WO2024258819A1 - Cytotoxicité néoplasique induite par glycosylation - Google Patents

Cytotoxicité néoplasique induite par glycosylation Download PDF

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Publication number
WO2024258819A1
WO2024258819A1 PCT/US2024/033347 US2024033347W WO2024258819A1 WO 2024258819 A1 WO2024258819 A1 WO 2024258819A1 US 2024033347 W US2024033347 W US 2024033347W WO 2024258819 A1 WO2024258819 A1 WO 2024258819A1
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compound
glycosyltransferase
neoplasia
cancer
udp
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Colleen HARRINGTON
Luc DE WAAL
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Broad Institute Inc
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Broad Institute Inc
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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/39—Heterocyclic compounds having sulfur as a ring hetero atom having oxygen in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin

Definitions

  • Glycosyltransferases are enzymes that establish natural glycosidic linkages. They catalyze the transfer of saccharide moieties from an activated nucleotide sugar (also known as the “glycosyl donor”) to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen-, carbon-, nitrogen-, or sulfur-based.
  • UDP- glycosyltransferases are important conjugation enzymes found in all kingdoms of life, catalyzing a sugar conjugation with small lipophilic compounds and playing a crucial role in detoxification and homeostasis. Glycosyltransferases are not implicated in apoptosis or cytotoxicity.
  • the present disclosure provides compounds that are converted into active compounds by the presence of a glycosyltransferase within a cell.
  • the present disclosure is partially based on the discovery that glycosyltransferases are able to glycosylate certain compounds to induce cytotoxicity of neoplastic cells.
  • Glycosyltransferases such as UGT3 A2 are often tissue specific, therefore only relegating the active compounds to tissue specific neoplasias that are characterized by expression of the glycosyltransferase. These neoplasia include Ewing sarcoma, rhabdomyosarcoma, and various lymphoid cancers.
  • Methods for treating a selected subject having a neoplasia, the method comprising administering to the subject a compound that is a substrate for a glycosyltransferase and forms a glycosylated compound upon interaction with the glycosyltransferase, wherein the subject is or was selected by characterizing the neoplasia for the presence of the glycosyltransferase.
  • the compound comprises an aromatic ring substituted with a hydroxyl or an amide group.
  • the compound is not doxorubicin or etoposide.
  • the compound may have the structure: a pharmaceutically acceptable salt thereof.
  • the disclosure also includes pharmaceutical compositions for the treatment of a neoplasia characterized by the presence of the glycosyltransferase; wherein the compound is a substrate for a glycosyltransferase and forms a glycosylated compound upon interaction with the glycosyltransferase.
  • the compound comprises an aromatic ring substituted with a hydroxyl or an amide group.
  • the compound is not doxorubicin or etoposide.
  • the compound may have the structure: a pharmaceutically acceptable salt thereof.
  • a method of killing a neoplastic cell comprising contacting the neoplastic cell with a compound that is a substrate for a glycosyltransferase and forms a glycosylated compound upon interaction with the glycosyltransferase, wherein the neoplastic cell is or was selected by characterizing the neoplastic cell for the presence of the glycosyltransferase.
  • the compound comprises an aromatic ring substituted with a hydroxyl or an amide group.
  • the compound is not doxorubicin or etoposide.
  • the compound may have the structure: a pharmaceutically acceptable salt thereof.
  • the compound or the glycosylated compound may be a substrate for a second protein target expressed by the neoplasia.
  • the glycosylated compound is a substrate for a second protein target expressed by the neoplasia.
  • the compound or glycosylated compound is a substrate for a second protein target expressed by the neoplasia and the subject is or was selected for characterizing the neoplasia for the presence of both the glycosyltransferase and the second protein target.
  • the second protein target may be an enzyme such as v-ATPase or a H + -ATPase or Carbonic anhydrase.
  • the enzyme is a kinase.
  • the UGT gene family is defined by a signature motif in the C-terminal domain where the uridine diphosphate (UDP)-sugar donor binds. Mammals use only 9 sugar nucleotide donors for glycosyltransferases: UDP -glucose, UDP -galactose, UDP-GlcNAc, UDP-GalNAc, UDP -xylose, UDP-glucuronic acid, GDP-mannose, GDP-fucose, and CMP-sialic acid.
  • the phosphate(s) of these donor molecules are usually coordinated by divalent cations such as manganese, however metal independent enzymes exist.
  • the glycosyltransferase may be a member of the UGT gene family or the SLC35B4 gene (or a protein or polypeptide expressed therefrom).
