WO2023207545A1 - 支链氨基酸转氨酶1的功能获得性突变体及其应用 - Google Patents

支链氨基酸转氨酶1的功能获得性突变体及其应用 Download PDF

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WO2023207545A1
WO2023207545A1 PCT/CN2023/086648 CN2023086648W WO2023207545A1 WO 2023207545 A1 WO2023207545 A1 WO 2023207545A1 CN 2023086648 W CN2023086648 W CN 2023086648W WO 2023207545 A1 WO2023207545 A1 WO 2023207545A1
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bcat1
mutant protein
activity
mutation
protein
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French (fr)
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雷群英
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复旦大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01042—Branched-chain-amino-acid transaminase (2.6.1.42)
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of medical technology, and relates to gain-of-function mutants of branched-chain amino acid transaminase 1 (BCAT1) and their applications, and more specifically to the preparation of BCAT1 and BCAT1 E61A mutation inhibitors for the prevention and/or treatment of BCAT1 and its BCAT1 E61A .
  • BCAT1 branched-chain amino acid transaminase 1
  • branched-chain amino acids are essential amino acids for the human body, and they are collectively called branched-chain amino acids (BCAA).
  • BCAA branched-chain amino acids
  • BCAT Branched-chain amino acid transaminase
  • Branched-chain amino acid transaminase is a PLP-dependent reversible reaction. Its main function is to transfer the amino group on the branched-chain amino acid to ⁇ -ketoglutarate ( ⁇ -KG) to produce the corresponding branched-chain ketoacid (BCKA) and glutamic acid. Its carbon skeleton BCKA is further decomposed in the BCKA dehydrogenase complex (BCKDH) to generate acetyl coenzyme A (Ac-CoA) and succinyl coenzyme A (Suc-CoA), thereby entering the citric acid cycle.
  • BCKA dehydrogenase complex BCKDH
  • BCAT or BCKDH mutations may cause maple disease, which seriously affects human health.
  • BCAT protein is not in-depth enough. Only some BCATs have been found to be overexpressed during the development of cancer, and no mutant forms that lead to functional activation of BCAT1 have been reported.
  • the present invention urgently needs to further develop various mutant forms of BCAT1, and accordingly provide targeted therapeutic drugs targeting these different mutant forms.
  • the purpose of the present invention is to provide a new gain-of-function mutant form, namely BCAT1 E61A mutant protein and its targeted therapeutic drugs.
  • a branched chain amino acid transferase 1 (BCAT1) mutant protein is provided, so The above-mentioned mutant protein has an E ⁇ A mutation at position 61 corresponding to human branched-chain amino acid transferase 1, and has branched-chain amino acid transferase activity.
  • the mutant protein is a gain-of-function mutant protein.
  • the ratio (A1/A0) of the transaminase activity A1 of the mutant protein to the transaminase activity A0 of the wild-type BCAT1 protein is about 1.5, more preferably about 1.47.
  • amino acid sequence of the human branched-chain amino acid transferase 1 is shown in SEQ ID No: 1.
  • the branched chain amino acid is selected from the following group: leucine, isoleucine, valine, or a combination thereof.
  • amino acid sequence of the mutant protein is shown in SEQ ID No: 2.
  • a second aspect of the present invention provides an isolated polynucleotide encoding the BCAT1 E61A mutant protein described in the first aspect of the present invention.
  • a third aspect of the present invention provides a vector containing the polynucleotide described in the second aspect of the present invention.
  • the vector is an expression vector.
  • the fourth aspect of the present invention provides a host cell containing the vector described in the third aspect of the present invention, or the nucleic acid of the host cell contains the isolated polynucleotide described in the second aspect of the present invention. .
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cells include cells derived from the following microorganisms:
  • Saccharomyces cerevisiae Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis Yeast (Saccharomyces pombe), Kluyveromyces marxiamus (Kluyveromyces marxiamus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipites, Huata Candida shehatae, Candida tropicalis, Escherichia coli.
  • the host cell includes Saccharomyces cerevisiae, Pichia pastoris or Myceliophthora thermophila.
  • the host cell expresses BCAT1 E61A mutant protein.
  • the fifth aspect of the present invention provides a method for preparing the BCAT1 E61A mutant protein described in the first aspect of the present invention, comprising the steps:
  • the sixth aspect of the present invention provides the use of a targeted inhibitor of the mutant protein described in the first aspect of the present invention for preparing a pharmaceutical composition or preparation, and the pharmaceutical composition or preparation is used to treat BCAT1 E61A Mutation-related diseases.
  • the disease associated with the BCAT1 E61A mutation is selected from the following group: metabolic disease, cancer, or a combination thereof.
  • the metabolic disease is selected from the group consisting of: insulin resistance, hypercholesterolemia, diabetes, maple disease, rheumatoid arthritis, crescentic glomerulonephritis, or a combination thereof.
  • the cancer is selected from the group consisting of pancreatic cancer, lung cancer, glioma, leukemia, intestinal cancer, gastric cancer, or a combination thereof.
  • the disease is a BCAT1 E61A positive disease.
  • the inhibitor is selected from the group consisting of small molecules, antibodies, polypeptides, oligonucleotides, aptamers, or combinations thereof.
