melanoma.
provided technologies provides benefits in a subject after administration or delivery of an agent.
tumor growth is reduced.
tumor size is reduced.
provided technologies provide tumor regression.
a condition, disorder or disease is associated with or characterized by deregulation of beta-catenin. In some embodiments, a condition, disorder or disease is associated with or characterized by increased levels of beta-catenin polypeptide. In some embodiments, a condition, disorder or disease is associated with or characterized by deregulation of c-Myc and/or N-Myc. In some embodiments, a condition, disorder or disease is associated with or characterized by increased levels of c-Myc and/or N-Myc polypeptides. In some embodiments, a condition, disorder or disease is not associated with or characterized by deregulation of beta-catenin.
a condition, disorder or disease is not associated with or characterized by increased levels of beta-catenin polypeptide. In some embodiments, a condition, disorder or disease is not associated with or characterized by deregulation of c-Myc and/or N-Myc. In some embodiments, a condition, disorder or disease is not associated with or characterized by increased levels of c- Myc and/or N-Myc polypeptides. In some embodiments, a condition, disorder or disease is associated with or characterized by deregulation of c-Myc and/or N-Myc and/or increased levels of c-Myc and/or N-Myc polypeptides, but not by deregulation of beta-catenin and/or increased levels of beta-catenin polypeptide.
a condition, disorder or disease comprises mutations.
a condition, disorder or disease, e.g., cancer comprises a KRAS mutation (e.g., G12D).
a condition, disorder or disease, e.g., cancer comprises an APC mutation. It has been reported that APC mutation may be associated with upregulation and/or hyperactivation of beta-catenin.
a condition, disorder or disease is cancer, e.g., colorectal cancer, comprising a KRAS mutation.
a condition, disorder or disease is cancer, e.g., colorectal cancer, comprising an APC mutation.
a condition, disorder or disease e.g., colorectal cancer
the present disclosure provides technologies for modulating level of expression and/or activity of a nucleic acid, e.g., a gene, a transcript, a polypeptide, and/or a product thereof in a system, comprising administering or delivering to the system a provided agent or a composition that comprises or delivers a provided agent.
level of expression of a nucleic acid, e.g., a gene, or a product thereof is modulated.
level of activity of a nucleic acid e.g., a gene, or a product thereof (e.g., a transcript, a polypeptide, etc.) is modulated.
level of a transcript and/or a product thereof e.g., a polypeptide
level of activity of a transcript and/or a product thereof e.g., a polypeptide
a transcript is a transcript of a nucleic acid, e.g., gene, described herein.
level of a polypeptide is modulated.
level of activity of a polypeptide is modulated.
a polypeptide is a encoded by a nucleic acid or a transcript described herein. In some embodiments, a level is increased. In some embodiments, a level is decreased.
a system is an in vitro system. In some embodiments, a system is an in vivo system. In some embodiments, a system is or comprises a cell, tissue or organ. In some embodiments, a system is or comprises one or more cancer cells. In some embodiments, a system is or comprises tumor. In some embodiments, a system is or comprises an organism. In some embodiments, a system is a subject. In some embodiments, a system is a human. In some embodiments, a system comprises beta-catenin.
a system expresses beta-catenin. In some embodiments, a system comprises beta-catenin and a partner. In some embodiments, a system expresses beta-catenin and a partner. In some embodiments, a level is regulated by beta-catenin. In some embodiments, a level is regulated by WNT activation. In some embodiments, a level is regulated by beta-catenin/WNT signaling. In some embodiments, a level is regulated by interaction of beta-catenin and a partner.
interaction of beta-catenin and a partner is modulated, e.g., reduced, prevented, etc., by an agent, e.g., a stapled peptide, as described herein.
a partner is TCF.
level of expression and/or activity of a nucleic acid and/or a product thereof is modulated.
a nucleic acid is AXIN2.
level of an AXIN2 transcript, e.g., mRNA is reduced.
level of an AXIN2 polypeptide is reduced.
a nucleic acid is SP5.
level of an SP5 transcript e.g., mRNA
level of an SP5 polypeptide is reduced.
a nucleic acid is CXCL12.
level of a CXCL12 transcript e.g., mRNA
level of a CXCL12 polypeptide is increased.
a nucleic acid is a member of a negatively enriched gene set observed in, or can be identified using technologies in, e.g., WO 2022/261257.
a nucleic acid is a member of BCAT_GDS748_UP gene set.
a nucleic acid is a member of BCAT.100_UP.V1_UP gene set. In some embodiments, a nucleic acid is a member of HALLMARK_WNT_BETA_CATENIN_SIGNALING gene set. In some embodiments, a nucleic acid is a member of RASHI_RESPONSE_TO_IONIZING_RADIATION_1 gene set. In some embodiments, a nucleic acid is a member of REACTOME_RRNA_PROCESSING gene set. In some embodiments, a nucleic acid is a member of HALLMARK_MYC_TARGETS_V1 gene set.
a nucleic acid is a member of HALLMARK_MYC_TARGETS_V2 gene set. In some embodiments, a nucleic acid is a member of HALLMARK_OXIDATIVE_PHOSPHORYLATION gene set. In some embodiments, a nucleic acid is a member of HALLMARK_E2F_TARGETS gene set. In some embodiments, a nucleic acid is a member of HALLMARK_TNFA_SIGNALING_VIA_NFKB gene set. In some embodiments, a nucleic acid is a member of HALLMARK_G2M_CHECKPOINT gene set.
a nucleic acid is a member of HALLMARK_SPERMATOGENESIS gene set.
a gene set is described in a Figure or an Example.
a nucleic acid is described in a Figure or an Example, or is a member of a gene set is described in a Figure or an Example. Description of various gene sets can be found publicly, e.g., www.gsea- msigdb.org/gsea/msigdb/.
one or more or some or a majority of but not all nucleic acids or genes in a gene set is impacted in the same way, but overall a gene set can be negatively or positively enriched.
the present disclosure provides technologies for detecting, monitoring and/or confirming efficacy of an agent, e.g., a stapled peptide, or a method, e.g., a method of treating a condition, disorder or disease, a method for modulating level of a transcript and/or a product and/or activity thereof, comprising assessing level of expression and/or activity of a nucleic acid, e.g., a gene, a transcript, a polypeptide, and/or a product thereof.
an agent e.g., a stapled peptide
a method e.g., a method of treating a condition, disorder or disease, a method for modulating level of a transcript and/or a product and/or activity thereof, comprising assessing level of expression and/or activity of a nucleic acid, e.g., a gene, a transcript, a polypeptide, and/or a product thereof.
the present disclosure provides technologies for detecting, monitoring and/or confirming efficacy of an agent, e.g., a stapled peptide, comprising administering the agent to a subject, and assessing level of expression and/or activity of a nucleic acid, e.g., a gene, a transcript, a polypeptide, and/or a product thereof, in the subject.
an agent e.g., a stapled peptide
a nucleic acid e.g., a gene, a transcript, a polypeptide, and/or a product thereof
the present disclosure provides technologies for detecting, monitoring and/or confirming efficacy of a method for treating a condition, disorder or disease in a subject, comprising assessing level of expression and/or activity of a nucleic acid, e.g., a gene, a transcript, a polypeptide, and/or a product thereof, in the subject.
a method is a method for treating a condition, disorder or disease associated with TCF-beta- catenin interaction in a subject.
a condition, disorder or disease is cancer as described herein.
the present disclosure provides technologies for selecting subjects for administration or delivery of an agent, e.g., stapled peptide agents described herein (e.g., for preventing or treating a condition, disorder or disease). In some embodiments, the present disclosure provides technologies for selecting subjects for continued administration or delivery of an agent, e.g., stapled peptide agents described herein (e.g., for preventing or treating a condition, disorder or disease) after one or more administrations or deliveries. In some embodiments, level of a transcript is assessed. In some embodiments, level of a polypeptide is assessed. In some embodiments, assessment is performed utilizing a sample or samples collected from a system or a subject. In some embodiments, a sample is collected during administration or delivery.
an agent e.g., stapled peptide agents described herein (e.g., for preventing or treating a condition, disorder or disease).
the present disclosure provides technologies for selecting subjects for continued administration or delivery of an agent, e.g., stapled peptide agents described here
a sample is collected after administration or delivery.
level of expression and/or activity of a nucleic acid and/or a product thereof is modulated.
a nucleic acid is AXIN2.
level of an AXIN2 transcript e.g., mRNA
level of an AXIN2 polypeptide is reduced.
a nucleic acid is SP5.
level of an SP5 transcript e.g., mRNA
level of an SP5 polypeptide is reduced.
a nucleic acid is DKK4.
level of an DKK4 transcript e.g., mRNA
level of an DKK4 polypeptide is reduced.
a nucleic acid is NOTUM.
level of an NOTUM transcript e.g., mRNA
level of an NOTUM polypeptide is reduced.
a nucleic acid is FGF20.
level of an FGF20 transcript e.g., mRNA
level of an FGF20 polypeptide is reduced.
a nucleic acid is Myc.
level of an Myc transcript e.g., mRNA
level of an Myc polypeptide is reduced.
Myc is N-Myc.
Myc is C-Myc.
a nucleic acid is CXCL12.
level of a CXCL12 transcript e.g., mRNA
level of a CXCL12 polypeptide is increased.
a nucleic acid is a member of a negatively enriched gene set observed in, or can be identified.
a nucleic acid is a member of a gene set as described herein.
administration or delivery continues. In some embodiments, administration or delivery continues as prior one(s). In some embodiments, administration or delivery continue with an adjusted dose level and/or regimen. In some embodiments, if desired reductions of expression and/or levels of transcripts and/or products thereof, and/or one or more desired negatively and/or positively enriched gene sets, are not observed, administration or delivery may be adjusted, and in some embodiments, discontinued.
desired reductions of expression and/or levels of transcripts and/or products thereof comprise reductions of expression and/or levels of transcripts and/or products thereof of one or more or a majority of or all of SP5, CCND2, WNT5B, AXIN2, NKD1, WNT6, DKK1, DKK4, NOTUM, FGF20, Myc, nucleic acids of BCAT_GDS748_UP, BCAT.100_UP.V1_UP, HALLMARK_WNT_BETA_CATENIN_SIGNALING, RASHI_RESPONSE_TO_IONIZING_RADIATION_1, REACTOME_RRNA_PROCESSING, HALLMARK_MYC_TARGETS_V1, HALLMARK_MYC_TARGETS_V2, HALLMARK_OXIDATIVE_PHOSPHORYLATION, HALLMARK_E2F_TARGETS, HALLMARK_TNFA_SIGNALING_VIA_NF
desired increase of expression and/or levels of transcripts and/or products thereof comprise increase of expression and/or levels of transcripts and/or products thereof of CXCL12.
desired gene set enrichments comprise negative enrichment of one or more or all of BCAT_GDS748_UP, BCAT.100_UP.V1_UP, HALLMARK_WNT_BETA_CATENIN_SIGNALING, RASHI_RESPONSE_TO_IONIZING_RADIATION_1, REACTOME_RRNA_PROCESSING, HALLMARK_MYC_TARGETS_V1, HALLMARK_MYC_TARGETS_V2, HALLMARK_OXIDATIVE_PHOSPHORYLATION, HALLMARK_E2F_TARGETS, and HALLMARK_TNFA_SIGNALING_VIA_NFKB.
RNAseq GSE analysis of I-66 treated PDX tumors showed modulation of various genes, consistent with blockade of tumor growth, e.g., CCND2 (down), CCNE1/2 (down), CDK2/4 (down), PLK1/4 (down), p16INK4A (up), p15INK4B (up).
BCL2 down-regulation was observed.
ATF3 up-regulation was observed.
up- regulation of MDR transporters, e.g., ABCB1/4 was observed.
comparison is made to a reference.
reduction, increase, enrichment (negative or positive), changes, etc. are typically made to a suitable reference.
reduction, increase, enrichment (negative or positive), changes, etc. are to a reference assessment, in some embodiments, of a reference sample.
a reference assessment is or comprises assessment conducted prior to an administration or delivery of an agent.
a reference sample is collected prior to an administration or delivery of an agent.
a reference assessment is or comprises assessment conducted during an administration or delivery of an agent.
a reference sample is collected during an administration or delivery of an agent.
a reference assessment is or comprises assessment conducted after an administration or delivery of an agent.
a reference sample is collected after an administration or delivery of an agent.
a reference assessment is or comprises assessment conducted after an earlier administration or delivery of an agent.
a reference sample is collected after earlier an administration or delivery of an agent.
a sample is an aliquot of material obtained or derived from a source of interest as described herein.
a source of interest is a biological or environmental source.
a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human).
a source of interest is or comprises biological tissue or fluid.
a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and/or combinations or component(s) thereof.
a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
a biological fluid may be or comprise a plant exudate.
a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
a biological sample is or comprises cells obtained from an individual.
a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
processing e.g., by removing one or more components of and/or by adding one or more agents to
a primary sample e.g., filtering using a semi-permeable membrane.
Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
a sample comprise cancer cells.
a sample is obtained from a tumor.
a sample is obtained from a tumor in a patient.
levels of two or more transcripts and/or products thereof may be assessed.
assessment is performed after one or more doses of agents, e.g., stapled peptides are administered or delivered to a subject.
profiles, e.g., reduction, increase, etc., of one or more transcripts and/or products thereof matches those described herein, administration or delivery to a subject may continue.
administration or delivery to a subject may be stopped and/or continued according to different dose levels and/or regimens.
Various technologies can be utilized in accordance with the present disclosure to formulate, distribute, administer or deliver provided technologies such as agents, peptides, compounds, compositions, etc.
administration may be ocular, oral, parenteral, topical, etc.
administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
bronchial e.g., by bronchial instillation
buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc
enteral intra-arterial, intradermal, intragastric
administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, provided technologies are administered intravenously. [0340] Among other things, the present disclosure provides various structural moieties including designed amino acid residues that can be utilized to optimize various properties and activities, stability, delivery, pharmacodynamics, pharmacokinetics, etc. to provide various dosage forms, dosage regimen, therapeutic windows, etc.
provided agents and compositions thereof may be utilized with improved dosage regimen and/or unit doses.
administration of provided agents are adjusted based on conditions, disorders or diseases and/or subpopulations.
administration and/or dosage regimen of provided technologies are adjusted according to certain biomarkers and genomic alterations.