  • the glycosyltransferase is a UDP Glycosyltrasferase protein (e.g., UDP glycosyltransferase family 3 member Al, UDP glycosyltransferase family 3 member A2).
  • the glycosyltransferase is UDP glycosyltransferase family 3 member A2, UDP -xylose, or UDP N-acetylglucosamine transporter.
  • the neoplasia is cancer.
  • the cancer may be cancer of the skin, bone, adrenal glands, kidney, spleen, or testicals.
  • the cancer is Ewing’s sarcoma or rhabdomyosarcoma.
  • a or “an” shall mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” mean one or more than one. As used herein “another” means at least a second or more.
  • numeric values include the endpoints and all possible values disclosed between the disclosed values.
  • the exact values of all half-integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range.
  • a range of from 0.1% to 3% specifically discloses a percentage of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%.
  • a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1% to 3%, or from 0.1% to 2.5%. It will be understood that the sum of all weight % of individual components will not exceed 100%.
  • agent is meant a small compound, polypeptide or polynucleotide.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, such as a 25% change, or a 40% change, or a 50% or greater change in expression levels.
  • ingredients include only the listed components along with the normal impurities present in commercial materials and with any other additives present at levels which do not affect the operation of the disclosure, for instance at levels less than 5% by weight or less than 1% or even 0.5% by weight.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • an effective amount or “therapeutically effective amount” of an agent is meant the amount of an agent e.g., a compound described herein) required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
  • Agents described herein include compounds that are a substrate for a glycosyltransferase such as Compounds 1-4. In some embodiments, the compounds are administered in an effective amount for the treatment or prophylaxis of a disease disorder or condition.
  • an effective amount of an agent is, for example, an amount sufficient to achieve alleviation or amelioration or prevention or prophylaxis of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition associated with cancer; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition (e.g., those associated with cancer); and remission (whether partial or total), whether detectable or undetectable, as compared to the response obtained without administration of the agent.
  • the antiparasitic agent slows the rate of cancer or decreases the neoplastic cells in a host subject or medium.
  • composition represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.
  • Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gel cap); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein (see below).
  • compositions of the disclosure include nutraceutical compositions (e.g., dietary supplements) unless otherwise specified.
  • reduces is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • reference is meant a standard or control condition such as a healthy control cell.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • subject is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).
  • a subject in need thereof is typically a subject for whom it is desirable to treat a disease, disorder, or condition as described herein.
  • a subject in need thereof may seek or be in need of treatment, require treatment, be receiving treatment, may be receiving treatment in the future, or a human or animal that is under care by a trained professional for a particular disease, disorder, or condition.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any nucleic acid sequence encoding a polypeptide described herein). In some embodiments, such a sequence is at least 60%, or at least 80% or at least 85% or at least 90% or at least 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIG. l is a schematic of the glycosylation mechanism thought to be responsible for activity of the compounds of the present disclosure.
  • the compound is a substrate for the glycosyltransferase which becomes glycosylated. Without wishing to be bound by theory, this glycosylation is required for sensitivity of the compounds of the present disclosure.
  • FIG. 2 provides a schematic of the PRISM screen used to identify Compound 1 with selective activity in Ewing sarcoma (left).
  • FIG. 2 also provides an exemplary measurement result from the PRISM screen illustrating the Log2 -Fold-Change (LFC) following 625 nM administration of Compound 1 comparing Ewing Sarcoma (EWS) and other characterized cell lines.
  • LFC Log2 -Fold-Change
  • FIG. 3 A provides the Pearson Correlation of gene expression and compound sensitivity in EWS cells with CRISPR knockout of the indicated gene.
  • FIG. 3B provides the log fold change (LFC) of sensitivity following knockout of genes and corresponding q-values as determined by differential representation methods for CRISPR knockout of genes.
  • FIG. 4 provides a measurement illustrating the lysotracker+ punctae per cell for several test conditions including administration of Compound 1.
  • FIG. 5 is a Western Blot of the CRISPR KO clones taken from pooled CRISPR knockout (KO) of UGT3A2.
  • the arrow indicates the two bands associated with UGT3A2 in each clone.
  • FIGs. 6A-6C provide the concentration of Compound 1 administered and the corresponding number of Lysotracker spots measured (which is correlated with live cells following administration).
  • FIG. 6A provides sensitivity with A673 Cas9 LacZ (which expressed the glycosyltransferase UGT3 A2)
  • FIG. 6B provides sensitivity with UGT3 A2 KO clone 1 (which did not express the glycosyltransferase UGT3 A2)
  • FIG. 6C provides sensitivity with UG3 A2 (which expressed the glycosyltransferase UGT3 A2).