  • the targeted inhibitor is selected from the following group: candesartan, losartan, tibolone or a combination thereof.
  • the pharmaceutical composition is used to inhibit tumor growth, inhibit tumor metastasis, or a combination thereof.
  • test kit which includes:
  • Detection reagent the detection reagent is used to detect whether there is E61A mutation in branched-chain amino acid transferase 1 (BCAT1).
  • the detection reagent is selected from the following group: primers (PCR forward primers TGCATCATCTTACCCCATCTG (SEQ ID No: 3), reverse primer: CCAGCTGTCCTTTCTATTTGCT (SEQ ID No: 4); annealing temperature 54 degrees, 734bp).
  • the kit further contains:
  • the second active ingredient includes: small molecule compounds, antibodies, nucleic acid molecules, or combinations thereof.
  • the second active ingredient is selected from the following group: shRNA, interfering RNA, siRNA, microRNA, or a combination thereof.
  • the kit further contains:
  • the third active ingredient includes: small molecule compounds, antibodies, nucleic acid molecules, or combinations thereof.
  • the third active ingredient is selected from the following group: shRNA, interfering RNA, siRNA, microRNA, or a combination thereof.
  • the kit contains the second active ingredient and the third active ingredient.
  • the eighth aspect of the present invention provides the use of a BCAT1 targeted inhibitor for preparing a pharmaceutical composition or preparation, and the pharmaceutical composition or preparation is used to inhibit the activity of RhoC.
  • the BCAT1 targeted inhibitor includes a targeted inhibitor that inhibits wild-type BCAT1, a targeted inhibitor that inhibits mutant BCAT1, or a combination thereof.
  • the mutant BCAT1 is BCAT1 E61A mutant protein.
  • the BCAT1 targeted inhibitor is selected from the following group: candesartan, losartan, tibolone, anti-BCAT1 antibodies or combinations thereof.
  • a ninth aspect of the present invention provides a combination of active ingredients, which combination includes:
  • the tenth aspect of the present invention provides a use of the combination of active ingredients described in the ninth aspect of the present invention for preparing a medicine, which is used to treat diseases related to high expression of BCAT1 or mutant BCAT1.
  • the disease is selected from the group consisting of metabolic diseases, cancer, or combinations thereof.
  • the metabolic disease is insulin resistance, hypercholesterolemia, diabetes, maple disease, or a combination thereof.
  • the cancer is selected from the group consisting of pancreatic cancer, lung cancer, glioma, leukemia, intestinal cancer, or a combination thereof.
  • the mutant BCAT1 is BCAT1 E61A mutant protein.
  • the present invention provides a method for inhibiting RhoC activity in vitro, including the steps:
  • the cells are selected from the group consisting of tumor cells.
  • the cells are selected from the following group: tumor cells TE1 and tumor cells MGC803.
  • the method is non-therapeutic and non-diagnostic.
  • the BCAT1 targeted inhibitor is selected from the following group: candesartan, losartan, tibolone, anti-BCAT1 antibodies or combinations thereof.
  • a twelfth aspect of the present invention provides a method for screening candidate therapeutic drugs, including the steps:
  • test substance is a potential candidate therapeutic drug.
  • the "significantly lower” refers to T1/T0 ⁇ 0.5, preferably ⁇ 0.25, and more preferably ⁇ 0.1.
  • the BCAT1 E61A mutant protein has the amino acid sequence shown in SEQ ID No: 2.
  • the method further includes:
  • the inhibitory effect includes: inhibition of tumor cell growth, tumor cell migration migration inhibition, or a combination thereof.
  • the method further includes: in the positive control group, in the presence of a positive compound, testing the activity T2 of the BCAT1 E61A mutant protein; wherein, in addition to the positive compound, other tests of the positive control group Conditions were the same as the test group;
  • the method is non-therapeutic and non-diagnostic.
  • the positive compound is selected from the following group: candesartan, losartan, tibolone, anti-BCAT1 antibody or a combination thereof.
  • the present invention provides a method for treating a disease, including the step of administering a therapeutically effective amount of a BCAT1 E61A targeted inhibitor to a subject in need of treatment.
  • the disease associated with the BCAT1 E61A mutation is selected from the following group: metabolic disease, cancer, or a combination thereof.
  • the metabolic disease is selected from the group consisting of: insulin resistance, maple disease, hypercholesterolemia, diabetes, or combinations thereof.
  • the cancer is selected from the following group: leukemia, gastric cancer, glioma, intestinal cancer, lung cancer, pancreatic cancer, or a combination thereof.
  • the disease is a BCAT1 E61A positive disease.
  • the targeted inhibitor is selected from the following group: candesartan, losartan, tibolone or a combination thereof.
  • the fourteenth aspect of the present invention provides the use of a BCAT1 E61A mutation detection reagent for preparing a kit for detecting whether the BCAT1 E61A mutation exists in a patient sample.
  • the kit is used to determine whether the patient suffers from a disease related to BCAT1 E61A mutation.
  • the kit contains a detection reagent, and the detection reagent is used to detect whether there is an E61A mutation in branched-chain amino acid transferase 1 (BCAT1).