Provided agents may deliver biological effects, e.g., therapeutic effects, via various mechanisms.
efficacy may be driven by AUC.
efficacy may be driven by Cmax.
a provided agent is utilized in combination with another therapy.
a provided agent is utilized in combination with another therapeutic agent.
another therapy or therapeutic agent is administered prior to an administration or delivery of a provided agent. In some embodiments, another therapy or therapeutic agent is administered at about the same time as an administration or delivery of a provided agent. In some embodiments, a provided agent and another agent is in the same pharmaceutical composition. In some embodiments, another therapy or therapeutic agent is administered subsequently to an administration or delivery of a provided agent. In some embodiments, a subject is exposed to both a provided agent and another therapeutic agent. In some embodiments, both a provided agent and another agent can be detected in a subject. In some embodiments, a provided agent is administered before another agent is cleared out by a subject or vice versa.
a provided agent is administered within the half-life, or 2, 3, 4, 5 or 6 times of the half-life, of another agent or vice versa.
a subject is exposed to a therapeutic effect of a provided agent and a therapeutic effect of another therapeutic agent.
an agent may provide an effect after an agent is cleared out or metabolized by a subject.
a procedure e.g., surgery, radiation, etc., may provide an effect after the procedure is completed.
another therapy is a cancer therapy.
another therapy is or comprises surgery.
another therapy is or comprises radiation therapy.
another therapy is or comprises immunotherapy.
another therapeutic agent is or comprises a drug. In some embodiments, another therapeutic agent is or comprises a cancer drug. In some embodiments, another therapeutic agent is or comprises a chemotherapeutic agent. In some embodiments, another therapeutic agent is or comprises a hormone therapy agent. In some embodiments, another therapeutic agent is or comprises a kinase inhibitor. In some embodiments, another therapeutic agent is or comprises a checkpoint inhibitor (e.g., antibodies against PD-1, PD-L1, CTLA-4, etc.). In some embodiments, a provide agent can be administered with lower unit dose and/or total dose compared to being used alone. In some embodiments, another agent can be administered with lower unit dose and/or total dose compared to being used alone.
a combination therapy provides improved results, e.g., when compared to each agent utilized individually. In some embodiments, a combination therapy achieves one or more better results, e.g., when compared to each agent utilized individually.
another agent is a checkpoint inhibitor, an EGFR inhibitor, a VEGF inhibitor, a VEGFR inhibitor, a kinase inhibitor, or an anti-cancer drug.
an additional agent is a checkpoint inhibitor. In some embodiments, an additional agent is an immune oncology agent.
an additional agent is an antibody against a checkpoint molecules.
an additional agent is an antibody of PD1, PDL-1, CTLA4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-s, C10orf54, etc.
an antibody is an anti-PD1 antibody.
an antibody is an anti-PD-L1 antibody.
an antibody is an anti-CTLA4.
another agent is an EGFR inhibitor, e.g., erlotinib, gefitinib, lapatinib, panitumumab, vandetanib, cetuximab, etc.
another agent is an VEGF and/or VEGFR inhibitor, e.g., pazopanib, bevacizumab, sorafenib, sunitinib, axitinib, ponatinib, regorafenib, vandetanib, cabozantinib, ramucirumab, lenvatinib, ziv-aflibercept, etc.
another agent is a kinase inhibitor.
another therapeutic agent is a chemotherapeutic agent.
another therapeutic agent is an anti-cancer drug, e.g., cyclophosphamide, methotrexate, 5- fluorouracil (5-FU), doxorubicin, mustine, vincristine, procarbazine, prednisolone, dacarbazine, bleomycin, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bortezomib, carboplatin, chlorambucil, cytarabine, daunorubicin, docetaxel, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, mitoxantrone, paclitaxel, pemetrexed,
an agent having the structure of formula I R N ⁇ L P1 ⁇ L AA1 ⁇ L P2 ⁇ L AA2 ⁇ L P3 ⁇ L AA3 ⁇ L P4 ⁇ L AA4 ⁇ L P5 ⁇ L AA5 ⁇ L P6 ⁇ L AA6 ⁇ L P7 ⁇ R C , I or a salt thereof, wherein: R N is a peptide, an amino protecting group or R’ ⁇ L RN ⁇ ; each of L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 is independently L, wherein L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 comprise: a first R’ group and a second R’ group which are taken together to form ⁇ L s ⁇ which is bonded to the atom to
R N is a peptide, an amino protecting group or R’ ⁇ L RN ⁇ ; each of L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 is independently L, wherein L P1 , L P2 , L P3 , L P4 , L P5 , L P6 , and L P7 comprise: a first R’ group and a second R’ group which are taken together to form ⁇ L s ⁇ which is bonded to the atom to which a first R’ group is attached and the atom to which a second R’
L P1 is or comprises ⁇ [X] p ⁇ X 1 ⁇ , wherein each X and X 1 is independently an amino acid residue, wherein p is 0-10, and X 1 is bonded to L AA1 .
L AA1 is an amino acid residue that comprises a side chain comprising an acidic group.
L P2 is or comprises ⁇ [X]pX 3 X 4 [X]p’ ⁇ , wherein each X, X 3 and X 4 is independently an amino acid residue, and each of p and p’ is independently 0- 10. 6.
L AA2 is an amino acid residue that comprises a side chain comprising an acidic group. 7.
L AA2 is X 5 .
L P3 is or comprises ⁇ [X]pX 6 X 7 [X]p’ ⁇ , wherein each X, X 6 and X 7 is independently an amino acid residue, and each of p and p’ is independently 0- 10.
L AA3 is an amino acid residue that comprises a side chain comprising an acidic group. 10.
L P4 is or comprises ⁇ [X]pX 7 X 8 [X]p’ ⁇ , wherein each X, X 7 and X 8 is independently an amino acid residue, and each of p and p’ is independently 0- 10.
R AA4 is an optionally substituted group selected from phenyl, 5- or 6-membered monocyclic heteroaryl having 1-4 heteroatoms, and 9- or 10- membered bicyclic heteroaryl having 1-4 heteroatoms. 12.
L P5 is or comprises ⁇ [X]pX 10 X 11 [X]p’ ⁇ , wherein each X, X 10 and X 11 is independently an amino acid residue, and each of p and p’ is independently 0-10.
R AA5 is an optionally substituted group selected from phenyl, 5- or 6-membered monocyclic heteroaryl having 1-4 heteroatoms, and 9- or 10- membered bicyclic heteroaryl having 1-4 heteroatoms.
the length of L P6 is a covalent bond.
R AA6 is an optionally substituted group selected from phenyl, 5- or 6-membered monocyclic heteroaryl having 1-4 heteroatoms, and 9- or 10- membered bicyclic heteroaryl having 1-4 heteroatoms.
L P7 is or comprises ⁇ X 14 ⁇ [X]p’ ⁇ , wherein p’ is 0-10, each of X and X 14 is independently an amino acid residue, and X 14 is bonded to L AA6 . 17.
R N is R’, wherein R’ is ⁇ C(O)R, ⁇ CO 2 R, or ⁇ SO 2 R. 18.
R C is ⁇ OR’ or ⁇ N(R’) 2 , wherein each R’ is independently R. 19.
An agent comprising one or more of: a first acidic group (e.g., of a first acidic amino acid residue); a second acidic group (e.g., of a second acidic amino acid residue); a first aromatic group (e.g., of a first aromatic amino acid residue); a second aromatic group (e.g., of a first aromatic amino acid residue); and a third aromatic group (e.g., of a third aromatic amino acid residue).
a first acidic group e.g., of a first acidic amino acid residue
a second acidic group e.g., of a second acidic amino acid residue
a first aromatic group e.g., of a first aromatic amino acid residue
a second aromatic group e.g., of a first aromatic amino acid residue
a third aromatic group e.g., of a third aromatic amino acid residue
An agent comprising: a first acidic group (e.g., of a first acidic amino acid residue); a second acidic group (e.g., of a second acidic amino acid residue); a first aromatic group (e.g., of a first aromatic amino acid residue); a second aromatic group (e.g., of a first aromatic amino acid residue); and a third aromatic group (e.g., of a third aromatic amino acid residue).
An agent comprising: a first acidic group (e.g., of a first acidic amino acid residue); a second acidic group (e.g., of a second acidic amino acid residue); a third acidic group (e.g., of a third acidic amino acid residue); a first aromatic group (e.g., of a first aromatic amino acid residue); a second aromatic group (e.g., of a first aromatic amino acid residue); and a third aromatic group (e.g., of a third aromatic amino acid residue). 22.
An agent comprising: a first acidic group (e.g., of a first acidic amino acid residue); a second acidic group (e.g., of a second acidic amino acid residue); a hydrophobic group (e.g., of a hydrophobic amino acid residue) a first aromatic group (e.g., of a first aromatic amino acid residue); a second aromatic group (e.g., of a first aromatic amino acid residue); and a third aromatic group (e.g., of a third aromatic amino acid residue).
An agent comprising: a first acidic group (e.g., of a first acidic amino acid residue); a second acidic group (e.g., of a second acidic amino acid residue); a third acidic group (e.g., of a third acidic amino acid residue); a hydrophobic group (e.g., of a hydrophobic amino acid residue) a first aromatic group (e.g., of a first aromatic amino acid residue); a second aromatic group (e.g., of a first aromatic amino acid residue); and a third aromatic group (e.g., of a third aromatic amino acid residue).
a first acidic group e.g., of a first acidic amino acid residue
a second acidic group e.g., of a second acidic amino acid residue
a third aromatic group e.g., of a third aromatic amino acid residue
the agent of any one of the preceding Embodiments wherein the distance between a first acidic group and a second acidic group is about the distance between the acidic groups of two acidic amino acid residues of a peptide motif, wherein there are two amino acid residues between the two acidic amino acid residues. 25. The agent of any one of the preceding Embodiments, wherein a first acidic amino acid residue is at position N and a second is at position N+3. 26.
the agent of any one of the preceding Embodiments wherein the distance between a first acidic group and a third acidic group is about the distance between the acidic groups of two acidic amino acid residues of a peptide motif, wherein there are three amino acid residues between the two acidic amino acid residues.
a first acidic amino acid residue is at position N and a third is at position N+4.
the distance between a first acidic group and a first aromatic group is about the distance between the acidic group of a first acidic amino acid residue and the aromatic group of an aromatic amino acid residue of a peptide motif, wherein there are six amino acid residues between the first acidic amino acid residue and the first aromatic amino acid residue.
a first acidic amino acid residue is at position N and a first aromatic amino acid residue is at position N+7.
a first aromatic amino acid residue is at position M and a second is at position M+3.
the distance between the first aromatic group and the third aromatic group is about the distance between the aromatic groups of two aromatic amino acid residues of a peptide motif, wherein there are three amino acid residues between the two aromatic amino acid residues. 35.
the agent of Embodiment 40 wherein there are two staples in the peptide motif. 42. The agent of Embodiment 40, wherein there are three staples in the peptide motif. 43. The agent of any one of the preceding Embodiments, wherein the agent is or comprise a peptide. 44. The agent of any one of the preceding Embodiments, wherein the peptide is a stapled peptides comprising two or more staples. 45. The agent of any one of the preceding Embodiments, wherein the peptide is a stapled peptides comprising three or more staples. 46.
the agent of any one of the preceding Embodiments, wherein the peptide is a stapled peptides comprising three and no more than three staples. 47. The agent of any one of the preceding Embodiments, wherein a first acidic group, a second acidic group, a third acidic group, a hydrophobic group, a first aromatic group, a second aromatic group and a third aromatic group, if present, are presented from N to C direction of a peptide. 48. The agent of any one of the preceding Embodiments, wherein the agent is or comprises a helix structure. 49.
An agent comprising: X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 , wherein: each of X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , and X 14 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group; X 5 comprises a side chain comprising an acidic or a polar group; and each of X 9 , X 12 and X 13 comprises a side chain comprising an optionally substituted aromatic group.
An agent wherein the agent is or comprises a peptide comprising: [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 , wherein: each of p0, p15, p16 and p17 is independently 0 or 1; each of X 0 , X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , and X 17 is independently an amino acid residue, wherein: X 2 comprises a side chain comprising an acidic or a polar group; X 5 comprises a side chain comprising
the agent comprises three or more staples within 10-20 amino acid residues.
52. The agent of any one of the preceding Embodiments, wherein three or more of X 0 , X 1 , X 3 , X 4 , X 7 , X 10 , X 11 and X 14 are each independently an amino acid residue suitable for stapling, or are each independently stapled.
53. The agent of any one of the preceding Embodiments, wherein X 1 and X 4 are each independently an amino acid residue suitable for stapling or are 54.
X 4 and X 11 are each independently an amino acid residue suitable for stapling.
56. The agent of any one of Embodiments 1-54, wherein X 4 and X 11 are connected by a staple.
57. The agent of any one of the preceding Embodiments, wherein X 10 and X 14 are each independently an amino acid residue suitable for stapling.
58. The agent of any one of Embodiments 1-56, wherein X 10 and X 14 are connected by a staple.
X 7 and X 10 are each independently an amino acid residue suitable for stapling. 60.
X 1 is PL3.
X 4 is a residue of an amino acid that comprises an olefin.
X 4 is B5. 70.
X 10 is a residue of an amino acid that comprises an optionally substituted carboxyl group, an optionally substituted amino group, an azidyl group, an optionally substituted alkynyl group, or an optionally substituted thiol group. 71.
X 10 is Lys, GlnR, TriAzLys, sAla, dLys, AsnR, hGlnR, iPrLys, TriAzOrn, DGlnR, Orn, 4PipA, sCH2S, [8FBB]Cys, [4FB]Cys, [mXyl]Cys, [oXyl]Cys, [pXyl]Cys, dOrn, dDab, NMeOrn, [2_6-naph]Cys, or [3_3-biph]Cys. 72.
X 10 is Lys. 73.
X 11 is PyrS2.
X 14 is a residue of an amino acid that comprises a carboxyl group, an amino group, an azidyl group, an alkynyl group, or a thiol group.
X 14 is GlnR, Lys, sAla, Gln, Cys, TriAzLys, AsnR, hGlnR, 4PipA, sAbu, Orn, GlnR, [4mampiperidine]GlnR, [39N2spiroundecane]GlnR, [29N2spiroundecane]GlnR, iPrLys, sCH2S, [diaminobutane]GlnR, [4aminopiperidine]GlnR, dGlnR. 78.
