WO2025192585A1 - Composé peptidique cyclique, son procédé de production et composé lieur - Google Patents

Composé peptidique cyclique, son procédé de production et composé lieur

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Publication number
WO2025192585A1
WO2025192585A1 PCT/JP2025/009072 JP2025009072W WO2025192585A1 WO 2025192585 A1 WO2025192585 A1 WO 2025192585A1 JP 2025009072 W JP2025009072 W JP 2025009072W WO 2025192585 A1 WO2025192585 A1 WO 2025192585A1
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Prior art keywords
peptide
cyclic
compound
cyclic peptide
peptide chain
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English (en)
Japanese (ja)
Inventor
直樹 梅澤
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Nagoya City University
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Nagoya City University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

  • the present invention relates to a cyclic peptide compound having a thioester structure and a method for producing the same.
  • Naturally occurring cyclic peptide compounds possess a variety of physiological activities.
  • cyclic peptide compounds such as urotensin II and somatostatin have vasoconstrictive effects and inhibit the secretion of growth hormone (GH) from the pituitary gland.
  • GH growth hormone
  • Patent Document 1 requires the use of a special tRNA synthetase and the incorporation of an unnatural amino acid into the peptide chain for cyclization, which is not necessarily easy.
  • the present invention provides novel cyclic peptide compounds that can be easily cyclized without requiring the use of special tRNA synthetases or unnatural amino acids, a method for producing the same, and linker compounds that can be used in the production method.
  • Pep1 is a peptide chain of 1 to 20 amino acids consisting of an arbitrary amino acid sequence
  • Pep2 is a peptide chain of 1 to 20 amino acids consisting of an arbitrary amino acid sequence
  • Pep3 is a peptide chain of 1 to 20 amino acids consisting of an arbitrary amino acid sequence.
  • a method for producing a cyclic peptide compound comprising a step of subjecting a peptide P having two cysteine residues to a thioester exchange reaction with a linker compound having a bis-thioester structure to obtain a cyclic peptide compound having a bis-thioester structure represented by formula (I) described below.
  • a linker compound having a bis-thioester structure represented by formula (I) described below.
  • the production method of the present invention provides novel cyclic peptide compounds that can be easily cyclized without requiring the use of special tRNA synthetases or unnatural amino acids.
  • the cyclic peptide compounds of the present invention are novel and can be used to constitute a compound library for searching for lead compounds for pharmaceuticals, for example.
  • the linker compounds of the present invention are useful in the synthesis of cyclic peptide compounds.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is a graph showing the results of a fluorescence polarization assay examining the binding of a peptide chain before cyclization and a cyclic peptide compound to MDM2 protein.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 22 is a graph showing the change over time in the area of each peak in the chromatogram of FIG. 21.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • FIG. 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • FIG. 1 shows the structures and yields of two cyclic peptides.
  • FIG. 29 shows the results of a fluorescence polarization assay using the peptides shown in FIG. 28.
  • FIG. 1 shows a method for screening by fluorescence polarization assay and the results of an example thereof.
  • FIG. 1 shows the results of screening by fluorescence polarization assay.
  • FIG. 1 shows the chemical formulas of linker compounds used in the examples.
  • FIG. 1 shows the results of screening by fluorescence polarization assay.
  • 1 is an HPLC chromatogram of the product obtained by thioester exchange reaction.
  • FIG. 1 shows the amino acid sequences of peptides used in the Examples.
  • cyclic peptide compound may be referred to as a “cyclic peptide.”
  • a first aspect of the present invention is a cyclic peptide compound having a bis-thioester structure represented by the following formula (I).
  • the cyclic peptide of this aspect may be referred to as "cyclic peptide (I).”
  • Cyclic peptide (I) is not limited to peptides cyclized by bonding a cysteine residue at the N-terminus and a cysteine residue at the C-terminus of the peptide chain, but also includes peptides cyclized intramolecularly by crosslinking two cysteine residues at any position in the peptide chain via a linker.