  • BCAT1 branched-chain amino acid transferase 1
  • FIG 1 shows that BCAT1 is highly expressed and BCAT1 E61A mutations occur in many cancers.
  • Figure 2 shows that the BCAT1 E61A mutation promotes enzymatic activity.
  • FIG. 3 shows that high expression of BCAT1 and BCAT1 E61A mutation promote cell growth.
  • FIG. 4 shows that high expression of BCAT1 and BCAT1 E61A mutation promote cell migration and invasion.
  • FIG. 5 shows that BCAT1 and BCAT1 E61A mutations affect cell migration and invasion through RhoC.
  • Figure 6 shows that the BCAT1 E61A mutation causes lung tissue hyperplasia and promotes branched-chain amino acid catabolism in vivo.
  • Figure 7 shows that BCAT1 E61A mutation promotes liver cancer initiation and progression.
  • FIG. 8 shows that the BCAT1 E61A mutation promotes myeloid leukemia.
  • Figure 9 shows that BCAT1 catabolite branched-chain ketoacids bind to RhoC and promote RhoC activity.
  • Figure 10 shows a model diagram of the mechanism of BCAT1 high expression and BCAT1 E61A mutation.
  • Figure 11 shows that candesartan directly binds to the enzyme activity site of BCAT1 and BCAT1 E61A mutant enzyme to inhibit enzyme activity.
  • Figure 12 shows the inhibition of candesartan on the activity and cell migration of RhoC, a key downstream factor of BCAT1.
  • Figure 13 shows that complementing BCAT1 catabolite branched-chain ketoacids effectively rescues the inhibition of RhoC activity in candesartan-treated cells.
  • Figure 14 shows that targeting the BCAT1-RhoC metabolic signaling axis inhibits gastric cancer cell metastasis in vivo.
  • Figure 15 shows the results of the screen for BCAT1 E61A inhibitors.
  • the inventor unexpectedly discovered the BCAT1 E61A gain-of-function activation mutant protein for the first time, that is, the BCAT1 E61A mutant protein.
  • Studies have shown that the catalytic activity of the BCAT1 E61A mutant protein of the present invention is significantly improved after mutation.
  • the present invention also develops targeted inhibitors for the mutant protein of the present invention, and verifies that these targeted inhibitors can be used to treat BCAT1 E61A mutation-related diseases such as tumors. On this basis, the present invention was completed.
  • experiments of the present invention prove that inhibiting BCAT1 E61A mutation can significantly alleviate tumors and other metabolism-related diseases caused by BCAT1 E61A mutation.
  • BCAT1-specific targeted inhibitors such as candesartan can directly bind to BCAT1 E61A and inhibit its activity.
  • BCAT1-specific targeted inhibitors such as candesartan can inhibit Tumors and other diseases caused by BCAT1 E61A mutations.
  • the term “optionally” or “optionally” means that the subsequently described event or circumstance may occur but does not necessarily occur, may have but is not required to occur, may be 1, 2 or 3.
  • RhoC stands for Ras Homolog Family Member C.
  • the Chinese name is Ras homologous family member C. It belongs to one of the subfamily members of the small G protein superfamily. As a molecular switch, it can open or close various intracellular signaling pathways and is also involved in many Physiological and pathological processes.
  • Branched-chain aminotransferase 1 catalyzes the first reversible reaction in the degradation of human branched-chain amino acids, catalyzing the transamination reaction of branched-chain amino acids and ketoglutarate to generate the corresponding branched-chain ketoacids and glutamic acid.
  • mutein of the invention protein of the invention
  • gain-of-function activating mutant of the invention gain-of-function activating mutein of the invention
  • BCAT1 E61A protein of the invention Used interchangeably, it means that there is an E ⁇ A mutation at position 61 corresponding to human branched-chain amino acid transferase 1 and it has branched-chain amino acid transferase activity.
  • this term includes not only mutant proteins having the E61A mutation based on the amino acid sequence of wild-type human BCAT1 (SEQ ID No: 1), but also mutant proteins that may contain additional mutations, as long as these additional mutations do not affect or substantially does not affect the transaminase function of BCAT1 E61A able.
  • E61A refers to having an amino acid mutation from glutamic acid (E) to leucine (A) at position 61 of the amino acid sequence.
  • E glutamic acid
  • A leucine
  • GCG GAG
  • amino acid sequence involved in the present invention is as follows:
  • the mutant protein of the present invention also includes conservative variants thereof, which refers to positions other than position 61 (E61A) compared with the amino acid sequence of the mutant protein of the present invention (SEQ ID No: 2). , at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
  • the present invention also provides targeted inhibitors for the mutant protein of the present invention and their applications.
  • the targeted inhibitor of the mutant protein of the present invention can be used to inhibit the activity of the BCAT1 E61A mutant protease, thereby treating diseases related to the BCAT1 E61A mutation.
  • the targeted inhibitor of the mutant protein of the present invention can be used as an active ingredient to prepare medicines to treat related diseases.
  • the targeted inhibitor includes (but is not limited to): small molecules, antibodies, polypeptides, oligonucleotides, aptamers, ADCs, or combinations thereof.
  • Targeted inhibitors of the invention are selected from the group consisting of candesartan, losartan, tibolone or combinations thereof.