X 14 is Glu. 79.
the agent of any one of the preceding Embodiments, wherein X 2 comprises a side chain comprising ⁇ COOH or a salt form thereof.
X 2 is Asp. 83.
X 3 comprises one or two hydrophobic side chains.
the agent of any one of the preceding Embodiments, wherein the side chain of X 3 is C 1-10 alkyl 85.
the agent of any one of Embodiments 1-84, wherein X 3 is Chg. 90.
the agent of any one of Embodiments 1-84, wherein X 3 is Leu. 91.
the agent of any one of Embodiments 1-84, wherein X 3 is Ala.
X 5 comprises a side chain comprising an acidic group.
X 5 comprises a side chain comprising ⁇ COOH or a salt form thereof.
the agent of any one of the preceding Embodiments, wherein X 5 is Asp. 95.
the agent of any one of the preceding Embodiments, wherein X 6 is 3COOHF.
the agent of any one of Embodiments 1-98, wherein X 6 is TfeGA. 101.
the agent of any one of Embodiments 1-98, wherein X 6 is Asp. 102.
the agent of any one of Embodiments 1-98, wherein X 6 is Glu. 103.
the agent of any one of the preceding Embodiments, wherein X 7 is Aib. 105.
the agent of any one of Embodiments 1-102, wherein X 7 is CyLeu. 107.
the agent of any one of Embodiments 1-102, wherein X 7 is Phe. 108.
X 12 is selected from 3Thi, 2F3MeF, Phe, nLeu, 2COOHF, CypA, 2ClF, Ala, Abu, Leu, hLeu, Npg, Cpa, Nva, Cba, ChA, 2FurA, 2OMeF, 2MeF, 2BrF, 2CNF, 2NO2F, 2PyrA, 3PyrA, 4PyrA, His, 1NapA, Val, Ile, Chg, DiethA, hnLeu, OctG, 2Thi, and 2cbmF. 119.
X 12 comprises a side chain which is or comprises an optionally substituted aromatic group.
the agent of any one of the preceding Embodiments, wherein X 12 is 3Thi. 121.
the agent of any one of Embodiments 1-119, wherein X 12 is 2Thi. 126.
the agent of any one of Embodiments 1-119, wherein X 12 is 2MeF. 127.
the agent of any one of Embodiments 1-119, wherein X 12 is 2FF. 128.
the agent of any one of Embodiments 1-119, wherein X 12 is 34ClF. 129.
the agent any one of the preceding Embodiments, wherein the side chain of X 13 comprises an optionally substituted aromatic group. 130.
the agent of any one of the preceding Embodiments, wherein X 13 is BtzA.
the agent of any one of Embodiments 1-129, wherein X 13 is 2NapA.
the agent of any one of the preceding Embodiments, wherein p18 is 1. 150.
the agent of any one of Embodiments 1-148, wherein p18 is 0. 151.
the agent of any one of the preceding Embodiments, wherein p19 is 1.
the agent of any one of Embodiments 1-148, wherein p19 is 0. 153.
the agent of any one of the preceding Embodiments, wherein p20 is 1. 154.
the agent of any one of the preceding Embodiments, wherein p21 is 1. 156.
the agent of any one of Embodiments 1-154, wherein p21 is 0. 157.
the agent of any one of the preceding Embodiments, wherein the C-terminal group is R C . 162.
the agent of any one of the preceding Embodiments, wherein the C-terminal group comprises a PEG moiety. 163.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is ⁇ OH. 164.
the agent of any one of Embodiments 164-167, wherein the R’ is optionally substituted C 1-10 aliphatic.
the agent of any one of Embodiments 164-167, wherein the R’ is optionally substituted C 1-10 alkyl.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is ⁇ N(R) 2 . 175.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is ⁇ N(R) 2 , wherein each R is independently ⁇ H or optionally substituted C 1–6 aliphatic. 176.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is ⁇ NH 2 . 177.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is ⁇ NHMe. 178.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is ⁇ NHEt. 179.
the agent of any one of Embodiments 1-160, wherein the C-terminal group is Serol. 180.
the agent of any one of the preceding Embodiments, wherein the peptide has the structure of: R N ⁇ [X] p ⁇ [X 0 ] p0 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 [X 15 ] p15 [X 16 ] p16 [X 17 ] p17 ⁇ [X] p’ ⁇ R C , or a salt thereof, wherein: each X is independently an amino acid residue; each p and p’ is independently 0-10; R N is independently a peptide, an amino protecting group or R’ ⁇ L RN ⁇ ; R C is independently a peptide, a
R C is ⁇ dAlaol, wherein the amino group of ⁇ dAlaol is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is 194.
R C is ⁇ Prool, wherein the amino group of ⁇ Prool is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is 195.
R C is ⁇ Throl, wherein the amino group of ⁇ Throl is bonded to the last ⁇ C(O) ⁇ of the peptide backbone (R C is 196.
R C is ⁇ Serol, wherein the amino group of ⁇ Serol is bonded to the last ⁇ C(O) ⁇ of the peptide backbone
R C is 197.
L RC is a covalent bond.
L RC is optionally substituted ⁇ CH 2 ⁇ . 201.
the agent of any one of Embodiments 197-200, wherein the R’ is optionally substituted C 1-10 aliphatic.
the agent of any one of Embodiments 197-200, wherein the R’ is optionally substituted C 1-10 alkyl.
the agent of any one of Embodiments 197-200, wherein the R’ is optionally substituted C 1-30 heteroaliphatic having 1-10 heteroatoms.
the R’ is optionally substituted C 1-30 heteroaliphatic having 1-10 oxygen atoms.
the agent of any one of Embodiments 197-200, wherein the R’ is optionally substituted C 6-30 aryl. 206.
the agent of any one of Embodiments 197-200, wherein the R’ is optionally substituted 5-30 membered heteroaryl having 1-10 heteroatoms. 207.
208. The agent of any one of the preceding Embodiments, wherein the peptide forms a structure that comprises a helix. 209. The agent of any one of the preceding Embodiments, wherein the peptide binds to beta-catenin. 210.
peptide binds to a polypeptide whose sequence is or comprising SEQ ID NO: 2, or a fragment thereof: SVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIIL ASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCL WTLRNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEAL VRT (SEQ ID NO: 2). 211.
An agent having the structure of or a salt thereof.
An agent having the structure of or a salt thereof.
An agent having the structure of or a salt thereof.
An agent having the structure of or a salt thereof.
An agent having the structure of or a salt thereof.
An agent having the structure of or a salt thereof. 236.
An agent having the structure of or a salt thereof.
the agent of any one of the preceding Embodiments, wherein a double bond of a staple bonded to the first stapled amino acid that is bonded to a staple with a double bond, counting from the N-terminus, is Z. 241.
the agent of any one of the preceding Embodiments, wherein a double bond of a staple bonded to the first stapled amino acid that is bonded to a staple with a double bond, counting from the C-terminus, is E. 242.
the agent of any one of the preceding Embodiments, wherein a double bond of a staple bonded to the first stapled amino acid that is bonded to a staple with a double bond, counting from the C-terminus, is Z. 243.
the agent of any one of the preceding Embodiments, wherein a double bond of a (i, i+7) staple is E. 244.
the agent of any one of the preceding Embodiments, wherein a double bond of a (i, i+7) staple is Z.
the agent of any one of the preceding Embodiments, wherein a double bond of a (i, i+2), (i, i+3) or (i, i+4) staple is E. 246.
the agent of any one of the preceding Embodiments, wherein a double bond of a (i, i+2), (i, i+3) or (i, i+4) staple is Z. 247.
the agent of any one of Embodiments 222-238, wherein a double bond of a staple bonded to the first amino acid from the N-terminus is Z. 248.
the agent of any one of Embodiments 222-246, wherein a double bond of a staple bonded to the 11th amino acid from the N-terminus is E. 249.
the agent of any one of Embodiments 222-246, wherein a double bond of a staple bonded to the 11 th amino acid from the N-terminus is Z.
the agent of any one of the preceding Embodiments, wherein a carbon atom bonded to two staples is of R configuration.
the agent of any one of any one of the preceding Embodiments, wherein a carbon atom bonded to two staples is of S configuration.
An agent having the structure of SP-1-1 or a salt thereof.
An agent having the structure of SP-1-2 or a salt thereof.
An agent having the structure of SP-1-3 or a salt thereof.
An agent having the structure of SP-1-4 or a salt thereof.
An agent having the structure of SP-1-5 or a salt thereof.
An agent having the structure of SP-1-6 or a salt thereof.
An agent having the structure of SP-1-7 or a salt thereof.
An agent having the structure of SP-1-8 or a salt thereof.
An agent having the structure of SP-2-1 or a salt thereof.
An agent having the structure of SP-2-2 or a salt thereof.
An agent having the structure of SP-2-3 or a salt thereof.
An agent having the structure of SP-2-4 or a salt thereof.
An agent having the structure of SP-2-5 or a salt thereof.
An agent having the structure of SP-2-6 or a salt thereof.
An agent having the structure of SP-2-7 or a salt thereof.
An agent having the structure of SP-2-8 or a salt thereof.
An agent having the structure of SP-3-1 or a salt thereof.
An agent having the structure of SP-3-2 or a salt thereof.
An agent having the structure of SP-4-1 or a salt thereof.
An agent having the structure of SP-4-2 or a salt thereof.
An agent having the structure of SP-4-3 or a salt thereof.
An agent having the structure of SP-4-4 or a salt thereof.
An agent having the structure of SP-4-5 or a salt thereof.
An agent having the structure of SP-4-6 or a salt thereof.
An agent having the structure of SP-4-7 or a salt thereof.
An agent having the structure of SP-4-8 or a salt thereof.
An agent having the structure of SP-5-1 or a salt thereof.
An agent having the structure of SP-5-2 or a salt thereof.
An agent having the structure of SP-5-3 or a salt thereof.
An agent having the structure of SP-5-4 or a salt thereof.
the agent of any one of Embodiments 359-364 characterized in that the agent shows 1 H NMR peaks that overlap with the peaks between about 5.1-5.7 in Figure 6 under the same or comparable conditions. 366.
the agent of any one of Embodiments 359-364 characterized in that the agent shows the same 1 H NMR peaks between about 5.1-5.7 as Figure 6 under the same or comparable conditions. 367.
the agent of any one of Embodiments 359-364 characterized in that in its 1 H NMR spectrum, the peaks corresponding to 1 H bonded to carbon atoms overlap with peaks in Figure 6 under the same or comparable conditions. 368.
the agent of any one of Embodiments 359-364 characterized in that its 1 H NMR spectrum overlaps with peaks in Figure 6 under the same or comparable conditions. 369.
the agent of any one of Embodiments 359-364 characterized in that the agent shows 1 H NMR peaks that overlap with the peaks between about 5.1-5.7 in Figure 9 under the same or comparable conditions. 370.
the agent of any one of Embodiments 359-364 characterized in that the agent shows the same 1 H NMR peaks between about 5.1-5.7 as Figure 9 under the same or comparable conditions. 371.
the agent of any one of Embodiments 359-364 characterized in that in its 1 H NMR spectrum, the peaks corresponding to 1 H bonded to carbon atoms overlap with peaks in Figure 9 under the same or comparable conditions. 372.
the agent of any one of Embodiments 359-364 characterized in that its 1 H NMR spectrum overlaps with peaks in Figure 9 under the same or comparable conditions. 373.
the agent of any one of Embodiments 359-364 characterized in that its 1 H NMR spectrum comprises the following peaks (methanol-d4, about 298K, about 900 MHz, chemical shift): 3.96, 6.34, 6.84, 6.36, 4.38, 4.31, 3.82, 4.21, 7.21, 7.24, 7.07, 7.24, 7.21, 4.03, 4.25, 7.97, 7.94, 7.42, 7.50, 4.40, 5.29, 5.31, 4.27, 4.31, 5.56, 5.36, 7.89, 7.38, 7.41, 7.89, 7.00, 4.30, 2.00, 2.10, 1.60, 1.65, 3.73, 4.15, 3.15, 4.22, 2.29, 2.41, 3.53, 3.25, 3.06, 2.95, 1.93, 2.38, 2.92, 3.45, 1.18, 1.36, 1.55, 1.84
the agent of any one of Embodiments 359-364 and 373-375 characterized in that 13 C NMR of the agent comprises the following peaks (methanol-d4, about 298K, about 226 MHz, chemical shift): 65.89, 137.36, 138.13, 57.28, 138.06, 132.12, 169.51, 62.78, 31.36, 70.51, 132.57, 139.58, 141.98, 156.51, 173.13, 175.55, 175.8, 174.47, 175.8, 177.56, 178.58, 174.39, 175.7, 177.0, 175.8, 175.44, 175.6, 176.97, 175.55, 178.08, 172.38, 59.94, 123.42, 125.19, 129.15, 56.93, 55.69, 57.70, 60.70, 130.39, 129.83, 128.08, 129.83, 130.39, 54.54, 60.94, 131.96
the agent of any one of Embodiments 359-361 and 382-384 characterized in that the agent shows 1 H NMR peaks that do not overlap with the peaks between about 5.1-5.7 in Figure 6 under the same or comparable conditions. 386.
the agent of any one of Embodiments 359-361 and 382-384 characterized in that the agent does not show the same 1 H NMR peaks between about 5.1-5.7 as Figure 6 under the same or comparable conditions. 387.
the agent of any one of Embodiments 359-361 and 382-384 characterized in that in its 1 H NMR spectrum, the peaks corresponding to 1 H bonded to carbon atoms do not all overlap with peaks in Figure 6 under the same or comparable conditions. 388.
the agent of any one of Embodiments 359-361 characterized in that its 1 H NMR spectrum does not overlap with peaks in Figure 6 under the same or comparable conditions. 389.
each olefin double bond in a staple is converted into a single bond.
each olefin double bond is converted into a single bond.
each olefin double bond is independently and optionally converted into ⁇ CHR’ ⁇ CHR’ ⁇ , wherein each R is independently ⁇ H, ⁇ R, ⁇ OR, ⁇ OH, ⁇ N(R) 2 , or ⁇ SR.
each olefin double bond is independently converted into optionally substituted ⁇ CH 2 ⁇ CH 2 ⁇ . 393.
the agent of any one of the preceding Embodiments having a purity of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. 396.
the agent of any one of the preceding Embodiments having a purity of about 90% or more. 397.
the agent of any one of the preceding Embodiments having a purity of about 95% or more. 398.
the agent of any one of the preceding Embodiments having a purity of about 98% or more. 399.
a composition comprising an agent of any one of the preceding Embodiments or a salt thereof. 400.
a pharmaceutical composition comprising or delivering an agent of any one of the preceding Embodiments, and a pharmaceutically acceptable carrier.