  • FIGs. 7A-7C provide the results of the cell viability assay for Compound 1 (FIG. 7A), Compound 2 (FIG. 7B), Compound 3 (FIG. 7C) as compared to A673 LacZ cells and the UGT3 A2 KO clone 1 (which did not express UGT3 A2).
  • FIG. 7D compares the Compound 1 with a glycosylated version thereof.
  • FIGs. 8A-8B provide the results of cell viability assay on RH30 cells as UGT3 A KO RH30 cells for Compound 1 (FIG. 8A) and Compound 2 (FIG. 8B).
  • FIG. 9A shows the mass spectroscopic analysis (measured by area counts) of media and cell lysate at several time points following administration of Compound 1. At each time point, from left to right, the data shows Compound 1 in Media, Compound 1 in Cell Lysate, Glycosylated product in Media, and Glycosylated Product in Cell Lysate.
  • FIG. 9B compares the concentration of glycosylated product measured at several time points in both WT and KO cells.
  • glycosyltransferases confers cytotoxic activity to certain compounds. These glycosyltransferases are found in many neoplasms including Ewing’s sarcoma, rhabdomyosarcoma, and lymphoid cancers.
  • UDP Glycosyltransferase protein e.g., UDP glycosyltransferase family 3 member Al, UDP glycosyltransferase family 3 member A2 (UGT3 A2)
  • UGT3A2 in particular is biased expression in skin (RPKM 4.5), bone marrow (RPKM 1.8), adrenal glands, kidney, spleen, and the testis.
  • UGT3 A2 polypeptide is typically a protein or fragment thereof associated with the UDP-glucuronosyltransferase 3 A2 protein which is in glycosyltransferase family 1 and related proteins with GTB topology, one of two protein topologies observed for nucleotidesugar dependent glycosyltransferases.
  • the UGT3 A2 polypeptide may haveat least 85% amino acid sequence identity to the sequence provided at NCBI Ref No. XP_011512290 which provides the isoform XI.
  • An exemplary UGT3A2 Homo sapiens amino acid sequence is
  • the glycosyltransferase may be a UDP glucuronosyltransferase, a P-1, 3- glucuronyltransf erase, a P-4-glycosyltransf erase, a P-3 -glycosyltransferases, a glycosyltransferases group 1 domain protein, a glycosyltransferase family 2 protein, a glycosyltransferasefamily 6 protein, a dolichyl D-mannosylphosphate dependent mannosyltransferases, an exostosin glycosyltransferase family protein, a polypeptide N-acetyl galactosaminyltransferase, a fucosyltransferase, a mannosyl-glycoprotein-N- acetylglucosaminyltransferase, a glycosyltransferase family 8 protein, a glycogen phosphorylase, a
  • the glycosyltransferase is a UDP glucouronosyltransferase such asUDP glucuronosyltransferase family 1 member A complex locus (UGT1 A), UDP glucuronosyltransferase family 1 member Al (UGT1A1), UDP glucuronosyltransferase family 1 member A2, pseudogene (UGT1 A2P), UDP glucuronosyltransferase family 1 member A3 (UGT1 A3), UDP glucuronosyltransferase family 1 member A4 (UGT1 A4), UDP glucuronosyltransferase family 1 member A5 (UGT1 A5), UDP glucuronosyltransferase family 1 member A6 (UGT1 A6), UDP glucuronosyltransferase family 1 member A7 (UGT1 A7), UDP glucuronosyltransfera
  • the glycosyltransferase is a member of UDP glycosyltransferase family 3 such as UDP glycosyltransferase family 3 member Al (UGT3 Al) or UDP glycosyltransferase family 3 member A2 (UGT3 A2).
  • UDP glycosyltransferase family 3 member Al UDP glycosyltransferase family 3 member Al
  • UDP glycosyltransferase family 3 member A2 UDP glycosyltransferase family 3 member A2
  • the compounds of the present disclosure provide activity to neoplasias expressing glycosyltransferases following administration.
  • the glycosyltransferase glycosylates the compound thereby conferring activity.
  • the glycosyltransferase may convert Compound 1 in the following manner:
  • the glycosyltransferase may convert Compound 2 (R112) in any of the following manners:
  • the glycosyltransferase may convert Compound 3 (ti oxoIone) as follows:
  • the glycosyltransferase may convert Compound 4 (Bafilomycin Al or Baf Al) as follows:
  • the compounds may have an ECso (e.g., as measured by a viability assay) of less than (or 0.1 nM to) 10 pM or less than 1 pM.