  • Candesartan is a non-peptide angiotensin II receptor antagonist that selectively and irreversibly blocks ATI receptors without obvious side effects. Mainly used to treat high blood pressure.
  • the structural formula of candesartan is shown in formula (I):
  • the targeted inhibitor of the present invention also known as the compound of the present invention
  • the targeted inhibitor of the present invention has excellent inhibitory activity against the BCAT1 E61A mutant protein
  • the targeted inhibitor of the present invention including the compound and its various crystal forms, and pharmaceutically acceptable salts
  • the pharmaceutical composition containing the targeted inhibitor of the present invention as the main active ingredient can be used to prevent and/or treat (stabilize, alleviate or cure) BCAT1 E61A mutant protein-related diseases.
  • the pharmaceutical composition of the present invention contains the targeted inhibitor of the present invention and a pharmaceutically acceptable excipient or carrier within a safe and effective amount.
  • the “safe and effective dose” refers to the amount of compound that is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg/agent of the targeted inhibitor of the present invention, and more preferably, contains 10-200 mg/agent of the targeted inhibitor of the present invention.
  • the "dose" is a capsule or tablet.
  • the pharmaceutical composition of the present invention contains the targeted inhibitor of the present invention and a pharmaceutically acceptable excipient or carrier within a safe and effective amount.
  • the “safe and effective dose” refers to the amount of compound that is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg/agent of the targeted inhibitor of the present invention, and more preferably, contains 10-200 mg/agent of the targeted inhibitor of the present invention.
  • the "dose" is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gel substances that are suitable for human use and must be of sufficient purity and low enough toxicity. "Compatibility” here means that each component of the composition can be blended with the targeted inhibitor of the present invention and with each other without significantly reducing the efficacy of the compound.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • the administration mode of the targeted inhibitor or pharmaceutical composition of the present invention is not particularly limited.
  • Representative administration modes include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) Humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarder, such as paraffin; (f) Absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl mono
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared using coatings and shell materials such as enteric casings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active compound can also be combined with the above One or more of the excipients are formed into microencapsulated form.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils,
  • compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions may contain, in addition to the active compound, suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances and the like.
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances and the like.
  • compositions for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the targeted inhibitor of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • the pharmaceutical composition When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds.
  • One or more of the other pharmaceutically acceptable compounds may be administered simultaneously, separately, or sequentially with the compounds of the invention.
  • a safe and effective amount of the targeted inhibitor of the present invention is applied to a mammal (such as a human) in need of treatment.
  • the dosage during administration is a pharmaceutically effective dosage.
  • the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the present invention also provides detection reagents and detection kits for detecting whether there is an E61A mutation in branched-chain amino acid transferase 1 (BCAT1).
  • the detection reagent or kit of the present invention can be used to detect the presence of BCAT1 E61A mutation in patient samples.
  • the detection reagent of the present invention is a nucleic acid detection reagent.
  • the kit of the present invention is used to determine whether a patient suffers from diseases related to BCAT1 E61A mutation.
  • the kit may further include instructions for use.
  • the instructions describe the usage of the kit or detection, diseases related to BCAT1 E61A mutation, etc.
  • the recombinant human BCAT1 E61A gain-of-function activation mutant protein has significantly enhanced enzyme activity compared with the wild type.
  • Candesartan, losartan, and tibolone are approved for clinical use. Compared with brand-new drugs, their safety is better guaranteed and they have significant advantages in clinical trials and other aspects.
  • Candesartan, losartan, and tibolone can strongly bind to and specifically inhibit branched-chain amino acid transaminase 1 (BCAT1) and its gain-of-function activation mutant protein.
  • BCAT1 branched-chain amino acid transaminase 1
  • BCAT1 branched-chain amino acid transaminase 1
  • the CDS product of BCAT1 with corresponding protective bases and enzyme cleavage sites was cloned by PCR.
  • the digested PCR products were connected to the prokaryotic expression vector pET28a and the eukaryotic expression vector pCDH-puro, respectively.
  • the eukaryotic expression vector was added with a flag tag for purification.
  • the ligation product was transformed into the DH5 ⁇ cloning strain, and then the plasmid was identified, sequenced and extracted. At the same time, it is necessary to use the plasmid as a template to perform PCR point mutation, and then also perform transformation, identification, sequencing and extraction of the plasmid.
  • the corresponding pET28a plasmid is transformed into BL21 (DE3), a competent cell for prokaryotic expression. Then take a single clone, shake 10ml, and then inoculate it in 1L LB medium at a ratio of 1:1000 for expansion culture. When the OD value is about 0.6, turn to 16°C, add 0.5mM final concentration IPTG to induce expression overnight, centrifuge the next day to collect the bacteria, use purification buffer (20mM Tris pH 8.0, 150mM NaCl) to resuspend, and sonicate at 250W power. The bacteria were disrupted at 3-second intervals, and the supernatant was collected by ultracentrifugation.
  • the corresponding pCDH-puro plasmid is transfected into 293T cells, and the cells are collected after 48 hours.
  • Use PBS buffer containing 0.5% NP-40 to lyse collect the supernatant by high-speed centrifugation, add flag-beads to enrich the protein and leave overnight, then centrifuge to wash the flag-beads, add the flag peptide to elute the protein, and collect the supernatant to complete the purification Good eukaryotic proteins.