  • L represents any divalent linking group
  • Pep1 represents a hydrogen atom, any monovalent functional group, or any peptide chain
  • Pep2 represents a single bond or any peptide chain
  • Pep3 represents a hydrogen atom, any monovalent substituent, or any peptide chain.
  • Pep1 is an arbitrary peptide chain
  • the types of individual amino acids constituting the peptide chain may be natural or unnatural, and the number of amino acids and the order of the amino acids may also be arbitrary.
  • the peptide chain portion on the N-terminal side of the first cysteine residue in the peptide chain before cyclization may constitute Pep1 of the cyclic peptide (I) after cyclization.
  • the number of amino acids is not particularly limited, and may be, for example, 1 to 20.
  • the peptide chain constituting Pep1 may be chemically modified by known methods. For example, a fluorescent dye may be attached as a chemical modification. Note that, from the viewpoint of facilitating cyclization to obtain cyclic peptide (I) as described below, it is preferable that the peptide chain constituting Pep1 does not contain a cysteine residue.
  • Pep1 may be a hydrogen atom. If the N-terminus of the peptide chain before cyclization is a cysteine residue, Pep1 of the cyclic peptide (I) formed by cyclization of the peptide chain may be a hydrogen atom. Furthermore, if the amino group of the N-terminal cysteine residue has been chemically modified, Pep1 of the cyclic peptide (I) formed by cyclization of the peptide chain may be a functional group derived from the chemical modification. A variety of chemical structures formed by chemically modifying the N-terminus of amino acids or peptides are generally known, and these known chemical structures may be used as any monovalent functional group in Pep1 of this embodiment.
  • Pep2 is an arbitrary peptide chain
  • the types of individual amino acids constituting the peptide chain may be natural or unnatural, and the number of amino acids and the order of the amino acids may also be arbitrary.
  • the peptide chain portion sandwiched between the first cysteine residue and the second cysteine residue in the peptide chain before cyclization may constitute Pep2 of the cyclic peptide (I) after cyclization.
  • the number of amino acids is not particularly limited, and may be, for example, 1 to 20.
  • the peptide chain constituting Pep2 may be chemically modified by known methods. Note that, from the viewpoint of facilitating cyclization to obtain cyclic peptide (I) as described below, it is preferable that the peptide chain constituting Pep2 does not contain any cysteine residues.
  • Pep2 may be a single bond.
  • Pep2 of the cyclic peptide (I) obtained by cyclizing the peptide chain may be a single bond.
  • Pep3 is an arbitrary peptide chain
  • the types of individual amino acids constituting the peptide chain may be natural or unnatural, and the number of amino acids and the order of the amino acids may also be arbitrary.
  • the peptide chain portion on the C-terminal side of the second cysteine residue in the peptide chain before cyclization may constitute Pep3 of the cyclic peptide (I) after cyclization.
  • the number of amino acids is not particularly limited, and may be, for example, 1 to 20.
  • the peptide chain constituting Pep3 may be chemically modified by known methods. Note that, from the viewpoint of facilitating cyclization to obtain cyclic peptide (I) as described below, it is preferable that the peptide chain constituting Pep3 does not contain a cysteine residue.
  • Pep3 can be any monovalent functional group other than a peptide chain.
  • the C-terminus of a peptide chain before cyclization is a cysteine residue
  • Pep3 of cyclic peptide (I) formed by cyclization of the peptide chain can be a hydroxyl group.
  • the hydroxyl group of the C-terminal cysteine residue is chemically modified
  • Pep3 of cyclic peptide (I) formed by cyclization of the peptide chain can be a functional group derived from the chemical modification.
  • a variety of chemical structures in which the C-terminus of an amino acid or peptide is chemically modified are generally known, and these known chemical structures can be used as the arbitrary monovalent functional group in Pep3 of this embodiment.