401. A composition selected from Table E2.
402. A pharmaceutical composition, comprising or delivering one or more or all peptide agents in a composition selected from Table E2 and a pharmaceutically acceptable carrier.
403. A composition selected from Table E3.
404. A pharmaceutical composition, comprising or delivering one or more or all peptide agents in a composition selected from Table E3 and a pharmaceutically acceptable carrier.
the composition of any one of the preceding Embodiments, comprising an agent comprising one or more staples each independently comprises one or more olefin double bond. 406.
composition of any one of the preceding Embodiments, wherein each ratio of the two stereoisomers of each olefin double bond in each staple is independently about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1 or more. 407.
the composition of any one of the preceding Embodiments, wherein a selectivity is favoring an E configuration. 408.
the composition of any one of the preceding Embodiments, wherein a selectivity is favoring a Z configuration. 409.
composition of any one of the preceding Embodiments characterized in that when the composition is administered to a system, level of a transcript of a nucleic acid or a product thereof is reduced compared to absence of the administration or administration of a reference composition. 411.
the composition of any one of the preceding Embodiments characterized in that when the composition is administered to a system comprising cancer cells, level of a transcript of a nucleic acid or a product thereof in the cancer cells is reduced compared to absence of the administration or administration of a reference composition. 412.
the composition of any one of Embodiments 410-415, wherein FGF20 transcript level is reduced. 417.
420. The composition of any one of Embodiments 410-419, wherein Axin2 transcript level is reduced. 421.
the composition of any one of Embodiments 410-421, wherein DKK4 transcript level is reduced. 423.
the composition of Embodiment 433, wherein the cancer cells are COLO320DM cells. 435.
the composition of Embodiment 433, wherein the cancer cells are patient-derived cancer cells. 436.
the composition of Embodiment 433, wherein the cancer cells are patient-derived colorectal cancer cells. 437.
the composition of Embodiment 436, wherein the colorectal cancer cells has an APC mutation.
the composition of Embodiment 438, wherein the reference is absence of the agent.
the composition of Embodiment 438, wherein the reference is administration of a reference agent. 441.
the composition of Embodiment 438, wherein the reference agent is I-470. 442.
the composition of any one of Embodiments 410-447, wherein one or each dose is independently about 20-80 mg/kg for a mouse. 449.
the composition of any one of Embodiments 410-448, wherein one or each dose is independently about 20 mg/kg for a mouse.
the composition of any one of Embodiments 410-448, wherein one or each dose is independently about 60 mg/kg for a mouse. 451.
a method comprising a) preparing a first compound comprising two moieties each of which independently comprises an olefin double bond; b) providing a second compound by stapling the two moieties by olefin metathesis of an olefin double bond of one moiety with an olefin double bond of the other to form a first-formed staple; c) add one or more additional moieties to the second compound to provide a third compound which comprising two moieties each of which independently comprises an olefin double bond; and d) providing a fourth compound by stapling the two moieties in the third compound by olefin metathesis of an olefin double bond of one moiety with an olefin double bond of the other to form a second- formed staple.
each moiety is independently an amino acid residue comprising a terminal olefin of any one of the preceding Embodiments. 453.
the method of any one of the preceding Embodiments, wherein a moiety in a first compound is an amino acid residue comprising two olefin double bond.
455. The method of any one of the preceding Embodiments, wherein one moiety in a first compound is B5. 456.
the method of any one of Embodiments, wherein the two moieties of the first compound is independently X 4 and X 11 . 457.
a first-formed staple is a (i, i+7) staple. 458.
the method of any one of the preceding Embodiments, wherein the first compound comprises ⁇ X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 ⁇ . 459.
the method of any one of the preceding Embodiments, wherein the first compound comprises ⁇ X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 ⁇ . 460.
the method of any one of the preceding Embodiments, wherein the first compound comprises a staple. 461.
Embodiment 460 wherein the staple is a (i, i+4) staple. 462.
the method of Embodiment 460, wherein the staple is between X 10 and X 14 . 463.
the method of any one of the preceding Embodiments, wherein one and only one amino acid residue comprises an olefin double bond is added to the second compound. 465.
the third compound is or comprises ⁇ X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 ⁇ . 466.
the method of any one of the preceding Embodiments, wherein the first- and second-formed staples are bonded to the same amino acid residue. 468.
the method of any one of the preceding Embodiments, wherein the two moieties in the third compound is independently X 1 and X 4 . 471.
a method comprising: contacting an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe- Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof, wherein each PG is independently protecting group for ⁇ COOH, with an agent that can promote olefin metathesis to provide an agent comprising -PL3- Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- wherein PL3 and B5, and B5 and PyrS2 are independently stapled.
Embodiment 476 The method of Embodiment 475, wherein an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form is Ac-PL3-Asp(PG)-Npg- B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support. 477.
a method comprising: contacting an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof, wherein each PG is independently protecting group for ⁇ COOH, with an agent that can promote olefin metathesis to provide an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- wherein B5 and PyrS2 is stapled. 481.
Embodiment 480 wherein an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib- Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof comprises -B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof. 482.
Embodiment 480 wherein an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib- Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof comprises -Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof. 483.
Embodiment 480 wherein an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib- Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof comprises -Asp-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof.
Embodiment 480 wherein an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib- Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof comprises -Asp-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt form thereof. 485.
Embodiment 480 wherein an agent comprising -B5-Asp(PG)-3COOHF(PG)-Aib- Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof is Fmoc-Asp-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt thereof. 486.
the method of any one of Embodiments 480-490, wherein the agent that can promote olefin metathesis is Hoveyda-Grubbs M720 complex.
Embodiments 480-493 comprising providing an agent that comprises - PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof, wherein there is a staple between B5 and PyrS2 495.
Embodiments 480-493 comprising providing an agent that comprises - PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt form thereof, wherein there is a staple between B5 and PyrS2 496.
Embodiments 480-493 comprising providing an agent that comprises Ac- PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt form thereof, wherein there is a staple between B5 and PyrS2. 497.
a method comprising: contacting an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe- Lys*3-PyrS2- or a salt form thereof, wherein there is a staple between B5 and PyrS2, and wherein each PG is independently a protecting group, with an agent that can promote olefin metathesis to provide an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof, wherein B5 and PyrS2, and B5 and PL3, are independently stapled.
Embodiment 498 The method of Embodiment 497, wherein an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof wherein there is a staple between B5 and PyrS2 comprises -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA- GlnR*3-Ala- or a salt form thereof wherein there is a staple between B5 and PyrS2.
Embodiment 499 The method of Embodiment 497, wherein an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof wherein there is a staple between B5 and PyrS2 comprises -PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA- GlnR*3-Ala-solid support or a salt form thereof wherein there is a staple between B5 and PyrS2.
an agent comprising -PL3-Asp(PG)-Npg-B5-Asp(PG)- 3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2- or a salt form thereof wherein there is a staple between B5 and PyrS2 is Ac-PL3-Asp(PG)-Npg-B5-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3- Ala-solid support or a salt form thereof wherein there is a staple between B5 and PyrS2.
the method of any one of Embodiments 497-503, wherein the agent that can promote olefin metathesis comprises ruthenium. 505.
the method of any one of Embodiments 497-503, wherein the agent that can promote olefin metathesis is Grubbs M102 complex. 506.
a method comprising: providing an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof; and reacting ⁇ NH 2 of Lys and ⁇ COOH of Glu to provide an agent comprising -Lys*3-PyrS2-3Thi-BztA- GlnR*3-. 508.
the method of Embodiment 507, wherein an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises -Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof. 509.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises -Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt form thereof.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof is Fmoc-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt thereof. 511.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises -Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises -Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt form thereof. 513.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises Fmoc-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt thereof. 514.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof. 515.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt form thereof. 516.
an agent comprising -Lys-PyrS2-3Thi-BztA-Glu- or a salt thereof comprises Fmoc-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt thereof. 517.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises -Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala- or a salt form thereof. 521.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises -Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt form thereof. 522.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises Fmoc-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA-GlnR*3-Ala-solid support or a salt thereof. 523.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA- GlnR*3-Ala- or a salt form thereof. 524.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi-BztA- GlnR*3-Ala-solid support or a salt form thereof. 525.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises Fmoc-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi- BztA-GlnR*3-Ala-solid support or a salt thereof.
an agent comprising -Lys*3-PyrS2-3Thi- BztA-GlnR*3- or a salt thereof comprises Fmoc-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys*3-PyrS2-3Thi- BztA-GlnR*3-Ala-solid support or a salt thereof.
a method comprising: de-protecting an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)- or a salt form thereof, wherein each of PGL and PGG is independently a protecting group, to provide an agent comprising -Lys- PyrS2-3Thi-BztA-Glu- or a salt form thereof. 528.
Embodiment 527 wherein an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises -Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala- or a salt form thereof.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises -Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala-solid support or a salt form thereof.
Embodiment 527 wherein an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof is Fmoc-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala-solid support or a salt thereof.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises -Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala- or a salt form thereof. 532.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises -Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala-solid support or a salt form thereof. 533.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises Fmoc-Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA-Glu(PGG)-Ala-solid support or a salt thereof. 534.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)-Ala- or a salt form thereof. 535.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)-Ala-solid support or a salt form thereof. 536.
an agent comprising -Lys(PGL)-PyrS2-3Thi-BztA- Glu(PGG)- or a salt thereof comprises Fmoc-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys(PGL)-PyrS2-3Thi- BztA-Glu(PGG)-Ala-solid support or a salt thereof. 537.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof is Fmoc-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt thereof.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof comprises -Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala- or a salt form thereof. 541.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof comprises -Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt form thereof. 542.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof comprises Fmoc-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu-Ala-solid support or a salt thereof.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof comprises -Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA-Glu- Ala-solid support or a salt form thereof. 545.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof comprises Fmoc-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA- Glu-Ala-solid support or a salt thereof.
an agent comprising -Lys-PyrS2-3Thi- BztA-Glu- or a salt thereof comprises Fmoc-Asp(PG)-3COOHF(PG)-Aib-Ala-Phe-Lys-PyrS2-3Thi-BztA- Glu-Ala-solid support or a salt thereof.
the de-protecting is performed in the presence of a Pd complex. 547.
the method of any one of Embodiments 475-550 comprising coupling Fmoc-BztA-OH or a salt thereof to a Glu residue. 552.
the method of any one of Embodiments 475-551 comprising coupling Fmoc-3Thi-OH or a salt thereof to a BztA residue. 553.
the method of any one of Embodiments 475-552 comprising coupling Fmoc-PyrS2-OH or a salt thereof to a 3Thi residue. 554.
the method of any one of Embodiments 475-553 comprising coupling Fmoc-Lys(PGL)-OH or a salt thereof to a PyrS2 residue. 555.
the method of any one of Embodiments 475-554 comprising coupling Fmoc-Phe-OH or a salt thereof to an optionally stapled Lys residue. 556.
the method of any one of Embodiments 475-555 comprising coupling Fmoc-Ala-OH or a salt thereof to a Phe residue. 557.
the method of any one of Embodiments 475-556 comprising coupling Fmoc-Aib-OH or a salt thereof to an Ala residue. 558.
the method of any one of Embodiments 475-557 comprising coupling Fmoc-3COOHF(PG)-OH or a salt thereof to an Aib residue. 559.
the method of any one of Embodiments 475-558 comprising coupling Fmoc-Asp(PG)-OH or a salt thereof to a 3HCOOHF(PG) residue. 560.
the method of any one of Embodiments 475-559 comprising coupling Fmoc-B5-OH or a salt thereof to an Asp(PG) residue. 561.
the method of any one of Embodiments 475-560 comprising coupling Fmoc-Npg-OH or a salt thereof to an optionally stapled B5 residue.
the method of any one of Embodiments 475-561 comprising coupling Fmoc-Asp(PG)-OH or a salt thereof to a Npg residue. 563.
the method of any one of Embodiments 475-562 comprising coupling Fmoc-PL3-OH or a salt thereof to an Asp(PG) residue. 564.
the method of any one of Embodiments 475-563 comprising capping amino group of a PL3 residue. 565.
the method of any one of Embodiments 475-564 comprising capping amino group of a PL3 residue with ⁇ Ac. 566.
the method of any one of Embodiments 475-565 comprising de-protecting PG. 567.
the method of any one of Embodiments 475-566 comprising cleaving an agent from a solid support. 568.
the method of any one of Embodiments 475-566 comprising cleaving an agent from a solid support by contacting the solid support with TFA. 569.