  • the glycosylated mechanism is shown in the schematic provided in FIG. 1.
  • the glycosylated compound may then interact with a different protein e.g., an enzyme, a kinase, v-ATPase, H + -ATPase, Carbonic anhydrase).
  • Compounds provided herein can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • the optically active forms can be obtained for example by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant).
  • certain of the disclosed compounds may exist in various stereoisomeric forms including stereoisomers, enantiomers, diastereomers, or racemates (z.e., the compound exists as a mixture containing two enantiomers and does not rotate polarized light).
  • Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well- known techniques and methods, such as chiral chromatography and separation methods based thereon.
  • the compound provided herein may also be present as geometric isomer which differ in the orientation of substituent atoms (e.g., to a carbon-carbon double bond, to a cycloalkyl ring, to a bridged bicyclic system).
  • Atoms (other than H) on each side of a carbon-carbon double bond may be in an E (substituents are on opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) configuration.
  • “R,” “S,” “S*,” “R*,” “E,” “Z,” “cis,” and “trans,” indicate configurations relative to the core molecule and may be used to indicate the geometric configuration of the presently disclosed compounds.
  • Certain of the disclosed compounds may exist in atropisomeric forms.
  • Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers.
  • the compounds disclosed herein may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture.
  • Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods.
  • the named or depicted stereoisomer may be typically more than 50% (e.g., at least 55%, 60%, 70%, 80%, 90%, 99%, or 99.9%) by weight (or mole fraction) relative to the other stereoisomers.
  • the depicted or named enantiomer is more than 50% (e.g., at least 55%, 60%, 70%, 80%, 90%, 99%, or 99.9%) by weight (or mole fraction) optically pure.
  • the depicted or named diastereomer is more than 50% (e.g., at least 55%, 60%, 70%, 80%, 90%, 99%, or 99.9%) by weight (or mole fraction) pure.
  • Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer.
  • Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers.
  • Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer.
  • percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer.
  • Solvates of the compounds described herein may form the aggregate of the compound or an ion of the compound with one or more solvents. Such solvents may not interfere with the biological activity of the solute.
  • suitable solvents include, but are not limited to, water, MeOH, EtOH, and AcOH.
  • Solvates wherein water is the solvent molecule are typically referred to as hydrates. Hydrates include compositions containing stoichiometric amounts of water, as well as compositions containing variable amounts of water.
  • the compounds described herein may be present as a pharmaceutically acceptable salt.
  • salts are composed of a related number of cations and anions (at least one of which is formed from the compounds described herein) coupled together e.g., the pairs may be bonded ionically) such that the salt is electrically neutral.
  • Pharmaceutically acceptable salts may retain or have similar activity to the parent compound (e.g., an ED50 within 10%) and have a toxicity profile within a range that affords utility in pharmaceutical compositions.
  • pharmaceutically acceptable salts may be suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio.
  • Salts are described in: Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, di chloroacetate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hippurate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, mucate, 2- naphthalenesulfonate
  • Representative basic salts include alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, aluminum salts, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, caffeine, and ethylamine.
  • Pharmaceutically acceptable acid addition salts of the disclosure can be formed by the reaction of a compound of the disclosure with an equimolar or excess amount of acid.
  • hemi-salts can be formed by the reaction of a compound of the disclosure with the desired acid in a 2:1 ratio, compound to acid.
  • the reactants are generally combined in a mutual solvent such as diethyl ether, tetrahydrofuran, methanol, ethanol, /.w-propanol, benzene, or the like.
  • the salts normally precipitate out of solution within, e.g., one hour to ten days and can be isolated by filtration or other conventional methods. It will be understood that in the event of any inconsistency between a chemical name and formula, both compounds with the indicated chemical name and compounds with the indicated chemical structure will be considered as embraced by the disclosure.
  • the compounds of the present disclosure include the compounds themselves, as well as their salts and their prodrugs, if applicable.
  • a salt for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate.
  • a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein.
  • Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion.
  • prodrugs include Ci-6 alkyl esters of carboxylic acid groups, which, upon administration to a subject, are capable of providing active compounds that may be glycosylated.
  • the compounds described herein are useful for the treatment and prophylaxis of neoplasia in a subject in need thereof.
  • the compounds described herein may also be compounds for use in the preparation of a medicament for the treatment of neoplasia in a subject in need thereof. These neoplasias may be characterized for the presence of the glycosyltransferase.