  • the carboxy group of the C-terminal amino acid can be substituted with a carboxamide group.
  • linking group (L) is not particularly limited as long as it functions as a linker capable of intramolecularly crosslinking two cysteine residues in a peptide chain.
  • a structurally flexible linker may be advantageous for crosslinking.
  • the linking group (L) may be, for example, an alkylene group having 1 to 20 carbon atoms or 1 to 10 carbon atoms.
  • the aromatic cyclic group or aliphatic cyclic group tends to impart rigidity to the linker. These groups preferably have 5 to 12 carbon atoms, more preferably 5 to 7 carbon atoms.
  • the aromatic cyclic group and the aliphatic cyclic group may be heterocyclic rings in which one or more of the carbon atoms constituting the ring are substituted with heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).
  • One or more of the hydrogen atoms possessed by the alkylene group, the aromatic cyclic group, and the aliphatic cyclic group may be substituted with a substituent (e.g., an alkyl group having 1 to 3 carbon atoms, a halogen atom, a hydroxyl group, a carboxy group, a carbonyl group, an amino group, etc.).
  • a substituent e.g., an alkyl group having 1 to 3 carbon atoms, a halogen atom, a hydroxyl group, a carboxy group, a carbonyl group, an amino group, etc.
  • a second aspect of the present invention is a method for producing a cyclic peptide compound, comprising the step of subjecting a peptide P having two cysteine residues to a thioester exchange reaction with a linker compound having a bisthioester structure to obtain a cyclic peptide compound represented by formula (I). This method allows the production of the cyclic peptide compound of the first aspect.
  • Peptide P before cyclization has two cysteine residues.
  • the first and second cysteine residues are cross-linked by a linker compound.
  • the types of individual amino acids that make up peptide P may be natural or unnatural, and the number of amino acids and the order of the amino acids are also arbitrary.
  • the sequence of peptide P may be a sequence in which, from the N-terminus to the C-terminus, Pep1, the first cysteine residue, Pep2, the second cysteine residue, and Pep3 are peptide-bonded in this order.
  • the explanation of each part is the same as that of the first embodiment, so it will be omitted.
  • a third or subsequent cysteine residue may be present in any of the Pep1-3 portions of peptide P, but since this may interfere with the cross-linking reaction between the first and second cysteine residues, it is preferable to protect the thiol groups (-SH) of the third or subsequent cysteine residues in advance by chemical modification to prevent interference with the cross-linking reaction.
  • -SH thiol groups
  • Peptide P may be chemically modified at its N-terminus or C-terminus.
  • the side chains of any amino acid residues constituting peptide P may be chemically modified at their respective known locations.
  • the thioester structures of the linker compound undergo a reversible thioester exchange reaction with the thiol groups of the two cysteine residues in peptide P. Because this thioester exchange reaction can be easily carried out in an aqueous solvent, it is preferable to use a linker compound represented by formula (II) below.
  • the thioester exchange reaction between peptide P and a linker compound is preferably carried out in a pH buffered aqueous solution at a pH of 7.5 to 9.0 or 8.0 to 8.5.
  • the thioester exchange reaction proceeds easily and reversibly at 20 to 40°C within the above pH range. From the perspective of increasing the yield of the desired cyclic peptide compound, the equilibrium can be shifted toward the cyclic peptide compound by adding an excess of linker compound relative to peptide P.
  • the thioester exchange reaction stops under acidic conditions, so it can be stopped at any time by adding acid to the reaction solution.
  • the reaction usually reaches equilibrium within a few minutes to a few hours. If the cyclic peptide compound produced in the reaction solution precipitates, the product can be recovered by centrifugation or filtration. If necessary, it can then be purified by known methods such as HPLC.
  • HPLC HPLC
  • a template substance may be added to the reaction solution.
  • the template substance is a substance that induces at least a portion of peptide P into a specific three-dimensional structure.