571 The method of any one of Embodiments 475-569, wherein each PG is t-butyl. 572.
Embodiment 582 wherein the stapling is performed via olefin metathesis of terminal olefins. 584.
the method of Embodiment 584, wherein the selectivity is about 1.5:1 or more. 586.
the method of Embodiment 584, wherein the selectivity is about 2:1 or more. 587.
the method of Embodiment 584, wherein the selectivity is about 3:1 or more.
the method of Embodiment 592, wherein the selectivity is independently about 1.5:1 or more. 594.
the method of Embodiment 592, wherein the selectivity is independently about 2:1 or more. 595.
the method of Embodiment 592, wherein the selectivity is independently about 3:1 or more. 596.
the method of Embodiment 592, wherein the selectivity is independently about 4:1 or more. 597.
the method of Embodiment 592, wherein the selectivity is independently about 9:1 or more. 598.
the method of Embodiment 592, wherein the selectivity is independently about 10:1 or more. 599.
the method of any one of Embodiments 584-598, wherein a selectivity is favoring an E isomer. 600.
the method of Embodiment 602, wherein the ratio after enrichment is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1 or more. 604.
the method of Embodiment 603, wherein the ratio is about 10:1 or more. 606.
the method of Embodiment 603, wherein the ratio is about 20:1 or more. 607.
the method of Embodiment 603, wherein the ratio is about 50:1 or more. 608.
the method of Embodiment 601, wherein configuration of each olefin double bond in each staple is independently enriched. 609.
the method of Embodiment 608, wherein the ratio for each olefin double bond after enrichment is independently about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1 or more. 610.
each ratio is independently about 5:1 or more. 611.
the method of Embodiment 609, wherein each ratio is independently about 10:1 or more. 612.
the method of Embodiment 609, wherein each ratio is independently about 20:1 or more. 613.
the method of Embodiment 609, wherein each ratio is independently about 50:1 or more. 614.
a chiral center is formed with about or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% stereoselectivity. 617.
the method of any one of the preceding Embodiments, wherein a chiral center is formed with about or at least about 80% or more stereoselectivity. 618.
the method of any one of the preceding Embodiments, wherein a chiral center is formed with about or at least about 85% or more stereoselectivity. 619.
a chiral center is formed with about or at least about 90% or more stereoselectivity.
the method of any one of the preceding Embodiments comprising collecting each peak with the correct mass as a product composition. 634.
the method of Embodiment 634, where the percentage is 10% or more. 636.
the method of Embodiment 634, where the percentage is 20% or more. 637.
the method of Embodiment 634, where the percentage is 50% or more. 638.
the method of Embodiment 634, where the percentage is 60% or more. 639.
the method of any one of Embodiments 634-644, wherein the peak is from UV detection. 646.
the method of any one of Embodiments 634-644, wherein the peak is from UV detection at 220 nm. 647.
the method of any one of the preceding Embodiments, wherein an agent of any one of Embodiments 362-381 are prepared. 648.
the method of any one of Embodiments 647-649 comprising comparing 1 H NMR of a prepared compound to that of any one of Embodiments 363-378. 651.
the method of any one of Embodiments 647-650 comprising comparing 13 C NMR of a prepared compound to that of any one of Embodiments 379-380. 652.
the method of any one of Embodiments 647-651 wherein when the data are determined to be of the same agent, the prepared agent is released, and/or when the data are determined to be of a different agent, the prepared agent is rejected.
a pharmaceutical composition comprising or delivering a composition of Embodiment 653 and a pharmaceutically acceptable carrier.
a method for modulating beta-catenin interaction with a partner in a system comprising contacting beta-catenin with an agent or composition of any one of the preceding Embodiments. 656.
a method for modulating beta-catenin interaction with a partner in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 657.
the method of nay one of Embodiments 655-656, wherein the partner is TCF7, LEF1, TCF7L1, TCF7L2, Axin1, Axin2, or APC. 658.
a method for modulating a TCF-beta-catenin interaction in a system comprising contacting beta- catenin with an agent or composition of any one of the preceding Embodiments. 659.
a method for modulating a TCF-beta-catenin interaction in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments.
a method for inhibiting beta-catenin dependent cell proliferation comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 661.
a method for decreasing beta-catenin polypeptide level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments.
Embodiment 662 The method of Embodiment 661, wherein CTNNB1 mRNA level is not increased. 663. The method of Embodiment 661, wherein CTNNB1 mRNA level is increased 664.
a method for increasing CTNNB1 mRNA level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 665. The method of Embodiment 664, wherein beta-catenin polypeptide level is not increased. 666. The method of Embodiment 664, wherein beta-catenin polypeptide level is increased. 667.
a method for decreasing c-Myc polypeptide and/or transcript level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 668.
a method for decreasing N-Myc polypeptide and/or transcript level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 672.
the method of any one of Embodiments 661-674, wherein L-Myc polypeptide level remain substantially the same. 676.
a method for decreasing Axin2 polypeptide and/or transcript level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 678. The method of any one of Embodiments 661-677, wherein Axin2 polypeptide level is reduced. 679. The method of any one of Embodiments 661-678, wherein Axin2 mRNA level is reduced. 680. A method for decreasing DKK4 polypeptide and/or transcript level in a system, comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments.
a method for decreasing Ki67 polypeptide and/or transcript level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 687.
a method for decreasing percentage of Ki67 polypeptide-positive cells in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 689.
a method for decreasing Notum polypeptide level in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments. 690.
a method for modulating WNT/beta-catenin pathway in a system comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments, wherein expression of a nucleic acid is modulated. 701. A method, comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments, wherein level of a transcript of a nucleic acid and/or a product thereof is modulated. 702. A method, comprising administering or delivering to the system an agent or composition of any one of the preceding Embodiments, wherein expression of a nucleic acid is modulated.. 703. The method of any one of Embodiments 655-702, wherein a system is an in vitro system.
a system is or comprises a tumor. 711.
a method for treating or preventing a condition, disorder or disease associated with beta-catenin in a subject comprising administering or delivering to the subject an effective amount of an agent or composition of any one of the preceding Embodiments. 713.
a method for treating a condition, disorder or disease in a subject comprising administering or delivering to the subject an effective amount of an agent or composition of any one of the preceding Embodiments. 714.
Embodiment 713 wherein the condition, disorder or disease is associated with or characterized by deregulation of beta-catenin. 715.
the method of Embodiment 713, wherein the condition, disorder or disease is not associated with or characterized by increased levels of beta-catenin. 718.
the method of any one of Embodiments 713-721, wherein the condition, disorder or disease is cancer. 723.
a method for treating or preventing a condition, disorder or disease associated with TCF-beta-catenin interaction in a subject comprising administering or delivering to the subject an effective amount of an agent or composition of any one of the preceding Embodiments. 728.
Embodiment 733 The method of Embodiment 728 or 729, wherein a second therapeutic agent or therapy is administered subsequently to an agent of any one of the preceding Embodiments. 734.
a second therapeutic agent is or comprises an antibody. 742.
a second therapeutic agent is or comprises a CTLA-4, PD-1 or PD-L1 inhibitor. 743.
the method of any one of the preceding Embodiments, wherein a second therapeutic agent is or comprises a cell. 744.
the method of any one of the preceding Embodiments, wherein the second therapeutic agent reduces one or more side effects of an agent or composition of any one of the preceding Embodiments. 745.
the agent or composition reduces one or more side effects of a second therapeutic agent. 746.
a second therapy is or comprises surgery. 747.
the method of any one of the preceding Embodiments, wherein a second therapy is or comprises chemotherapy.
the method of any one of the preceding Embodiments, wherein a second therapy is or comprises radiotherapy. 749.
the method of any one of the preceding Embodiments, wherein a second therapy is or comprises hormone therapy.
the method of any one of the preceding Embodiments, wherein a second therapy is or comprises stem cell or bone marrow transplant. 751.
the method of any one of the preceding Embodiments, wherein a second therapy is or comprises immunotherapy. 752.
a second therapy is or comprises T- cell therapy.
a second therapy is or comprises CAR T-cell therapy.
a second therapy is or comprises administering to the subject a population of immune cells.
the agent or composition reduces one or more side effects of a second therapy.
unit dose of a second therapy or therapeutic agent is reduced compared to when it is administered alone. 757.
Embodiments 763-771 wherein a sample is from a biopsy. 773.
a nucleic acid is selected from gene set BCAT_GDS748_UP, BCAT.100_UP.V1_UP, HALLMARK_WNT_BETA_CATENIN_SIGNALING, RASHI_RESPONSE_TO_IONIZING_RADIATION_1, REACTOME_RRNA_PROCESSING, HALLMARK_MYC_TARGETS_V1, HALLMARK_MYC_TARGETS_V2, HALLMARK_OXIDATIVE_PHOSPHORYLATION, HALLMARK_E2F_TARGETS, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_G2M_CHECKPOINT, HALLMARK_SPERMATOGENESIS, or HALLMARK_EPITHELIAL_MESENCHYMAL.
the method of any one of Embodiments 763-782, wherein a nucleic acid is DKK4. 784.
the method of any one of Embodiments 763-782, wherein a nucleic acid is NOTUM. 785.
the method of any one of Embodiments 763-782, wherein a nucleic acid is FGF20. 786.
the method of any one of Embodiments 763-776, wherein the nucleic acid is CXCL12. 787.
a composition, agent, compound or method of any one of the preceding Embodiments, wherein ruthenium level of the composition, or a preparation or composition of the agent or compound, is no more than about 1000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 ppm (weight/weight). 818.
a composition, agent, compound or method of any one of the preceding Embodiments, wherein ruthenium level of the composition, or a preparation or composition of the agent or compound, is no more than about 10 ppm.
EXEMPLIFICATION [0348] Those skilled in the art appreciate that various technologies are available for manufacturing and assessing provided agents including various peptides such as stapled peptides in accordance with the present disclosure, for example, many technologies for preparing small molecules and peptides can be utilized to prepare provided agents, and various assays are available for assessing properties and/or activities of provided agents. Described below are certain such useful technologies. As demonstrated herein, in some embodiments, it is confirmed that provided technologies can exhibit nanomolar cell-based activity in protein- protein interaction (PPI), transcriptional regulation, proliferation assays, etc. In some embodiments, it is confirmed that provided technologies possess favorable pharmacokinetic properties.
PPI protein- protein interaction
in vivo dosing of provided technologies confirms on-target pharmacodynamic modulation of ⁇ -catenin activity and strong anti-tumor activity in multiple human xenograft models, which confirm that provided technologies are useful for treating various conditions, disorders or diseases as described herein.
Example 1 Preparation of compounds.
peptides can be prepared using various peptide synthesis technologies in accordance with the present disclosure. In many embodiments, peptides were prepared using Fmoc-based synthesis, often on suitable solid phase. For various stapled peptides, amino acid residues were stapled through suitable chemistry, e.g., olefin metathesis for amino acids that comprise olefin groups.
Example 2 Provided technologies can provide improved properties and/or activities.
Various technologies can be utilized to assess technologies of the present disclosure. For example, in some embodiments, solubility, LogD, interactions with targets, etc. of various agents such as stapled peptides were assessed using technologies described in WO 2022/261257 (e.g., those in its Examples).
solubility, shake flask LogD, and binding to beta-catenin were assessed.
an assay for assessing interactions, e.g., with beta-catenin was fluorescence polarization as described in WO 2022/261257.
an assay was Nano-BRET target engagement assay that assessed beta-catenin/TCF4 engagement as described in WO 2022/261257.
an assay was a reporter assay as reported in Example 4 of WO 2022/261257.
an assay was a COLO320DM proliferation assay as reported in Example 4 of WO 2022/261257. Certain results are presented in Tables below as examples.
Table E Certain data of various compositions as examples. Structural information and compositions of stapled peptides are described in Table E2.
Compound ID 2. beta-Catenin FP IC50 (nM): A ⁇ 50 nM; 50 nM ⁇ B ⁇ 200 nM; 200 nM ⁇ C ⁇ 750 nM; 750 nM ⁇ D ⁇ 1000 nM; E > 1000 nM 3.
NanoBRET Abs IC50 (uM) A ⁇ 1.5 uM; 1.5 uM ⁇ B ⁇ 3.0 uM; 3.0 uM ⁇ C ⁇ 10.0 uM; D > 10.0 uM 4.
Table E2 Certain peptides and compositions thereof as examples. Peptides are stapled unless indicated otherwise (among other things, the present disclosure also provides unstapled versions of such peptides, optionally protected with one or more protection group (e.g., protection of N-terminus, C-terminus, side chains, etc.), and intermediates thereof). As appreciated by those skilled in the art, stapling may provide more than one stereoisomers (e.g., E/Z of double bonds and/or diastereomers). In some embodiments, a double bond in a staple is E. In some embodiments, a double bond in a staple is Z.
isomers are listed separately (typically based on reverse phase HPLC peaks (e.g., detected by UV (e.g., at 220 nm) and/or MS) in the order of elution: each earlier eluted peak is assigned a smaller ID number than each later eluted peaks (if any); in some cases, a peak may contain two or more isomers; in some cases, isomers are not separated (or single isomer), e.g., when there is one peak on HPLC).
Compositions utilized in various assays are typically of stapled peptides; the present disclosure also provides peptides prior to stapling and compositions thereof.
a general HPLC method Xselect CSH C18 column 1.7um 2.1x50mm 130 ⁇ ; Column temperature 40 °C; Flow 0.6 mL/min; 0.1% formic acid in both acetonitrile and water, 7.2 min gradient from 5 to 95% acetonitrile. In some embodiments, a different gradient and/or a C8 column were used.
N-terminal cap is connected via R 1 to the amino group (R 1 ) of the first amino acid (AA1).
a N-Term cap may be properly considered as part of AA1. From there, each carboxylate (R 2 ) of an amino acid is connected to the amino group (R 1 ) of the subsequent amino acid, until the carboxylate (R 2 ) of the final amino acid is connected to R 1 of a C-terminal group.
R 1 of the monomer in brackets is attached to R 3 of the amino acid.
R 3 For the amino acid Dap, with two potential branch points (R 3 and R4), if two branches are indicated, the R 1 of the first branch is connected to R 3 , and R 1 of the second branch connected to R4.
R 3 groups of each of those amino acids are linked to each other.
the R 3 groups of those amino acids are linked to each other.
R 1 is attached to the branching amino acid adjacent to it in the sequence, and the R 2 group of the branching monomer is attached to R 3 of the amino acid with no branching monomer designated.
PyrS2 is tied together with either R4, R5, R6, or one arm of B5, and if PL3 is present, it is typically tied to the other arm of B5.
a N-terminal group contains an olefin, it is tied to either AA3, or a branching group off of AA3.
olefins have been hydrogenated to ⁇ CH 2 ⁇ CH 2 ⁇ after olefin metathesis; if it is indicated “C-term only”, then only the C-terminal side staple, e.g., in many cases PyrS2/R5 olefin staple, has been hydrogenated to ⁇ CH 2 ⁇ CH 2 ⁇ .