  • Enzyme kinetic experiments were performed using prokaryotic expression of BCAT1 and BCAT1 E61A mutant proteins.
  • eukaryotic expressed BCAT1 and BCAT1 E61A mutant proteins were used to conduct enzyme activity experiments.
  • BCAT1 mutant cell lines (KATO III and TE1) and wild-type cell lines (AGS and KYSE180) based on LC-MS, it was found that the overall metabolic level of BCAT1 mutant cell lines was significantly different from that of wild-type cell lines. upregulated (as shown in Figure 2d-e).
  • BCAT1 and its BCAT1 E61A promoted cell growth in various tumor cell lines (as shown in Figure 3a). Knocking out BCAT1 and its BCAT1 E61A reduced cell clonogenesis in various tumor cell lines (as shown in Figure 3b).
  • BCAT1 or BCAT1 E61A mutation can promote tumor growth.
  • Knocking out BCAT1 and its BCAT1 E61A reduced cell migration in various tumor cell lines (as shown in Figure 4a). Overexpression of BCAT1 and its BCAT1 E61A promoted cell migration in various tumor cell lines (as shown in Figure 4b). Knocking out BCAT1 and its BCAT1 E61A in tumor cell lines resulted in weakened cell invasion (as shown in Figure 4c). Overexpression of BCAT1 and its BCAT1 E61A in tumor cell lines promoted cell invasion (as shown in Figure 4d).
  • BCAT1 or BCAT1 E61A mutation can promote tumor migration and invasion.
  • BCAT1 promotes tumor migration and invasion by enhancing RhoC activity.
  • mice were constructed using CRISPR-Cas9 technology. And the pathological classification of mice was studied.
  • the metabolic flux of branched-chain amino acids was tracked by isolating and culturing epidermal fibroblasts from transgenic mice while adding valine-13C5 or leucine - 13C6,15N1 in low-BCAA medium.
  • Wild-type and Bcat1 E61A point mutation heterozygous and homozygous mice were intraperitoneally injected with diethylnitrosamine (DEN) 2 mg/kg on the 14th day of life, and 4 weeks after administration, intraperitoneally injected with carbon tetrachloride (CCl4) 1mL/kg, twice a week for a total of 9 weeks, to perform liver cancer modeling (as shown in Figure 7a). After modeling, no significant changes were recorded in the appearance, spleen, lungs and other important organs, body weight and food intake, liver/body weight ratio and serum albumin level (as shown in Figure 7b-g).
  • DEN diethylnitrosamine
  • CCl4 carbon tetrachloride
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • the Bcat1 E61A mutation caused mice to die and have giant spleens, lymph nodes, and livers (shown in Figure 8a-b).
  • MPO+CD34+ shown in Figure 8c
  • PH3 Phospho-Histone H3
  • BCAT1 and its BCAT1 E61A promote BCKA production and increase compartmentalized concentration to promote RhoC activity and further promote tumor migration and invasion (as shown in Figure 10).
  • the inhibitory activity of the compounds was further measured through the change curve of ⁇ OD value with time.
  • BCAT1 E61A enzyme activity As shown in Figure 15a, among more than 600 compounds, the vast majority of compounds have no inhibitory effect on BCAT1 E61A enzyme activity. However, a few have certain inhibitory effects on BCAT1 E61A enzyme activity, including candesartan, chloride Sartan, tibolone.
  • candesartan, losartan and tibolone have significant targeted inhibitory activity on BCAT1 E61A enzyme activity.
  • the order according to the inhibitory effect on BCAT1 E61A enzyme activity is as follows: Candesartan > Losartan > Tibolone.
  • the inhibitory activity of candesartan against wild-type and mutant BCAT1 E61A was determined by varying the concentration of candesartan.
  • candesartan can inhibit the enzymatic activity of BCAT1 or BCAT1 E61A in a concentration-dependent manner.
  • candesartan can directly bind to BCAT1 or BCAT1 E61A protein, and candesartan binds to BCAT1 E61A protein more strongly.
  • Example 5 Candesartan inhibits BCAT1 or BCAT1 E61A activity at the in vivo level to reduce cell migration.
  • Candesartan inhibits the increase in RhoC activity caused by overexpression of BCAT1 or BCAT1 E61A at the in vivo level.
  • BCAT1 or BCAT1 E61A were counted and treated with and without candesartan (150 ⁇ M final concentration). The cells were lysed after 18 hours and their RhoC activity levels were analyzed.
  • RhoC activity results showed that overexpression of BCAT1 or BCAT1 E61A in the untreated candesartan control group significantly increased RhoC activity, and overexpression of BCAT1 or BCAT1 E61A in the candesartan experimental group significantly inhibited RhoC activity (as shown in Figure 12a Show).
  • Fibroblasts were counted from wild-type, heterozygous Bcat1 E61A and homozygous Bcat1 E61A isolated from transgenic mice with and without treatment with candesartan (150 ⁇ M final concentration). Cells were lysed after 18 hours and analyzed for RhoC activity levels.