  • Pharmaceutical dosage forms are provided as well, which may comprise a compound of the present disclosure (e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4) and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • a compound of the present disclosure e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4
  • pharmaceutically acceptable carriers, diluents, or excipients e.g., a pharmaceutically acceptable carriers, diluents, or excipients.
  • Unit dosage forms also referred to as unitary dosage forms, often denote those forms of medication supplied in a manner that does not require further weighing or measuring to provide the dosage (e.g., tablet, capsule, pellet, caplet).
  • the compositions of the present disclosure may be present as unit dosage forms.
  • a unit dosage form may refer to a physically discrete unit suitable as a unitary dosage for human subjects and other species, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients.
  • Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, and gel cap.
  • the compounds described herein, including crystallized forms, polymorphs, and solvates thereof may be present in a unit dosage form.
  • Useful pharmaceutical carriers, excipients, and diluents for the preparation of the compositions hereof can be solids, liquids, or gases. These include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the pharmaceutically acceptable carrier or excipient does not destroy the pharmacological activity of the disclosed compound and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g., binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, and aerosols.
  • the carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil. Water, saline, aqueous dextrose, and glycols are examples of liquid carriers, particularly (when isotonic with the blood) for injectable solutions.
  • formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution and rendering the solution sterile.
  • suitable pharmaceutical excipients include starch, cellulose, chitosan, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.
  • compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, and buffers.
  • suitable pharmaceutical carriers and their formulation are described in Remington’s Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for administration to the recipient.
  • Non-limiting examples of pharmaceutically acceptable carriers and excipients 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; 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 polyethylene glycol and propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents
  • Cyclodextrins such as a-, P-, and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2-and 3-hydroxypropyl-cyclodextrins, or other solubilized derivatives can also be used to enhance delivery of the compounds described herein.
  • the compositions of the disclosure are formulated in pellets or tablets for an oral administration. According to this type of formulation, they comprise lactose monohydrate, cellulose microcrystalline, crospovidone/povidone, aroma, compressible sugar and magnesium stearate as excipients.
  • the compositions are in the form of pellets or tablets, they are for instance 1 mg, 2 mg, or 4 mg pellets or tablets. Such pellets or tablets are divisible so that they can be cut to suit the posology according to the disclosure in one or two daily takes.
  • the compositions of the disclosure are formulated in injectable solutions or suspensions for a parenteral administration.
  • the injectable compositions are produced by mixing therapeutically efficient quantity of torasemide with a pH regulator, a buffer agent, a suspension agent, a solubilization agent, a stabilizer, a tonicity agent and/or a preservative, and by transformation of the mixture into an intravenous, sub-cutaneous, intramuscular injection or perfusion according to a conventional method. Possibly, the injectable compositions may be lyophilized according to a conventional method.
  • suspension agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, xanthan gum, sodic carboxymethylcellulose and polyethoxylated sorbitan monolaurate.
  • solubilization agent examples include polyoxy ethylene- solidified castor oil, polysorbate 80, nicotinamide, polyethoxylated sorbitan monolaurate, macrogol and ethyl ester of caste oil fatty acid.
  • the stabilizer includes sodium sulfite, sodium metalsulfite and ether, while the preservative includes methyl p-hydroxybenzoate, ethyl p- hydroxybenzoate, sorbic acid, phenol, cresol and chlorocresol.
  • An example of tonicity agent is mannitol.
  • the pharmaceutical composition further comprises a viscosity enhancing agent.
  • the viscosity enhancing agent includes methylcellulose, hydroxy ethylcellulose, hydroxypropylmethylcellulose and smart hydrogel.
  • the viscosity enhancing agent is hydroxyethylcellulose.
  • the pharmaceutical composition comprises 0.01-1.0% (w/v) viscosity enhancing agent.
  • the intranasal pharmaceutical composition comprises 0.05% (w/v) hydroxyethylcellulose.
  • the pH of the pharmaceutical composition is from 4.0 to 7.5. In other embodiments, the pH of the pharmaceutical composition is from 4.0 to 6.5. In another embodiment the pharmaceutical composition has a pH of from 5.5 to 6.5. In further embodiments, the pharmaceutical composition has a pH of from 6.0 to 6.5. In various implementations, the pH of said aqueous solution or liquid formulation is from pH 3 to pH 7, from pH 3 to pH 6, from pH 4 to pH 6, or from pH 5 to pH 6. These pH ranges may be achieved through the incorporation of one or more pH modifying agents, buffers, and the like. In some embodiments, a pH modifier such as acetic acid, is present in a final concentration of at least 0.001% or at least 0.01% or between 0.01%-0.2% by weight of the composition.