C-term only only the C-terminal side staple, e.g., in many cases PyrS2/R5 olefin staple, has been hydrogenated to ⁇ CH 2 ⁇ CH 2 ⁇ .
these are separable isomers or compositions comprising one or more isomers.
a peptide comprising an amino acid residue starting with “Dap7” or “DapAc7” the olefin of that amino acid residue is tied together with one arm of B5 via olefin metathesis, while the R 3 group of that stapling amino acid residue is tied to the R 3 of another amino acid residue, e.g., GlnR*3 residue, elsewhere in the peptide.
various peptides e.g., stapled peptides, comprising residues of amino acids described herein can provide higher affinity than reference peptides that comprise a reference amino acid, e.g., a natural amino acid such as Asp or Glu, but are otherwise identical.
a reference amino acid e.g., a natural amino acid such as Asp or Glu
Example 3 Provided technologies can provide high selectivity and purity.
the present disclosure provides various technologies for preparing stapled peptides, including those comprising multiple staples. As described herein, in some embodiments, two or more staples are formed in one step. For example, in some embodiments, two or more staples are formed in a metathesis reaction.
all staples formed by metathesis are formed in a metathesis reaction. In some embodiments, each of such staples are formed through olefin metathesis of terminal olefins. In some embodiments, multiple staples are formed after full lengths of peptides have been achieved. In some embodiments, one or more staples comprising double bonds are formed after full lengths of peptides have been achieved. In some embodiments, all staples comprising double bonds are formed after full lengths of peptides have been achieved. In some embodiments, one or more staples formed through metathesis are formed after full lengths of peptides have been achieved. In some embodiments, all staples formed through metathesis are formed after full lengths of peptides have been achieved.
staples are formed in two or more staples.
two or more staples comprising olefin are formed in two or more steps.
two or more staples are formed in two or more metathesis steps.
two or more metathesis steps utilize different conditions, e.g., different catalysts.
each staple is formed in a separate step.
each staple comprising a double bond is formed in a separate step.
each staple comprising an olefin is formed in a separate step.
each staple formed by olefin metathesis is formed in a separate metathesis step.
stepwise stapling provides improved levels of selectivity to form a desired product (e.g., I-66) over other compounds, e.g., stereoisomers (e.g., for I-66, I-67).
a desired product e.g., I-66
stereoisomers e.g., for I-66, I-67
I-66 was prepared as described below, and over 10:1 I-66:I-67 ratio was observed.
the present disclosure provides a composition comprising I-66, wherein the ratio of I-66 to I-67 is about or at least about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
the present disclosure provides a composition comprising I-66 and I-67, wherein the ratio of I-66 to I-67 is about or at least about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
the ratio is about or at least about 5:1. In some embodiments, the ratio is about or at least about 10:1. In some embodiments, the ratio is about or at least about 20:1. In some embodiments, the ratio is about or at least about 30:1. In some embodiments, the ratio is about or at least about 50:1. In some embodiments, the ratio is about or at least about 80:1. In some embodiments, the ratio is about or at least about 90:1. In some embodiments, the ratio is about or at least about 100:1. In some embodiments, I-66 is provided in a salt form, e.g., a pharmaceutically acceptable salt form. In some embodiments, I-66 is provided in multiple forms including multiple salt forms.
I-67 is provided in a salt form, e.g., a pharmaceutically acceptable salt form. In some embodiments, I-67 is provided in multiple forms including multiple salt forms.
I-66 was synthesized by manual SPPS on Rink amide MBHA resin (98 g, 0.51 mmol/g loading, 50 mmol total). Deprotection steps were performed by treating the resin with 20% piperidine in DMF (v/v, 1000 mL) for thirty minutes with agitation via nitrogen bubbling. The resin was drained and washed with DMF four times.
the resin was drained and washed with DMF four times. The cycles of Fmoc deprotection and amino acid addition continued as above. A repeat coupling step was performed for Fmoc-Npg-OH.
the B5/PyrS2 staple was closed by treating the resin with Hoveyda-Grubbs M720 catalyst (15.7 g, 25 mmol) and 1,4-benzoquinone (13.5 g, 125 mmol) in dichloroethane. The reaction proceeded at room temperature for 2 h with nitrogen bubbling, the catalyst was drained, and the treatment with M720 catalyst and 1,4-benzoquinone was repeated one more time before continuing with linear peptide synthesis.
the PL3/B5 staple was closed by treating the resin with Grubbs catalyst M102 (20.6 g, 25 mmol) in dichloroethane at room temperature for 2 h with nitrogen bubbling. The catalyst solution was drained, and the treatment with Grubbs catalyst M102 was repeated another two times.
the peptide was cleaved by treating the resin with 95:5 TFA : water (800 mL, v/v) for 2 hours, and the peptide was precipitated by pouring the cleavage cocktail into cold methyl tert-butyl ether. The precipitated peptide was filtered, washed with cold MTBE twice, and dried under vacuum.
the peptide was first purified by dissolving in DMF, and loading onto a Luna C810 um 100 ⁇ column (flow rate: 20 mL/min) with a gradient of 45% to 75% acetonitrile in water (with 0.075% TFA) over 50 minutes.
Product-containing fractions were dried, and the isolated peptide was subjected to a second purification, and was dissolved in 30% acetonitrile in water and loaded on a Kromasil C85 ⁇ m 100 ⁇ column (20 mL/min), first flowing 0.4M ammonium acetate over the column for 25 min, then eluting with a gradient of 50% to 70% acetonitrile in water with 0.5% acetic acid over 50 minutes.
the product-containing fractions were lyophilized to provide I- 66 (40:1 I-66:I-67, 4997 mg, 2.41 mmol, 4.8% yield) plus a second lot of I-66 (8:1 I-66:I-67, 2015 mg, 0.97 mmol, 1.9% yield).
retention time of I-66 is 15.3 min and retention time of I-67 is 16.2 min.
such a protocol provides improved resolution compared to a reference protocol by which I-66 and I-67 may elute as one peak or may otherwise not sufficiently separated.
I-67 eluted together as the second peak and the mixture may be designated as I-67.
ratios can also be assessed using other technologies, e.g., NMR.
such a preparation of I-66 or preparations corresponding thereto were assessed in various biological assays and was confirmed to possess various properties and activities; see, e.g., Examples 11-18.
NMR spectra such as those in Figure 6 and Figure 9 to Figure 18 may contain peaks of certain impurities and/or residue 1 H in a NMR solvent. In some embodiments, NOE was observed between the peaks at about 5.45-5.6 and at about 5.2-5.35. Fractions can be further purified to provide improved purity.
I-66 and/or I-67 prepared herein may be utilized as standard/reference to assess and/or characterize other compounds and/or other preparations of I-66 and/or I-67 (e.g., different batches prepared by the same or different methods).
I-470 is similarly prepared.
I-470 differs from I-66 in that I-470 has Glu2 and Glu5 while I-66 has Asp2 and Asp5.
a preparation of I-470 is prepared as described below. In a preparation, I-470 was synthesized by manual SPPS on Rink amide MBHA resin (25.5 g, 0.51 mmol/g loading, 13 mmol total).
Deprotection steps were performed by treating the resin with 20% piperidine in DMF (v/v, 200 mL) for thirty minutes with agitation via nitrogen bubbling. The resin was drained, and washed with DMF four times. An amino acid to be coupled was dissolved in DMF (800 mL), and a coupling agent indicated below and either diisopropylethylamine (DIEA), or HOAt, were added in the equivalents listed below. Coupling proceeded for 30 minutes at room temperature with nitrogen bubbling, and the amino acid solution drained and the resin washed with DMF four times. After Aib addition, prior to Fmoc deprotection, the resin was washed with DMF five times, and dichloromethane five times.
DIEA diisopropylethylamine
the peptide was cleaved by treating the resin with 95:5 TFA : water (200 mL, v/v) for 2 hours, and the peptide was precipitated by pouring the cleavage cocktail into cold methyl tert-butyl ether. The precipitated peptide was filtered, washed with cold MTBE twice, and dried under vacuum. The peptide was first purified by dissolving in DMF, and loading onto a Luna C810 um 100 ⁇ column (flow rate: 20 mL/min) with a gradient of 0% to 80% acetonitrile in water (with 0.075% TFA) over 50 minutes.
a preparation of the present disclosure is a mixture of trans : cis olefin isomers at a staple. In some embodiments, a preparation of the present disclosure is a mixture of trans : cis olefin isomers at a staple, e.g., a B5/PyrS2 staple.
the present disclosure provides a compound having the structure of or a salt thereof. In some embodiments, the present disclosure provides a compound having the structure of
the present disclosure provides a compound having the structure of or a salt thereof. In some embodiments, the present disclosure provides a compound having the structure of
a compound has the same retention time as I-66 prepared above under the same or comparable HPLC conditions.
a HPLC condition separates I-66 and I-67.
the compound when co-injected with a I-66 preparation described herein, the compound elute as a single peak as I-66.
the compound is characterized in that in its 1 H NMR spectrum, it shows peaks that overlap with those between about 5.1-5.7 in Figure 6. In some embodiments, the compound is characterized in that in its 1 H NMR spectrum, it has the same peak pattern as Figure 6 between about 5.1-5.7. In some embodiments, the compound has the same NMR spectra as I-66 under the same or comparable conditions. In some embodiments, the compound has the same 1 H NMR spectra as I-66 under the same or comparable conditions, e.g., DMSO-d6, 373 K. Those skilled in the art appreciate that peaks of certain 1 H, such as those bonded to nitrogen and oxygen, may shift in 1 H NMR for the same compound during different assessments.
the compound has such a structure that for its 1 H NMR, peaks of 1 H bonded to carbon are found in Figure 6 under the same or comparable conditions (DMSO-d6, 373 K). In some embodiments, the compound has such a structure that its 1 H NMR peaks are found in Figure 6 under the same or comparable conditions (DMSO-d6, 373 K). In some embodiments, the compound has such a structure that its 1 H NMR peaks for 1 H bonded to carbon atoms are found in Figure 6 under the same or comparable conditions (DMSO-d6, 373 K).
integration of peak(s) in Figure 6 that correspond(s) to each 1 H bonded to carbon in the compound is independently about 1 (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, about 0.2-1.8, about 0.5- 1.5, about 0.7-1.5, 0.8-1.2, etc.) when integration of the triplet at about 5.45 to about 5.6 is set as 1.
integration of peak(s) in Figure 6 that correspond(s) to each 1 H in the compound is independently about 1 (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, about 0.2-1.8, about 0.5-1.5, about 0.7-1.5, 0.8-1.2, etc.) when integration of the triplet at about 5.45 to about 5.6 is set as 1.
An integration of Figure 6 is presented in Figure 7 as an example.
the compound is characterized in that in its 1 H NMR spectrum, it shows peaks that overlap with those between about 5.1-5.7 in Figure 9.
the compound is characterized in that in its 1 H NMR spectrum, it has the same peak pattern as Figure 9 between about 5.1-5.7.
the compound has the same NMR spectra (e.g., one or more or all of Figure 9, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17, and Figure 18) as I-66 under the same or comparable conditions, e.g., methanal-d4, 298 K.
the compound has the same 1 H NMR spectra (e.g., Figure 9) as I-66 under the same or comparable conditions, e.g., methanal-d4, 298 K.
the compound has the same 13 C NMR spectra (e.g., Figure 12) as I-66 under the same or comparable conditions, e.g., methanal-d4, 298 K.
the compound has such a structure that for its 1 H NMR, peaks of 1 H bonded to carbon are found in Figure 9 under the same or comparable conditions (methanol-d4, 298 K).
the compound has such a structure that its 1 H NMR peaks are found in Figure 9 under the same or comparable conditions (methanol-d4, 298 K).
the compound has such a structure that its 1 H NMR peaks for 1 H bonded to carbon atoms are found in Figure 9 under the same or comparable conditions (methanol-d4, 298 K). In some embodiments, the compound has such a structure that its 13 C NMR peaks are found in Figure 9 under the same or comparable conditions (methanol-d4, 298 K).
integration of peak(s) in Figure 9 that correspond(s) to each 1 H bonded to carbon in the compound is independently about 1 (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, about 0.2-1.8, about 0.5- 1.5, about 0.7-1.5, 0.8-1.2, etc.) when integration of the triplet at about 5.45 to about 5.6 is set as 1.
integration of peak(s) in Figure 9 that correspond(s) to each 1 H in the compound is independently about 1 (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, about 0.2-1.8, about 0.5-1.5, about 0.7-1.5, 0.8-1.2, etc.) when integration of the triplet at about 5.45 to about 5.6 is set as 1.
An integration of peaks in Figure 9 is included in Figure 9 as an example.
1 H NMR of a compound comprises the following peaks (or peaks corresponding to the following peaks) (methanol-d4, about 298K, about 900 MHz, chemical shift): 3.96, 6.34, 6.84, 6.36, 4.38, 4.31, 3.82, 4.21, 7.21, 7.24, 7.07, 7.24, 7.21, 4.03, 4.25, 7.97, 7.94, 7.42, 7.50, 4.40, 5.29, 5.31, 4.27, 4.31, 5.56, 5.36, 7.89, 7.38, 7.41, 7.89, 7.00, 4.30, 2.00, 2.10, 1.60, 1.65, 3.73, 4.15, 3.15, 4.22, 2.29, 2.41, 3.53, 3.25, 3.06, 2.95, 1.93, 2.38, 2.92, 3.45, 1.18, 1.36, 1.55, 1.84, 1.53, 3.27, 3.31, 3.27, 3.44, 2.74, 2.96, 1.92, 2.07, 1.37, 2.38, 1.80, 1.88, 2.
chemical shifts of 1 H bonded to carbon of a compound are (methanol-d4, about 298K, about 900 MHz): 3.96, 6.34, 6.84, 6.36, 4.38, 4.31, 3.82, 4.21, 7.21, 7.24, 7.07, 7.24, 7.21, 4.03, 4.25, 7.97, 7.94, 7.42, 7.50, 4.40, 5.29, 5.31, 4.27, 4.31, 5.56, 5.36, 7.89, 7.38, 7.41, 7.89, 7.00, 4.30, 2.00, 2.10, 1.60, 1.65, 3.73, 4.15, 3.15, 4.22, 2.29, 2.41, 3.53, 3.25, 3.06, 2.95, 1.93, 2.38, 2.92, 3.45, 1.18, 1.36, 1.55, 1.84, 1.53, 3.27, 3.31, 3.27, 3.44, 2.74, 2.96, 1.92, 2.07, 1.37, 2.38, 1.80, 1.88, 2.71, 2.81, 3.20, 2.05, 1.97, 2.17, 0.
1 H NMR of a compound comprises the following peaks (or peaks corresponding to the following peaks) (methanol-d4, about 298K, about 900 MHz, chemical shift): 8.32, 7.73, 8.17, 8.19, 8.25, 8.44, 8.89, 8.10, 8.42, 7.45, 7.39, 8.15, 8.86, and 7.69; in some embodiments, one or more or all such peaks independently correspond to 1 H bonded to nitrogen and may shift or may not be present.