  • the template substance may physically or chemically interact with peptide P to provide a three-dimensional structure into which at least a portion of peptide P fits, in other words, a three-dimensional structure that induces at least a portion of peptide P to easily interact with the template substance.
  • peptide P fitting into the template substance means that peptide P exhibits an uneven shape that conforms to the unevenness of the three-dimensional structure.
  • the template substance may physically or chemically interact with peptide P to provide a functional group or atomic group that bonds with a portion of the amino acid residues of peptide P.
  • the interaction between the portion of the amino acid residues and the template substance may be one or more of hydrogen bonding, covalent bonding, electrostatic interaction, and hydrophobic interaction.
  • peptide P is induced into a specific three-dimensional structure, and cysteine residues within peptide P are more likely to be cross-linked by the linker compound.
  • the specific three-dimensional structure is more likely to be maintained or stabilized in the resulting cyclic peptide.
  • a pharmaceutical target molecule e.g., protein, peptide, nucleic acid, lipid
  • MDM2 is a protein that interacts with the cancer-suppressing p53 protein.
  • X-ray crystal structure analysis has reported that the peptide portion of p53, consisting of amino acid residues 17 to 29, adopts an ⁇ -helical structure, which fits into a groove on the surface of MDM2, forming a three-dimensional structure in which the two bind (P. H. Kussie et al., Science, 1996, 274, 5289, 948-953).
  • the side chains of F19, W23, and L26 of p53 fit into a deep recess on the surface of MDM2.
  • the reaction solution was cooled to 0 °C, followed by the addition of 1-hydroxybenzotriazole monohydrate (HOBt ⁇ H O, 404 mg, 2.64 mmol) and O-benzotriazol-1-yl N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1.00 g, 2.64 mmol).
  • HOBt ⁇ H O 404 mg, 2.64 mmol
  • HBTU O-benzotriazol-1-yl N,N,N',N'-tetramethyluronium hexafluorophosphate
  • reaction solution was stirred at 0°C for 10 min, followed by the addition of a DMF (8 mL) solution of compound 2 (320 mg, 1.28 mmol). The mixture was stirred at 0°C for 5 min and then at room temperature for 28 h. The residue was dissolved in ethyl acetate/hexane (3:1, v/v, 100 mL) and washed with 0.5 M aqueous KHSO 4 (100 mL), saturated aqueous NaHCO 3 (100 mL), and saturated brine (100 mL). The organic phase was dried over Na 2 SO 4 , and the solvent was evaporated under reduced pressure.
  • the white solid residue was purified by silica gel column chromatography (hexane ⁇ ethyl acetate/hexane (1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, v/v)) to obtain the title compound (white solid, 151 mg, 87%).
  • the aqueous phase was then extracted three times with CH2Cl2 (40 mL).
  • the combined organic phases were dried over Na2SO4 , and the solvent was evaporated under reduced pressure.
  • DIPEA remained in the residue, so it was dissolved in CH2Cl2 (50 mL) and washed with 0.5 M aqueous citric acid solution (50 mL).
  • the aqueous phase was extracted three times with CH2Cl2 (50 mL).
  • the combined organic phase was washed with saturated brine (80 mL), dried over Na2SO4 , and the solvent was evaporated under reduced pressure.
  • the residue was crystallized from acetonitrile to give the title compound (white solid, 376 mg, 78%).
  • the aqueous phase was then extracted three times with CH2Cl2 (40 mL).
  • the combined organic phases were dried over Na2SO4 , and the solvent was evaporated under reduced pressure.
  • the yellow solid residue was purified by silica gel column chromatography (ethyl acetate ⁇ ethyl acetate/methanol (99:1, v/v)), and the obtained yellow solid residue was crystallized from acetonitrile to obtain the title compound (white solid, 180 mg, 36%).
  • linker compounds L1 to L3 synthesized above are listed in FIG.