  • a pH modifier such as acetic acid
  • compositions of this disclosure may include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solid or liquid compositions used for application to skin and other tissues where the compositions may be used.
  • compositions may contain: additional antimicrobials, moisturizers and hydration agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyestuffs, coloring agents, powders, viscosity-controlling agents and water, and optionally including anesthetics, anti-itch actives, botanical extracts, conditioning agents, darkening or lightening agents, glitter, humectants, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinals.
  • the composition of the disclosure is formulated with the above ingredients so as to be stable for a long period of time, as may be beneficial where continual or long-term treatment is intended.
  • beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable.
  • a disease, disorder, or condition may be palliated which includes that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.
  • any biomarker e.g., a glycosyltransferase such as UGT3A, a second protein target such as an ezyme including kinases or v-ATPases
  • expression of a biomarker may be determined by comparison to a reference such as a healthy control cell or a baseline measurement.
  • expression of a biomarker may be determined by comparison to the reference and the cell is considered to express the biomarker if there is a small difference (e.g., the neoplasia copy number is within 10% of the copy number of the reference, the neoplasia copy number is within 5% of the reference) between expression in the cancer cell and the reference.
  • Genomics may be used to determine expression and relative expression levels.
  • the cell may be considered to not express a biomarker if the number of copies of the biomarker per cellular genome is less than 1 or less than 2' 1 or less than 2' 2 or less than 2' 3 or less than 2' 4 or less than 2' 5 .
  • the cell may be considered to express a biomarker if the number of copies of the biomarker per cellular genome is greater than 1 or greater than 2' 1 or greater than 2' 2 or greater than 2' 3 or greater than 2' 4 or greater than 2' 5 .
  • the method involves characterizing the neoplasia as expressing any indicated biomarker (e.g., prior to administration of the compounds of the present disclosure) by identifying the cancer as having increased expression of any indicated biomarker.
  • the method comprises administering the compounds of the present disclosure.
  • the method may comprise characterizing the neoplasia in a subject in need thereof as expressing a glycosyltransferase and administering the compounds of the present disclosure.
  • the method does not comprise the characterization step.
  • the cell is a cancer cell.
  • the hyperproliferative disease, disorder, or condition may be selected from bladder, brain, breast, cervical, colorectal, endometrial, esophageal, gallbladder, gastric, glioblastoma, kidney, leukemia (e.g., acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), liver (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma, angiosarcoma, hemangiosarcoma, hepatoblastoma), lung (e.g., nonsmall cell lung cancer, small cell lung cancer, mesothelioma), melanoma, ovarian, pancreatic, prostate, multiple myelo
  • the cancer may be a hematopoietic cancer, such as acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.
  • hematopoietic cancer such as acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma.
  • the method of prophylaxis or treatment of a neoplasia of a subject in need thereof may comprise administration to the subject a compound (e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4) or composition of the present disclosure.
  • a compound e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4
  • composition of the present disclosure may comprise administration to the subject a compound (e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4) or composition of the present disclosure.
  • the neoplasia is associated with a hyperproliferative disease, disorder or condition such as a cancer including acute leukemia (including MLL acute leukemia, MLL partial tandem duplicate acute leukemia, NPM mutated acute leukemia, MOZ acute leukemia, NUP98 acute leukemia, and CALM acute leukemia), chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, polycythemia vera, malignant lymphoma (including B-cell lymphoma), myeloma (including multiple myeloma), brain tumor, cancer of the head and neck, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, gallbladder and bile duct cancer, liver cancer, hepatocellular cancer, pancreatic cancer, colon cancer, rectal cancer, anal cancer, chorionepithelioma, endometrial cancer, cervical cancer, ova
  • the neoplasia is a neoplasia of the skin, bone, adrenal glands kidney, spleen, or testicals.
  • the neoplasia is Ewing’s sarcoma or rhabdomyosarcoma.
  • the neoplasia is a lymphoid cancer such as acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, or post-transplant lymphoproliferative disorders; the hematological malignancies and related conditions of myeloid lineage are selected from acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CM ML], hypereosinophilic syndrome, polycythaemia ver
  • Methods for preventing the growth of a population of neoplasia in a medium may comprise contacting the medium with a compound of the present disclosure (e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4).
  • a compound of the present disclosure e.g., compounds that are a substrate for a glycosyltransferase such as Compounds 1-4.