13 C NMR of a compound comprises the following peaks (or peaks corresponding to the following peaks) (methanol-d4, about 298K, about 226 MHz, chemical shift): 65.89, 137.36, 138.13, 57.28, 138.06, 132.12, 169.51, 62.78, 31.36, 70.51, 132.57, 139.58, 141.98, 156.51, 173.13, 175.55, 175.8, 174.47, 175.8, 177.56, 178.58, 174.39, 175.7, 177.0, 175.8, 175.44, 175.6, 176.97, 175.55, 178.08, 172.38, 59.94, 123.42, 125.19, 129.15, 56.93, 55.69, 57.70, 60.70, 130.39, 129.83, 128.08, 129.83, 130.39, 54.54, 60.94, 131.96, 129.37,
chemical shifts of 13 C of a compound are (methanol-d4, about 298K, about 226 MHz): 65.89, 137.36, 138.13, 57.28, 138.06, 132.12, 169.51, 62.78, 31.36, 70.51, 132.57, 139.58, 141.98, 156.51, 173.13, 175.55, 175.8, 174.47, 175.8, 177.56, 178.58, 174.39, 175.7, 177.0, 175.8, 175.44, 175.6, 176.97, 175.55, 178.08, 172.38, 59.94, 123.42, 125.19, 129.15, 56.93, 55.69, 57.70, 60.70, 130.39, 129.83, 128.08, 129.83, 130.39, 54.54, 60.94, 131.96, 129.37, 129.54, 135.08, 55.56, 131.26,
NMR NMR
solvent 13 C 49.00.
NMR e.g., 1 H, 13 C, COSY, TOCSY, NOESY, HSQC, HMBC, etc.
sample concentration was about 10mg/750 uL.
temperature is about 298 K.
a strong NOE peak between the olefin protons of the PL3-B5 staple was observed. In some embodiments, this peak was stronger than the NOE peak between neighboring aromatic protons of 3Thi, and/or of the p and m protons in the phenyl group of Phe.
NMR results such as 1 H NMR results (e.g., chemical shifts, integration of peaks, etc.,), 13 C NMR results, multidimensional (e.g., 2D) NMR results, etc., may have typical error ranges.
peaks corresponding to two or more 1 H may overlap. In some embodiments, such peaks may be integrated together for assessment of numbers of 1 H.
peaks corresponding to two or more 13 C may overlap.
NMR of a preparation of I-66 described above are the same or comparable with or without the addition of the compound at a detectable level (e.g., the same amount of I-66) under the same or comparable conditions.
1 H and/or 13 C NMR of a preparation of I-66 described above are the same or comparable with or without the addition of the compound at a detectable level (e.g., the same amount of I-66, or about 0.1-10, 0.2-5, 0.5-2, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3.1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc. of I-66).
1 H NMR are considered the same or comparable when peaks corresponding to 1 H bonded to carbon have comparable chemical shift, peak shapes and/or integration.
13 C NMR are considered the same or comparable when peaks have comparable chemical shift.
peaks from impurities and solvents are properly excluded when comparing NMR. In some embodiments, peaks from impurities, solvents, 1 H bonded to oxygen, nitrogen, etc. are properly excluded when comparing NMR. Those skilled in the art appreciate that spectra may be properly considered to be the same if those skilled in the art can reasonably conclude that such spectra are comparable to each other and are of the same compound. [0375] In some embodiments, a compound has the same retention time as I-67 prepared above under the same or comparable HPLC conditions.
a HPLC condition separates I-66 and I-67.
the compound when co-injected with a I-67 preparation described herein, the compound elute as a single peak as I-67.
the compound has the same 1 H NMR peaks between about 5.0-6.0 as I-67 under the same or comparable conditions.
the compound has the same NMR spectra as I-67 under the same or comparable conditions. In some embodiments, the compound has the same 1 H NMR spectra as I-67 under the same or comparable conditions, e.g., DMSO-d6, 373 K.
NMR of a preparation of I-67 described above are the same or comparable with or without the addition of the compound at a detectable level (e.g., the same amount of I-67) under the same or comparable conditions. In some embodiments, 1 H NMR of a preparation of I-67 described above are the same or comparable with or without the addition of the compound at a detectable level (e.g., the same amount of I- 67).
ratio of the compound to a stereoisomer of the compound is about or at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
ratio of the compound to each stereoisomer of the compound is independently about or at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
ratio of all compounds that are the compound or a salt thereof to all compounds that is a stereoisomer of the compound or a salt of the stereoisomer is about or at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
ratio of all compounds that are the compound or a salt thereof to all compounds that is a stereoisomer of the compound or a salt of the stereoisomer is about or at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
the ratio is about or at least about 2:1.
the ratio is about or at least about 3:1.
the ratio is about or at least about 4:1.
the ratio is about or at least about 5:1.
the ratio is about or at least about 10:1. In some embodiments, the ratio is about or at least about 20:1. In some embodiments, the ratio is about or at least about 30:1. In some embodiments, the ratio is about or at least about 50:1. In some embodiments, the ratio is about or at least about 80:1. In some embodiments, the ratio is about or at least about 90:1. In some embodiments, the ratio is about or at least about 100:1. [0376] In some embodiments, various data herein, e.g., HPLC, NMR, etc., can be utilized for assessing preparations, e.g., in terms of identify and/or purity of prepared agents.
HPLC and/or NMR data of a preparation are compared to, e.g., those of I-66 described herein, to confirm identify, purity, etc. In some embodiments, such comparison are utilized to release or reject preparations. In some embodiments, comparation is made to a reference, e.g., an I-66 preparation that has been characterized by HPLC, NMR, etc. to possess the correct identity, purity, etc., as described herein. [0377] In some embodiments, a preparation of I-66 comprises
a preparation of I-67 comprises or a salt thereof. In some embodiments, a preparation of I-66 or a preparation of I-67 comprises a first compound
ratio of the first compound to the second compound is about or at least about 2:1, 3:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
ratio of all compounds that are the first compound or a salt thereof to all compounds that are the second compound or a salt thereof is about or at least about 2:1, 3:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
ratio of the second compound to the first compound is about or at least about 2:1, 3:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
ratio of all compounds that are the second compound or a salt thereof to all compounds that are the first compound or a salt thereof is about or at least about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
the ratio is about or at least about 2:1.
the ratio is about or at least about 3:1.
the ratio is about or at least about 4:1.
the ratio is about or at least about 5:1.
the ratio is about or at least about 10:1.
the ratio is about or at least about 20:1. In some embodiments, the ratio is about or at least about 30:1. In some embodiments, the ratio is about or at least about 50:1. In some embodiments, the ratio is about or at least about 80:1. In some embodiments, the ratio is about or at least about 90:1. In some embodiments, the ratio is about or at least about 100:1.
a ratio is a molar ratio; in some embodiments, a ratio is a weight ratio; in some embodiments, a ratio is a volume ratio; and in some embodiments, a ratio is according to an assessment.
a ratio is of peak area of UV trace at a certain wavelength, e.g., 220 nm.
I-66 was prepared following the procedures below as examples. [0379] 4 kg Rink Amide MBHA resin (5.06 kg/kg-limiting reagent; in some embodiments, > 0.8 mmol/g) were added to a reactor. Resin was washed using a suitable solvent, e.g., DMF. 16 L DMF was added, followed by 4L piperidine, and reactor contents were agitated for a suitable period of time (e.g., about 5-15 min). Reactor contents were filtered and solvent discarded.
piperidine Treatment of piperidine was repeated with a suitable period of time (e.g., about 30-60 min). Reactor contents were filtered and solvent discarded, and resin was washed with DMF, in some embodiments, multiple times. In some embodiments, drain solvent was analyzed by absorbance (specification abs ⁇ 0.1 AU) or by pH (specification ⁇ 7) to assess completion. In some embodiments, ninhydrin test was performed on a resin sample. [0380] In a separate reactor, 0.79 kg Fmoc-Ala-OH hydrate (Limiting Reagent), 0.56 kg OxymaPure, and 16 L DMF were added. The reactor contents were agitated at ambient temperature.
a suitable period of time e.g., about 30-60 min.
Reactor contents were filtered and solvent discarded, and resin was was washed with DMF, in some embodiments, multiple times.
drain solvent was analyzed by absorbance (specification abs ⁇ 0.1 AU) or by pH
N,N'- Diisopropylcarbodiimide (1.2 mol/mol-LR) was added, and reactor contents were agitated at ambient temperature for about 30 - 90 min. The contents were added to resin. DMF (e.g., 4 L) was utilized to wash the residue contents and the wash was added to resin. Resin and other contents were agitated at ambient temperature for a suitable time, e.g., about 15-24 hrs. Ninhydrin test was utilized to assess completeness. After the reaction was done the solution was removed, and resin was washed multiple times, in some embodiments, with various solvents (e.g., in some embodiments, DMF (6x), MeOH (2x), MTBE (2x)).
Resin was further treated with diisopropylethylamine and acetic anhydride for a period of time (e.g., about 30-60 min). The solution was removed and resin was washed with DMF multiple times, in some embodiments, until pH is no more than 8.
Fmoc-Glu(Oallyl)-OH 1.97 kg Fmoc-Glu(Oallyl)-OH (2 mol/mol-LR), 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'-Diisopropylcarbodiimide (2.2 mol/mol-LR);
Fmoc-BztA-OH 2.13 kg Fmoc-BztA-OH (2 mol/mol-LR), 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'-Diisopropylcarbodiimide (2.2 mol/mol-LR);
Fmoc-3Thi-OH 1.89 kg Fmoc-3Thi-OH (2 mol/mol-LR), 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'- Diisopropylcarbodiimide (
Fmoc-B5-OH 2.08 kg Fmoc-B5-OH (2 mol/mol-LR), 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'- Diisopropylcarbodiimide (2.2 mol/mol-LR);
Fmoc-Npg-OH 1.76 kg Fmoc-Npg-OH (2 mol/mol-LR), 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'- Diisopropylcarbodiimide (2.2 mol/mol-LR);
Fmoc-Asp(tBu)-OH 1.97 kg Fmoc-Asp(tBu)-OH, 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'- Diisopropylcarbodiimide (2.2 mol/mol-LR).
Resin was then washed extensively, e.g., using toluene.
0.08 kg Hoveyda- Grubbs Catalyst M720 (0.05 mol/mol-LR), 0.03 kg p-benzoquinone and 1L toluene were added in a glove box. Agitate reactor contents under nitrogen at ambient temperature until the catalyst was fully dissolved. The reagent was added to resin, and reactor contents were agitated under nitrogen at ambient temperature for a period of time, e.g., about 20 - 28 h. In some embodiments, such a metathesis was repeated. Resin was extensively washed with toluene, dichloromethane and DMF.
PL3 was added: 1.81 kg Fmoc-PL3-OH (2 mol/mol-LR), 0.68 kg OxymaPure (2 mol/mol-LR), 0.83 L N,N'-Diisopropylcarbodiimide (2.2 mol/mol-LR). Reactor contents were agitated at ambient temperature for a period of time, e.g., 4 - 24 hrs. In some embodiments, LC/MS was utilized to monitor reaction. The resin was contacted with diisopropylethylamine and acetic anhydride.
the mixture was added to resin, and reactor contents were agitated under nitrogen at ambient temperature for a period of time, e.g., 20 - 28 h.
the reaction may be repeated. in some embodiments, metathesis were performed three times.
Resin was then washed, e.g., with DCM, DMF, 2-propanol, Methyl tert-butyl ether (MTBE), etc. Resin may be dried under vacuum. In some embodiments, 95% TFA in water was utilized to de-protect and cleave products from resin. In some embodiments, cleavage solution was neutralized.
a crude product was purified to provide high purity preparations (e.g., 95% or more I-66 optionally with Ru levels no more than 10 ppm).
high purity preparations e.g., 95% or more I-66 optionally with Ru levels no more than 10 ppm.
Various purification technologies e.g., precipitation, chromatography, etc. may be utilized in accordance with the present disclosure.
reverse phase LC was utilized.
agents e.g., I-66, can also be prepared as described below.
Various technologies can be utilized to perform each of the steps in accordance with the present disclosure including those described above.
Example 4 Provided technologies can provide high selectivity.
the present disclosure provides technologies with high selectivity for forming staples comprising olefin double bonds.
Fmoc-azidolysine-PyrS2-3Thi-BztA-propargylglycine-Ala-protide resin was synthesized using standard solid phase peptide synthesis procedures. The triazole staple was closed by treating the resin with one equivalent of copper (I) iodide, one equivalent of sodium ascorbate, ten equivalents of diisopropylethylamine, and ten equivalents of 2,6-lutidine in dichloromethane at room temperature for 48 h.
the resin was washed for 5 min with DCM 2X, MeOH 1X, H 2 O 2X, 50% H 2 O/MeOH 2X, and MeOH 2X. In some embodiments, it was observed there was a small layer of insoluble material floating on top of the reactor, which was eliminated by aspiration through a hose connected to a pump. Then, continued with washes with NMP 2X, DCM 1X, and MeOH 1X.
the first staple was closed by treating the resin with 5 mol% Hoveyda-Grubbs M720 catalyst (CAS 301224-40-8) and 10 mol% benzoquinone in dichloromethane at reflux for 48 h. After 48 h, the catalyst solution was drained, the resin washed with dichloromethane 3X, dried, and then treated again with 5 mol% Hoveyda-Grubbs M720 catalyst (CAS 301224-40-8) and 10 mol% benzoquinone in dichloromethane at reflux for 48 h. [0392] After analysis by LCMS, complete reaction was observed with no identifiable starting material. The desired product is detected in > 95%, no other isomer by-product is observed. Double bond configuration is assigned based on analysis of NMR data, reported selectivity, etc., and can also be assessed by other technologies, e.g., crystallography.
the second staple was closed by treating the resin with 30 mol% Grubbs I M102 (CAS 172222-30-9) and 60 mol% benzoquinone in dichloromethane at reflux for 24 h. After 24 h, the catalyst solution was drained, the resin washed with dichloromethane 3X, dried, and then treated again with 30 mol% Grubbs I M102 (CAS 172222-30-9) and 60 mol% benzoquinone in dichloromethane at reflux for 24 h. The crude product was cleaved and deprotected, and was analyzed by LCMS and showed 82% (UV at, e.g., 210- 400 nm) of I-335.