  • RhoC activity results showed that both heterozygous Bcat1 E61A and homozygous Bcat1 E61A in the untreated candesartan control group significantly increased RhoC activity, while in the experimental group treated with candesartan, the heterozygous Bcat1 E61A and homozygous Bcat1 E61A RhoC activity was significantly inhibited (as shown in Figure 12b).
  • the tumor cell lines TE1 and MGC803 were treated with candesartan at a final concentration of 50-250 ⁇ M. Cells were lysed after 18 hours and analyzed for RhoC activity levels.
  • RhoC activity results showed that candesartan inhibited RhoC activity in a concentration-dependent manner at the cellular level (as shown in Figure 12c).
  • Candesartan inhibits cell migration in a concentration-dependent manner at the in vivo level
  • Candesartan was added to the tumor cell lines TE1 and MGC803 at a final concentration of 50-250 ⁇ M to treat the cells. After 18 hours, approximately 100,000 cells/cell were counted for a Transwell experiment to analyze the effect of candesartan on cell migration.
  • the tumor cell lines TE1 and MGC803 were treated with candesartan at a final concentration of 50-250 ⁇ M. Add BCAA and ⁇ -KG, or BCKA and Glu respectively at the same time. Cells were lysed after 18 hours and analyzed for RhoC activity levels.
  • RhoC activity showed that the replenishment of BCKA and Glu could significantly rescue the inhibition of RhoC activity by candesartan (as shown in Figure 13).
  • Stable overexpression control empty plasmid, BCAT1 wild-type plasmid or BCAT1 E61A mutant gastric cancer cell line SGC7901 were inoculated intraperitoneally into 5-week-old nude mice.
  • Nude mice were inoculated intraperitoneally with candesartan, a diet containing 1/5 branched-chain amino acids, or knockdown of RhoC or ARHGEF1 in the above-mentioned stable cell lines. After inoculation, the appearance and weight of the mice were observed and recorded. After 5 weeks, the mice in each treatment group were anesthetized and sacrificed to detect peritoneal tumor formation, and the peritoneal tumor index (PCI) was calculated.
  • PCI peritoneal tumor index
  • mice inoculated with gastric cancer cells overexpressing wild-type BCAT1 and BCAT1 E61A mutant had obvious ascites (as shown in Figure 14a), and both significantly promoted the dissemination and metastasis of gastric cancer cells in the peritoneal cavity (as shown in Figure 14b) and inoculation The weight of the tumor (as shown in Figure 14c).
  • the present invention reports for the first time the important function of BCAT1 E61A in the process of tumor development and is a cancer driver gene mutation. It was found that the BCAT1 E61A mutation site exists in clinical tumor samples. This mutation can significantly enhance the enzymatic function of BCAT1, leading to a significant increase in the metabolite BCKA, thereby promoting the activity of downstream RhoC.