  • the synthesis routes for linker compounds L1 to L3 and the yields of each product are shown in Figure 2.
  • the yields of each product when linker compounds L4 to L9 were synthesized in the same manner are shown in Figure 2.
  • Compounds 5d, 5e, 5f, 5g, 5h, and 5i used in the synthesis of linker compounds L4 to L9 were synthesized by the same method as compounds 5a to 5c, with some changes made to the starting compounds.
  • Peptide P was synthesized using an automated microwave peptide synthesizer (Liberty Lite, CEM, Japan) in solid phase.
  • the amino acid sequence of the synthesized peptide was determined by substituting two amino acid residues at any position with cysteine residues, inserting cysteine residues at any position, or adding cysteine residues to either end of the peptide sequence, based on the 16 amino acid residues (positions 14-29) of p53 as the reference sequence.
  • the sequence of peptide "P9,” described below, contains the same amino acids (except alanine) as the reference sequence, but the order of the amino acids is completely different.
  • the synthesized peptides will be referred to as "P1" and distinguished by different numbers.
  • P1–P5 and P7–P10 were prepared using Rink Amide Protide resin (0.56 mmol/g, CEM).
  • the primary solvent was dimethylformamide (DMF).
  • Deprotection was performed using 20% piperidine/dimethylformamide (DMF), 0.2 M Fmoc-amino acid/dimethylformamide (DMF), and condensation was performed using 0.5 M N,N'-diisopropylcarbodiimide (DIC) in dimethylformamide (DMF), and 0.5 M oxyma pure in dimethylformamide (DMF).
  • DIC N,N'-diisopropylcarbodiimide
  • the N-terminus was acetylated using acetic anhydride and triethylamine in DMF after solid-phase peptide synthesis.
  • Cl-TCP(Cl) ProTide resin (0.36 mmol/g, CEM) was used, and the first amino acid was introduced using 1.0 M DIPEA/0.125 M potassium iodide in dimethylformamide (DMF). Additionally, instead of 0.5 M oxyma pure in DMF, a 0.5 M oxyma pure/0.05 M DIPEA solution was used. After peptide synthesis was completed, the resin was washed with CH2Cl2 and MeOH and finally dried under vacuum.
  • DMF dimethylformamide
  • the peptides P1 to P10 synthesized above are listed in FIG.
  • the amino acid sequences of the peptides P1 to P10 synthesized above (SEQ ID NOs: 1 to 10) are shown in the sequence listing.
  • a cyclic peptide compound (SEQ ID NO: 1) was obtained as follows. To a 50 mL centrifuge tube, 200 mM Tris-HCl buffer (pH 8.0, 7.61 mL), 20 mM TCEP aqueous solution (3.04 mL), 3 M NaCl aqueous solution (1.52 mL), water (17.7 mL), and a DMSO solution of P1 (10 mM; 304 ⁇ L) were sequentially added and mixed (total volume: 30.5 mL, see table below).
  • a cyclic peptide compound (SEQ ID NO: 1) was obtained as follows. To a 50 mL centrifuge tube, 200 mM Tris-HCl buffer (pH 8.0, 6.92 mL), 20 mM TCEP aqueous solution (2.77 mL), 3 M NaCl aqueous solution (1.38 mL), water (16.1 mL), and a DMSO solution of P1 (50 mM; 0.277 mL) were added sequentially and mixed.
  • a cyclic peptide compound (SEQ ID NO: 2) was obtained as follows. To a 50 mL centrifuge tube, 200 mM Tris-HCl buffer (pH 8.0, 7.00 mL), 20 mM TCEP aqueous solution (2.80 mL), 3 M NaCl aqueous solution (1.40 mL), water (16.24 mL), and a DMSO solution of P2 (50.0 mM; 0.280 mL) were added sequentially and mixed.
  • the expression plasmid was transformed into Escherichia coli strain BL21 (DE3) Codon plus (STRATAGENE).