Example 5 Two more peaks of olefin isomers were detected on as 13% and 5% of total area by HPLC, respectively. Double bond configuration is assigned based on analysis of NMR data, reported selectivity, etc., and can also be assessed by other technologies, e.g., crystallography. [0394] Example 5. Provided technologies can provide various advantages. [0395] Among other things, provided technologies can provide various advantages. In some embodiments, provided technologies can provide various advantages. In some embodiments, provided technologies can provide improved target binding profiles and/or activity profiles. For example, various stapled peptides, e.g., I-66, can provide strong binding to beta-catenin and modulation of gene expression.
various stapled peptides e.g., I-66
provided technologies e.g., I-66
provided technologies e.g., I-66
provided technologies e.g., I-66
can significantly reduce phospho-APC binding it was confirmed that provided technologies (e.g., I-66) can significantly reduce E-cadherin binding.
provided technologies e.g., I-66
direct inhibition of endogenous beta-catenin/TCF interaction was confirmed by co-immunoprecipitation (co-IP) assays as described herein.
co-IP co-immunoprecipitation
the present disclosure confirms that provided technologies can inhibit transcription of endogenous Wnt pathway target genes driven by the beta-catenin/TCF interaction.
provided technologies can modulate transcription and levels of various transcripts, in some embodiments, with selectivity. For example, in some embodiments, provided technologies inhibit beta-catenin driven transcription selectively in HAP1 WT cells.
provided technologies can reduce level of beta-catenin in nuclei.
provided technologies can block beta-catenin nuclear localization.
provided technologies can reduce level of beta-catenin nuclear translocation. For example, as confirmed in Figure 2, provided technologies can reduce levels of nuclear beta-catenin in various cells including COLO320DM cells (10 uM, 24 hr).
provided technologies can inhibit proliferation of various cells including various cancer cells.
provided technologies modulate WNT specific transcription.
provided technologies induce cell cycle arrest.
provided technologies induce G1 cell cycle arrest.
provided technologies increased proportion of cells in G1 phase of cell cycle.
provided technologies reduced proportion of cells in S phase of cell cycle.
COLO320DM which is a colorectal cell line comprising various mutations such as APC, TP53, etc.
WNT specific transcription such as of AXIN2 and CXCL12
provided technologies can significantly down- regulate Cyclin D2 and up-regulate p27.
changes of various genes e.g., AXIN2, CXCL12, etc., were observed to be consistent with changes with shRNA-knockdown of CTNNB1.
shRNA- resistant rescuing cDNA (shR-cDNA) of CTNNB1 were able to rescue beta-catenin knockdown and various observed effects thereof.
provided technologies are useful for treating various conditions, disorders or diseases including cancer. Among other things, it was confirmed that provided technologies can provide in vivo efficacy as demonstrated in various animal models. Certain useful models and/or protocols are described below as examples.
COLO320DM human colorectal cancer cells (ATCC, CCL-220), which comprise various mutations, e.g., APC and TP53, etc., were expanded in RPMI 1640 media (10% FBS) and inoculated subcutaneously, 10 7 cells per animal in 100 uL PBS/Matrigel (1:1) mixture, to male NU/J mice (JAX#2019) at 8 weeks of age.
Tumor volume was measured by electronic caliper every 2-3 days until tumor volume reached 2000 mm 3 and estimated as (length ⁇ width 2 )/2.
Animals were euthanized by CO 2 asphyxiation on the designated terminal day for each study, and plasma, tumors, tissues, etc., were excised for further analysis. Certain data are presented in Figure 4 as examples. [0405] As confirmed, technologies of the present disclosure can provide robust anti-tumor efficacy.
TGI tumor growth inhibitions
NOTUM may be utilized as a biomarker, e.g., for assessing a treatment, selecting patient population, determining whether to continue treatment, etc.
assessment of human plasma samples from normal and patients confirms that NOTUM levels are correlated with stage of diseases and may be suitable for clinical applications, e.g., as a target engagement biomarker.
various suitable in vivo pharmacokinetic and/or pharmacodynamic properties and/or activities have been confirmed.
Figure 5 provided technologies can be effectively delivered to tumors.
prolonged tumor exposure to I-66 was observed after a single dose, and tumor exposure was about 2 ⁇ 10 fold above in vitro IC50 for proliferation. It was also observed that I-66 tumor PK exceeded plasma PK at 96 hr time point.
I-66 provided significantly longer time periods during which tumor exposure was about or above in vitro IC50 for proliferation when compared to, e.g., Peptide A (Ac-PL3-Asp-Npg-B5-Asp-3COOHF-Ala-Ala-Phe-Leu- PyrS2-2F3MeF-BztA-Gln-NH2; PL3 and B5, and B5 and PyrS2 are stapled).
Peptide A A useful protocol for assessment is described below as an example. [0408] Experiments were carried out under an Institutional Animal Care and Use Committee-approved protocol, and institutional guidelines for the proper and humane use of animals were followed.
PK pharmacokinetic
a single intraperitoneal (IP) dose of I-66 and I-470 at 100 mg/kg demonstrated comparable plasma AUC in mouse.
long residence time in tumor was observed (e.g., t 1/2 > 48 hours in a tumor (SubQ, COLO320DM), single IV dose of 60 mg/kg).
tumor maintains levels above cell-based IC 50 for >7 days (tumor (SubQ, COLO320DM), single IV dose of 60 mg/kg, cell-based proliferation IC 50 for COLO320DM ⁇ 0.6 uM; cell-based IC 50 for FGF20 qPCR ⁇ 0.1 uM, AXIN2 qPCR ⁇ 0.3 uM).
tissue half-life for plasma is about 42 hours, colon about 124 hours, and muscle about 65 hours.
Robust and durable anti-tumor effects by provided technologies were confirmed in additional tumor models. In some embodiments, such effects were observed in a Patient-Derived Xenograft (PDX) cancer models.
PDX Patient-Derived Xenograft
a model is a mouse PDX colon cancer model. In some embodiments, this model has APC mutations (Tyr935Ter His1490LeufsTer20) and high AXIN2 expression. In some embodiments, for AXIN2 expression, LogCPM is about 2.5 or greater. Among other things, strong anti- tumor activities and durable tumor growth inhibition were confirmed.
TGI 103% on day 45 was observed for animals dosed at 50 mg/kg. No significant body weight loss was observed.
Certain data are presented in Figure 8, (A), as examples.
provided technologies were assessed in a mouse model carrying a patient-derived xenograft colorectal tumor. Again, robust anti-tumor effects were confirmed.
Certain data are presented in Figure 8, (B), as examples.
Vehicle vs. I-66, p 0.008.
animals were dosed and/or observed for longer time, e.g., beyond 24 days. No significant body weight loss was observed.
vehicle is 10mM sodium phosphate dibasic, 6% w/w PEG-400, 10 mg/mL L-Arginine.
a subject has Tyr935Ter APC mutation.
a subject has His1490Leu APC mutation.
a subject has Tyr935Ter His1490LeufsTer20 APC mutations.
RNA transcripts can modulate expression of various nucleic acids and/or levels of products thereof, e.g., RNA transcripts, polypeptides, etc.
tumor RNA- sequencing analysis confirmed that I-66 can provide, among other things, strong on-target Wnt/beta-catenin pathway modulation in COLO320DM tumors.
Certain negatively enriched gene sets are presented below as examples.
a negatively enriched gene is CCND2, WNT5B, AXIN2, NKD1, WNT6, DKK1, or DKK4.
top Negatively Enriched Gene Sets include BCAT_GDS748_UP, BCAT.100_UP.V1_UP, HALLMARK_WNT_BETA_CATENIN_SIGNALING, RASHI_RESPONSE_TO_IONIZING_RADIATION_1, REACTOME_RRNA_PROCESSING, HALLMARK_MYC_TARGETS_V1, HALLMARK_MYC_TARGETS_V2, HALLMARK_OXIDATIVE_PHOSPHORYLATION, HALLMARK_E2F_TARGETS, HALLMARK_TNFA_SIGNALING_VIA_NFKB. I-66 vs. I-470.
the top 5 negatively enriched gene sets were HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_MYC_TARGETS_V2, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_MYC_TARGETS_V1, and HALLMARK_OXIDATIVE_PHOSPHORYLATION (i.p.30 mg/kg, 48hr post single dose, COLO320DM tumor).
top 3 negatively enriched gene sets were HALLMARK_EPITHELIAL_MESENCHYMAL, HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_MYC_TARGETS_V2, (24 hr treatment in COLO320DM cells).
structures, interactions, etc. are characterized and assessed using X-Ray crystallography and structure determination.
atomic coordinates and structure factors are deposited in the Protein Data Bank (e.g., for beta-catenin/I-66, 8EK2, etc.).
the structure confirmed that various amino acid residues in I-66 interact with various amino acid residues in beta- catenin, for example: PL3-1 with Val349, Asp2 with Lys312 and Gly307, Npg3 with Tyr306, Asp5 with Asn387 and Trp383, 3COOHF6 with Arg342 and Lys345, Ala8 with Trp383, Phe9 with Lys345 and Trp383, 3Thi12 with Trp383 and Asn415, and BztA13 with Gln379, Leu382, Val416, Asn415, and Trp383.
PDX murine model was established in athymic nude-Foxn1 nu female mice, for example, in some embodiments, with CRC patient tumor with APC mutation (Tyr935Ter), amplified HER2, wild type KRAS and beta-catenin and high AXIN2 expression.
PDX patient-derived colorectal cancer xenografts
APC mutations for patient-derived colorectal cancer xenografts (PDX) with APC mutations, mixture of tumor (30mm 3 ) and Matrigel was implanted to female BALB/c nude mice (6-8 weeks).
mice Female Athymic Nude-Foxn1nu mice (6-8 weeks) were used.
tumors were subcutaneously implanted in the flank of an animal. Subsequently, mice were randomized when the average tumor volume reached 150 mm 3 , and intraperitoneally injected with formulated stapled peptides with corresponding dose levels (Injection Volume:10ml/kg). Tumor volume was measured by electronic caliper every 2-3 days until tumor volume reached 2000 mm 3 and estimated as (length ⁇ width 2 )/2. Body weights were weighed every 2-3 days.
TGI Tumor growth inhibition
TGI% [1 ⁇ (TVi ⁇ TV0)/(TVvi ⁇ TVv0)] ⁇ 100% (TVi: average tumor volume of a dosing group on day i, TV0: average tumor volume of a dosing group on day 0, TVvi: average tumor volume of a vehicle group on day i, TVv0: average tumor volume of a vehicle group on day0).
Animals were euthanized by CO 2 asphyxiation on the designated terminal day, and plasma, tumors, tissues, etc. were excised for further analysis. For in-life blood collection, 50 uL of blood was collected via submandibular bleed at indicated time points.
Example 6 For terminal sample collection, animals were euthanized by CO 2 asphyxiation on the designated terminal day for each study, and plasma, tumors, tissues were collected and stored at -80° C. Blood was transferred to K2EDTA and gently manually mixed by inversion 8-10 times. After inversion, sample tubes were centrifuged at 3500 RPM for 10 minutes at 2-4° C. The resultant plasma was collected and frozen immediately over dry ice and stored at ⁇ 80° C. [0419] Various useful technologies for assessing agents of the present disclosure and results therefrom are described in the Examples of WO 2022/261257 and are incorporated herein by reference. [0420] Example 6. Provided technologies can modulate levels of various transcripts and products thereof.
the present disclosure provides technologies for modulating levels and/or activities of transcripts of various nucleic acids (e.g., genes) and/or products encoded thereby.
levels and/or activities of transcripts and levels and/or activities of products, e.g., polypeptides, encoded by such transcripts may be modulated independently, or may be increased, decreased or maintained at the same or comparable levels independently.
provided technologies can reduce levels of total beta-catenin while increasing levels of CTNNB1 mRNA.
provided technologies can reduce levels of both c-Myc and N-Myc transcript and polypeptide levels.
a reduction is observed 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days post a last dose.
provided technologies e.g., I-66, does not lead to reduction in COLO320DM CDX Myc levels (e.g., 60 mg/kg, IV, 48 or 168 hours post single dose).
L-Myc transcript and/or polypeptide levels are not significantly changed.
Figure 21 demonstrates that provided technologies can reduce Axin2 transcript and polypeptide levels.
levels and/or activities of certain transcripts and/or products (e.g., polypeptides) thereof are largely unaffected under certain treatments (e.g., for DKK4 using certain dosage regimens in a CRC PDX tumor model).
such levels may be affected under some other treatments (e.g., in some embodiments, decrease of DKK4 polypeptide level was observed 7 days post 2 doses of I-6660 mg/kg is a CRC PDX tumor model).
certain biomarkers exhibit dose dependence and a range of sensitivities with repeat dosing of an agent, e.g., I-66.
levels of transcripts and/or polypeptides encoded by such genes prior to treatment may be utilized as biomarkers for selecting patients.
changes of levels of transcripts and/or polypeptides encoded by such genes before and prior treatment are useful as PD biomarkers.
various effects of provided technologies can be observed about or at least about 2 and/or 7 days post administration. In some embodiments, it is post administration of a first dose. In some embodiments, it is post administration of the second dose. In some embodiments, multiple doses are administered.
biomarkers are selected based on assessment of stapled peptide agents in various in vivo models such as patient-derived xenograft models.
useful biomarkers include mutations (e.g., APC) and transcript and/or polypeptide levels (e.g., of Axin2).
secondary antibodies used were Alexa Fluor 680 secondary antibody (A32734, Thermo Fisher Scientific) and anti-mouse Alexa Fluor 800 secondary antibody (A32730, Thermo Fisher Scientific). Protein bands were visualized and quantified using the Odyssey CLx Imaging System (LI-COR) and ImageStudio software (LI- COR) and expressed as normalized values to the corresponding housekeeping protein band.
LI-COR Odyssey CLx Imaging System
LI- COR ImageStudio software
RNA-seq Preparation For RNA-seq of PDX samples, library preparation and sequencing were performed by a vendor with TruSeq stranded mRNA library kit on Novaseq S4 Platform with PolyA enrichment. On average over 60 million 2x150 base pair reads were produced per sample.
RNA-seq Data Analysis Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.39. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.7.7a www.ncbi.nlm.nih.gov/pmc/articles/PMC3530905/.

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EP23904731.9A 2022-12-15 2023-12-15 Geklammerte peptide und verfahren dafür Pending EP4633656A1 (de)

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