  • the inventors also verified the key role of BCAT1 mutation in the occurrence and development of cancer, especially that this mutation can cause leukemia and promote the occurrence and development of chemically induced liver cancer.
  • the present invention conducted enzyme activity inhibition screening experiments on a variety of different compounds, and screened out several inhibitors such as candesartan that significantly target the enzyme activity of the BCAT1 mutant protein. These targeted inhibitors can be used to inhibit BCAT1 Mutant-related tumors.

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Abstract

本发明提供了支链氨基酸转氨酶1(BCAT1)的活化功能获得性突变体及其应用。具体地，本发明提供了一种人BCAT1E61A功能获得性活化突变体蛋白(即BCAT1E61A突变蛋白)以及针对所述突变蛋白的靶向抑制剂。与野生型蛋白相比，本发明的靶向抑制剂不仅对突变蛋白有更为显著的抑制作用，而且有效抑制浓度(IC50)显著低于现有BCAT1抑制剂，作用靶点的特异性更强。

Description

支链氨基酸转氨酶1的功能获得性突变体及其应用 技术领域
本发明涉及医药技术领域，涉及支链氨基酸转氨酶1(BCAT1)的功能获得性突变体及其应用，更具体地涉及和BCAT1及BCAT1E61A突变抑制剂在制备预防和/或治疗BCAT1及其BCAT1E61A突变介导的相关代谢疾病药物中的用途。
背景技术
亮氨酸、异亮氨酸和缬氨酸是人体必需氨基酸，它们被统称为支链氨基酸(BCAA)。研究发现支链氨基酸代谢异常通常会导致枫糖尿病(maple syrup urine disease)以及多种癌症(包括胰腺癌、肺癌、胶质瘤以及多种白血病)等疾病。限制或者低支链氨基酸的饮食能缓解这类相关代谢疾病。
支链氨基酸转氨酶(BCAT)是催化支链氨基酸分解代谢第一步的关键酶，主要有两种亚型，一个是定位于细胞胞浆中的BCAT1，另一个是定位于线粒体中的BCAT2。
支链氨基酸转氨酶为PLP依赖的可逆反应，主要作用是将支链氨基酸上的氨基转给α-酮戊二酸(α-KG)，产生对应的支链酮酸(BCKA)和谷氨酸。其碳骨架BCKA在BCKA脱氢酶复合体(BCKDH)进一步分解会生成乙酰辅酶A(Ac-CoA)和琥珀酰辅酶A(Suc-CoA)，从而进入柠檬酸循环。
研究表明，BCAT或者BCKDH突变或造成枫糖尿病，从而严重影响人体健康。然而，目前对于BCAT蛋白的研究尚不够深入，仅发现了一些BCAT在癌症发生发展过程中存在过表达，未报道过导致BCAT1功能活化的突变形式。
因此，本发明迫切需要进一步BCAT1的各种不同的突变形式，并相应地提供靶向这些不同突变形式的靶向治疗药物。
发明内容
本发明目的就是提供一种新的功能获得性突变形式即BCAT1E61A突变蛋白及其靶向治疗药物。
在本发明第一方面，提供了一种支链氨基酸转移酶1(BCAT1)突变蛋白，所 述的突变蛋白在对应于人支链氨基酸转移酶1的第61位存在E→A的突变，并且具有支链氨基酸转移酶活性。
在另一优选例中，所述突变蛋白为功能获得性突变蛋白。
在另一优选例中，所述突变蛋白的转氨酶活性A1与野生型BCAT1蛋白的转氨酶活性A0之比(A1/A0)为约1.5，更佳地为约1.47。
在另一优选例中，所述的人支链氨基酸转移酶1的氨基酸序列如SEQ ID No:1所示。
在另一优选例中，所述的支链氨基酸选自下组：亮氨酸、异亮氨酸、缬氨酸、或其组合。
在另一优选例中，所述突变蛋白的氨基酸序列如SEQ ID No:2所示。
本发明第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码本发明第一方面所述的BCAT1E61A突变蛋白。
本发明第三方面，提供了一种载体，所述载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体为表达载体。
本发明第四方面，提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体，或所述宿主细胞的核酸中含有本发明第二方面所述的分离的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述宿主细胞包括来源于以下微生物的细胞：
酿酒酵母(Saccharomyces cerevisiae)、毕赤酵母(Pichia pastoris)、摩纳酵母(Saccharomyces monacensis)、贝酵母(Saccharomyces bayanus)、巴氏酵母(Saccharomyces pastorianus)、卡氏酵母(Saccharomyces carlsbergensis)、粟酒裂殖酵母(Saccharomyces pombe)、马克斯克鲁维酵母(Kluyveromyces marxiamus)、乳酸克鲁维酵母(Kluyveromyces lactis)、脆壁克鲁维酵母(Kluyveromyces fragilis)，树干毕赤酵母(Pichia stipites)、休哈塔假丝酵母(Candida shehatae)、热带假丝酵母(Candida tropicalis)、大肠杆菌(Escherichia coli)。
在另一优选例中，所述宿主细胞包括酿酒酵母、毕赤酵母或嗜热毁丝霉。
在另一优选例中，所述宿主细胞表达BCAT1E61A突变蛋白。