  • the neoplasia is associated with a hyperproliferative disorder such as a cancer including acute leukemia (including MLL acute leukemia, MLL partial tandem duplicate acute leukemia, NPM mutated acute leukemia, MOZ acute leukemia, NUP98 acute leukemia, and CALM acute leukemia), chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, polycythemia vera, malignant lymphoma (including B-cell lymphoma), myeloma (including multiple myeloma), brain tumor, cancer of the head and neck, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, gallbladder and bile duct cancer, liver cancer, hepatocellular cancer, pancreatic cancer, colon cancer, rectal cancer, anal cancer, chorionepithelioma, endometrial cancer, cervical cancer, ovarian cancer, bladder
  • the neoplasia is a neoplasia of the skin, bone, adrenal glands kidney, spleen, or testicals.
  • the neoplasia is Ewing’s sarcoma or rhabdomyosarcoma.
  • the neoplasia is a lymphoid cancer such as acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, or post-transplant lymphoproliferative disorders; the hematological malignancies and related conditions of myeloid lineage are selected from acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CM ML], hypereosinophilic syndrome, polycythaemia ver
  • the neoplasia is a neoplasia of the skin, bone, adrenal glands kidney, spleen, or testicals.
  • the neoplasia is Ewing’s sarcoma or rhabdomyosarcoma.
  • the neoplasia is a lymphoid cancer such as acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin's lymphomas, hairy cell leukemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, or post-transplant lymphoproliferative disorders; the hematological malignancies and related conditions of myeloid lineage are selected from acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CM ML], hypereosinophilic syndrome, polycythaemia ver
  • the compounds or compositions of the present disclosure may be administered at least once a day for at least one week.
  • the composition is administered at least twice a day for at least two days.
  • the composition is administered approximately daily, at least daily, twice a week, weekly, or for once a month.
  • the composition is administered for several months, such as at least two months, six months, or one year or longer.
  • compositions may be further suited for long-term use, which may be particularly beneficial for preventing recurring infection, or for preventing infection or conditions in at-risk or susceptible patients, including immune compromised patients.
  • long-term use may involve treatment for at least two years, three years, four years, or even five or more years.
  • the compounds and pharmaceutical compositions can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects).
  • Examples of other drugs to combine with the compounds described herein include pharmaceuticals for the treatment of neoplasias (e.g., alkylating agents, antimetabolites, antibiotics, platinum complex compounds, camptothecin derivatives, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, phospholipid kinase inhibitors, monoclonal antibodies, interferons, hormones, angiogenic inhibitors, immune checkpoint inhibitors, epigenetics-associated molecular inhibitor, a protein post-translational modification inhibitor, a proteasome inhibitor).
  • neoplasias e.g., alkylating agents, antimetabolites, antibiotics, platinum complex compounds, camptothecin derivatives, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, phospholipid kinase inhibitors, monoclonal antibodies, interferons, hormones, angiogenic inhibitors,
  • the compounds of the present disclosure may be combined with one or more of a cytostatic agent, cisplatin, doxorubicin, taxotere, taxol, etoposide, irinotecan, camptostar, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5- fluorouracil, methoxtrexate, temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, Iressa, Tarceva, antibodies to EGER, Gleevec.TM., intron, ara-C, adriamycin, cytoxan, gemcitabine, Uracil mustard, Chlormethine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Strept
  • Combination methods can involve the use of the two (or more) agents formulated together or separately, as determined to be appropriate.
  • two or more drugs are formulated together for the simultaneous or near simultaneous administration of the agents.
  • the composition is a contained in a kit, which may contain the compositions of the present disclosure packaged to facilitate dispensing and/or administration of the compositions disclosed herein.
  • the packaging or dispenser may include a bottle, tube, spray bottle, or other dispenser.
  • the composition is packaged in a concentrated form, and diluted to a desired concentration upon use by the end user.
  • the composition may be formulated and packaged in a manner suitable for long-term storage to maintain efficacy of the composition.
  • a gene expression correlation screen was performed on Ewing Sarcoma (EWS) A673 cells following administration of Compound 1 :
  • CRISPR KO cells were generated of Ewing Sarcoma A673 cells.
  • a schematic of the PRISM Screen is shown in FIG. 2. Briefly, pools of cells are treated for 5 days with compounds, then cells are lysed and mRNA is isolated. The barcode sequences associated with characterized CCLE cell lines are amplified by PCR and detected by a Luminex scanner. The quantity of each barcode remaining after treatment serves as a readout to generate cell line sensitivity signatures for each compound.
  • FIG. 3 A provides the Pearson Correlation coefficients correlating gene expression of EWS specific genes and sensitivity to Compound 1.
  • FIG. 3B correlates the Shrunken log2 fold changes (LFC) for each q-value for each open reading frame (ORF) assigned by differential representation methods.