  • the E. coli was cultured in 2 L of LB medium. After harvesting, the cells were suspended in 40 mL of Buffer A (see below) and sonicated on ice (TOMY UD-20; output: 5, duty: 50, 2 min ⁇ 7 times). The cells were then centrifuged (26,740 g, 30 min, 4°C). The supernatant was passed through a 0.45 ⁇ m pore size Acrodisc filter for de-particleization.
  • GST glutathione-s-transferase
  • a column packed with 15 mL of Glutathione Sepharose 4B (GE Healthcare) was equilibrated with 5 CV of buffer A, and the dialyzed sample was passed through the column. The column was then washed with 1.3 CV of buffer A to obtain the sample.
  • the GST-removed MDM2 was finally purified by gel filtration chromatography using a Superdex 75pg 16/60 (GE Healthcare) column at room temperature. The flow rate was 1.2 mL/min. The column was equilibrated with 1.2 CV of Buffer A, and the sample was injected to obtain the final purified product.
  • Buffer A 20mM Tris-HCl (pH 8.0), 150mM NaCl Eluent: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 20 mM Glutathione
  • SEQ ID NO: 11 The amino acid sequence of MDM2 obtained above (SEQ ID NO: 11) is shown in the sequence listing.
  • HPLC HPLC ⁇ HPLC conditions
  • the mobile phase for HPLC was a mixture of 0.1% TFA aqueous solution (solvent A) and 0.1% TFA acetonitrile solution (solvent B).
  • the HPLC column used was an Inertsil ODS-3 5 ⁇ m, 4.6 x 250 mm (GL Sciences), and absorbance was detected at 275 nm.
  • a DMSO solution of the linker molecule (15.0 mM; 1.00 ⁇ L) and a 0.160 mM MDM2 stock solution (64.5 ⁇ L, in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl) were added and shaken at 37 °C for 2 h (see table below). 60.0 ⁇ L of the reaction mixture was then directly injected onto a HPLC column for analysis.
  • MDM2 functioned as a template, promoting the production of cyclic peptides with a three-dimensional structure that favors interaction with MDM2.
  • Fluorescence Polarization Assay A 96-well, half-area, black, flat-bottom polystyrene microplate (CORNIG, 3694) was used. The stock solutions were prepared as follows: Assay buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.01% Tween-20) was prepared by mixing 200 mM Tris-HCl buffer (pH 8.0) (7.50 mL), 3 M NaCl (750 ⁇ L), 0.022% Tween-20, and H2O (6.75 mL).
  • Tris-HCl buffer containing 150 mM NaCl 100 mM Tris-HCl buffer containing 150 mM NaCl was prepared by mixing 200 mM Tris-HCl buffer (5.00 mL), 3 M NaCl (500 ⁇ L), and H2O (4.50 mL).
  • Peptide DMSO solutions were prepared by serially diluting a 20 mM peptide DMSO solution to various concentrations. These solutions (45 ⁇ L) were further diluted with H 2 O (55 ⁇ L) to obtain 45% DMSO stock solutions.
  • the MDM2 stock solution was 0.16 mM in Tris-HCl buffer (20 mM, pH 8.0) containing 150 mM NaCl.
  • a 5% DMSO stock solution of the fluorescent peptide Flu-P4 (5-carboxyfluorescein-LTFEHYWAQLTS-NH 2 ) was prepared by mixing a 150 ⁇ M Flu-P4 DMSO solution (10 ⁇ L) with DMSO (740 ⁇ L) to obtain a 2.0 ⁇ M DMSO solution. This DMSO solution (50 ⁇ L) was further diluted with H 2 O (950 ⁇ L) to obtain a 100 nM aqueous solution (5% DMSO).
  • TCEP stock solution was prepared by dissolving TCEP in 100 mM Tris-HCl (pH 8.0) and 150 mM NaCl buffer.