  • LFC Shrunken log2 fold changes
  • ORF open reading frame assigned by differential representation methods.
  • knockout of two genes associated with glycosylation, SLC35B4 and UGT3A2 conferred resistance to Compound 1 in addition to various v-ATPase subunits implicating glycosyltrasferases and V-ATPase in the mechanism of action for Compound 1.
  • Lysotracker (LTR) Staining was performed on test conditions on several UGT3 A2 knockout clones. Briefly, on the day before staining, 35,000 cells were plated in 150 pL in in a 96 well plate. Several test conditions were measured: untreated cells, starvation (treatment with 0.2% by weight fetal bovine serum (FBS)), bafilomycin Al (Baf Al) treatment and compound 1 treatment at either 400 nM, 2 pM or 10 pM in media. After 5 hours in each test condition, live cells were stained with Lysotracker Red DND-99 and Hoechst for 30 minutes (100 nM Lysotracker Red DND-99, 1 :2000 Hoeschst stain by volume).
  • FBS fetal bovine serum
  • Baf Al bafilomycin Al
  • FIG. 4 provides the LTR+ punctae/cell for each test condition. indicates statistical significance (p ⁇ 0.05) as compared to the untreated condition.
  • FIG. 5 provides the Western Blot pull down assay of several isolated KO clones following pooled KO of UGT3A2.
  • the arrow marks two bands associated with UGT3 A2.
  • clone 1 has lost expression of UGT3A2 while clone 10 does not have UGT3A2 knock out.
  • the Cas9 LacZ control also includes expression of UGT3A2.
  • FIGs. 6A-6C correlates the amount of Compound 1 administered to the number of live cells measured.
  • FIGs. 6A-6C correlates the amount of Compound 1 administered to the number of live cells measured.
  • both A673 Cas9 LacZ (FIG. 6A) and Clone 10 (FIG. 6C) both of which express UGT3 A2 are sensitive to Compound 1 administration in a dose dependent manner.
  • Clone 1 (FIG. 6B) which does not express UGT3 A2, lost sensitivity to Compound 1.
  • FIGs. 6B which does not express UGT3 A2
  • Compound 1 (No -OH) lacks the hydroxyl at the at the 4 position of the naphtho[2,3-c]furan-l(3H)-one moiety which is the site of UGT3A2 glycosylation in Compound 1.
  • FIGs. 7A-7C provide the results of the cell viability assay for Compound 1 (FIG. 7A), Compound 2 (FIG. 7B), Compound 3 (FIG. 7C) as compared to A673 LacZ cells and the UGT3 A2 KO clone 1 (which did not express UGT3A2).
  • FIG. 7D provides the cell viability assay data comparing Compound 1 and the glycosylated version of Compound 1 having the structure:
  • FIG. 8 A shows the results with Compound 1 administration
  • FIG. 8B shows the results with Compound 2 (R112) administration.
  • A673 Ewing sarcoma cells were seeded in wells of a 12-well plate (100,000 cells/well). The cells were treated with 10 pM of Compound 1 in dimethyl sulfoxide (DMSO) in media:
  • BRD9645 At each time point (0-72 hours), media were collected and cells with phosphate buffered saline (PBS)and lysed with 500 pL cold acetonitrile: methanol (1 : 1). The media and cell lysate samples were kept at -80°C until analysis.
  • PBS phosphate buffered saline
  • the cell lysate samples were centrifuged at 14,000 rpm for 8 min.
  • FIG. 9A and 9B provide the measured relative amounts of Compound 1 and Glycosylated Compound 1 found in cell lysate and cell media according to this protocol.
  • FIG. 9B compares WT and KO cells.

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Abstract

La divulgation concerne des agents qui sensibilisent des cellules néoplasiques au moyen d'une glycosylation par une glycosyltransférase. Dans le mécanisme de cytotoxicité selon la présente divulgation, les composés deviennent glycosylés, ce qui entraîne une cytotoxicité compétitive. Des exemples de composés comprennent : un composé 1 (BRD9645), un composé 2 (R112), un composé 3 (tioxolone), un composé 4 (Baf A1).
PCT/US2024/033347 2023-06-15 2024-06-11 Cytotoxicité néoplasique induite par glycosylation Ceased WO2024258819A1 (fr)

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Citations (3)

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US20160312292A1 (en) * 2013-12-06 2016-10-27 Celgene Corporation Methods for treating hematological cancers and the use of biomarkers as a predictor of clinical sensitivity to immunodulatory therapies
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