  • assay buffer (53.8 ⁇ L), MDM2 stock solution (16.2 ⁇ L), 45% DMSO stock solution of peptide (10.0 ⁇ L), 5% DMSO stock solution of Flu-P4 (10.0 ⁇ L), and TCEP stock solution (10.0 ⁇ L) were sequentially added (total 100 ⁇ L, see table below).
  • the mixture was then stirred (double orbital, 500 rpm) for 1 minute and centrifuged at 1000 rpm for 5 minutes. After leaving the mixture to stand for 1 hour, fluorescence polarization (FP) was measured using a CLARIO star Plus (BMG LABTECH JAPAN Co., Ltd., Saitama City, Saitama Prefecture). The final concentrations of each component are shown in the table below.
  • the results of the fluorescence polarization assay are shown in Figure 20.
  • the fluorescent peptide Flu-P4 used in this assay is known to bind to MDM2, competes with the cyclic peptide added to the same assay system, and exhibits a weakened fluorescence polarization (measurement value) when separated from MDM2.
  • the IC50 an index of the binding strength between peptide P1 and MDM2, was 1720 nM.
  • the IC50 for the binding of the cyclic peptide (peptide P1 reacted with linker compound L1) to MDM2 was 768 nM. This result indicates that the cyclic peptide has a stronger interaction with MDM2 than P1.
  • cyclic (P1 + L3) a cyclic peptide cyclized by peptide P1 and linker compound L3 in a 1:1 ratio
  • cyclic (2 P1 + 2 L3) a cyclic peptide cyclized by peptide P1 and linker compound L3 in a 2:2 ratio
  • the results shown in the figure indicate that adding MDM2 to the reaction mixture increased the yield of intramolecularly cyclized cyclic peptides and decreased the yield of intermolecularly cyclized cyclic peptides.
  • Figure 28 shows the structures and yields of two cyclic peptides whose production increased in the presence of MDM2 in the above experiments.
  • the cyclic peptide cyclized by a 1:1 ratio of peptide P1 to linker compound L1 is referred to as "cyclic (P1 + L1)”
  • the cyclic peptide cyclized by a 1:1 ratio of peptide P1 to linker compound L2 is referred to as “cyclic (P1 + L2).
  • Figure 29 shows the results of a fluorescence polarization assay using these cyclic peptides and the solution conditions.
  • the expanded chemical formulas of linker compounds L1 to L9 are shown in Figure 32, and the amino acid sequences of peptides P1 to P15 are shown in Figure 35.
  • the amino acid sequences of peptides P1 to P9 in Figure 35 correspond to SEQ ID NOS: 1 to 9 in the Sequence Listing, and the amino acid sequences of peptides P11 to P15 correspond to SEQ ID NOS: 12 to 16 in the Sequence Listing.

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Abstract

Ce composé peptidique cyclique présente une structure bisthioester représentée par la formule (I). Dans la formule, "L" représente un groupe de liaison divalent arbitraire; "Pep1" représente un atome d'hydrogène, un groupe fonctionnel monovalent arbitraire, ou une chaîne peptidique arbitraire; "Pep2" représente une liaison simple, ou une chaîne peptidique arbitraire; et "Pep3" représente un substituant monovalent arbitraire, ou une chaîne peptidique arbitraire.
PCT/JP2025/009072 2024-03-14 2025-03-11 Composé peptidique cyclique, son procédé de production et composé lieur Pending WO2025192585A1 (fr)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
梅澤 直樹 ほか, 一時的環状化を用いた、細胞内で作用するｐ５３／ＭＤＭ２阻害ペプチドの開発, 日本薬学会第１４２年会要旨集, 04 March 2022, 27PO4-pm2-04, non-official translation (UMEZAWA, Naoki et al., Development of p53/MDM2 inhibitor peptides that act in cells using transient cyclization, Proceedings of the 142nd Annual Meeting of the Pharmaceutical Society of Japan) entire text *

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