本发明第五方面，提供了一种制备本发明第一方面所述的BCAT1E61A突变蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达本发明第一方面所述的BCAT1E61A突变蛋白；和
分离表达产物，从而获得本发明第一方面所述的BCAT1E61A突变蛋白
本发明第六方面，提供了一种本发明第一方面所述的突变蛋白的靶向抑制剂的用途，用于制备一药物组合物或制剂，所述药物组合物或制剂用于治疗BCAT1E61A突变相关的疾病。
在另一优选例中，所述的BCAT1E61A突变相关的疾病选自下组：代谢疾病、癌症或其组合。
在另一优选例中，所述的代谢疾病选自下组：胰岛素抵抗、高胆固醇血症、糖尿病、枫糖尿病、类风湿性关节炎，新月体性肾小球肾炎、或其组合。
在另一优选例中，所述的癌症选自下组：胰腺癌、肺癌、胶质瘤、白血病、肠癌、胃癌、或其组合。
在另一优选例中，所述疾病是BCAT1E61A阳性的疾病。
在另一优选例中，所述抑制剂选自下组：小分子、抗体、多肽、寡核苷酸、适体、或其组合。
在另一优选例中，所述的靶向抑制剂选自下组：坎地沙坦、氯沙坦、替勃龙或其组合。
在另一优选例中，所述药物组合物用于抑制肿瘤的生长、抑制肿瘤的转移、或其组合。
本发明第七方面，提供了一种试剂盒，所述的试剂盒包括：
(a)第一活性成分或含有所述第一活性成分的第一药物组合物，所述第一活性成分为针对本发明第一方面所述的突变蛋白的靶向抑制剂；和
(b)检测试剂，所述检测试剂用于检测支链氨基酸转移酶1(BCAT1)是否存在E61A突变。
在另一优选例中，所述的检测试剂选自下组：引物(PCR正向引物 TGCATCATCTTACCCCATCTG(SEQ ID No:3),反向引物:CCAGCTGTCCTTTCTATTTGCT(SEQ ID No:4)；退火温度54度,734bp)。
在另一优选例中，所述的试剂盒还含有：
(c)第二活性成分或含有所述第二活性成分的第二药物组合物，所述的第二活性成分用于降低RhoC的表达和/或活性。
在另一优选例中，所述的第二活性成分包括：小分子化合物、抗体、核酸分子、或其组合。
在另一优选例中，所述的第二活性成分选自下组：shRNA、干扰RNA、siRNA、microRNA、或其组合。
在另一优选例中，所述的试剂盒还含有：
(d)第三活性成分或含有所述第三活性成分的第三药物组合物，所述的第三活性成分用于降低BCAT1的表达。
在另一优选例中，所述的第三活性成分包括：小分子化合物、抗体、核酸分子、或其组合。
在另一优选例中，所述的第三活性成分选自下组：shRNA、干扰RNA、siRNA、microRNA、或其组合。
在另一优选例中，所述的试剂盒含有所述的第二活性成分和第三活性成分。
本发明第八方面，提供了一种BCAT1靶向抑制剂的用途，用于制备一药物组合物或制剂，所述药物组合物或制剂用于抑制RhoC的活性。
在另一优选例中，所述的BCAT1靶向抑制剂包括抑制野生型BCAT1的靶向抑制剂、抑制突变型BCAT1的靶向抑制剂、或其组合。
在另一优选例中，所述的突变型BCAT1为BCAT1E61A突变蛋白。
在另一优选例中，所述的BCAT1靶向抑制剂选自下组：坎地沙坦、氯沙坦、替勃龙、抗BCAT1的抗体或其组合。
本发明第九方面，提供了一种活性成分的组合，所述组合包括：
(i)第一活性成分或含有所述第一活性成分的药物组合物，所述第一活性成分为针对本发明第一方面所述的突变蛋白的靶向抑制剂；和
(ii)第二活性成分，所述的第二活性成分用于降低RhoC的表达和/或活性。
本发明第十方面，提供了一种本发明第九方面所述的活性成分的组合的用途，用于制备一药物，所述药物用于治疗BCAT1高表达或突变型BCAT1相关的疾病。
在另一优选例中，所述的疾病选自下组：代谢疾病、癌症或其组合。
在另一优选例中，所述的代谢疾病选胰岛素抵抗、高胆固醇血症、糖尿病、枫糖尿病、或其组合。
在另一优选例中，所述的癌症选自下组：胰腺癌、肺癌、胶质瘤、白血病、肠癌、或其组合。
在另一优选例中，所述的突变型BCAT1为BCAT1E61A突变蛋白。
本发明第十一方面，提供了一种体外抑制RhoC活性的方法，包括步骤：
(a)在BCAT1靶向抑制剂存在下，培养表达BCAT1蛋白和RhoC蛋白的细胞，从而抑制所述所述细胞中的RhoC活性。
在另一优选例中，所述细胞选自下组：肿瘤细胞。
在另一优选例中，所述细胞选自下组：肿瘤细胞TE1、肿瘤细胞MGC803。
在另一优选例中，所述方法是非治疗和非诊断的。
在另一优选例中，所述的BCAT1靶向抑制剂选自下组：坎地沙坦、氯沙坦、替勃龙、抗BCAT1的抗体或其组合。
本发明第十二方面，提供了一种筛选候选治疗药物的方法，包括步骤：
(a)在测试组中，在测试物存在下，测试BCAT1E61A突变蛋白的活性T1；并且在空白对照组中，在测试物不存在下，测试BCAT1E61A突变蛋白的活性T0；其中，除了测试物之外，所述测试组和空白对照组的其他测试条件相同；
(b)将测试组的活性T1与空白对照组的活性T0进行比较；
其中，如果所述的T1显著低于T0，则提示所述测试物为潜在的候选治疗药物。
在另一优选例中，所述的“显著低于”指T1/T0≤0.5，较佳地≤0.25，更佳地≤0.1。
在另一优选例中，所述的BCAT1E61A突变蛋白具有SEQ ID No:2所示的氨基酸序列。
在另一优选例中，所述方法还包括：
(c)测试所述的潜在的候选治疗药物对肿瘤细胞的抑制作用。
在另一优选例中，所述抑制作用包括：对肿瘤细胞生长的抑制、对肿瘤细胞迁 移的抑制、或其组合。
在另一优选例中，所述方法还包括：在阳性对照组中，在阳性化合物存在下，测试BCAT1E61A突变蛋白的活性T2；其中，除了阳性化合物之外，所述阳性对照组的其他测试条件与测试组相同；
并将测试组的活性T1与阳性对照组的T2进行比较。
在另一优选例中，所述方法是非治疗和非诊断的。
在另一优选例中，所述的阳性化合物选自下组：坎地沙坦、氯沙坦、替勃龙、抗BCAT1的抗体或其组合。
本发明第十三方面，提供了一种治疗疾病的方法，包括步骤：向需要治疗的受试者施用治疗有效量的BCAT1E61A靶向抑制剂。
在另一优选例中，所述的BCAT1E61A突变相关的疾病选自下组：代谢疾病、癌症或其组合。
在另一优选例中，所述的代谢疾病选自下组：胰岛素抵抗、枫糖尿病、高胆固醇血症、糖尿病、或其组合。
在另一优选例中，所述的癌症选自下组：白血病、胃癌、胶质瘤、肠癌、肺癌、胰腺癌、或其组合。
在另一优选例中，所述疾病是BCAT1E61A阳性的疾病。
在另一优选例中，所述的靶向抑制剂选自下组：坎地沙坦、氯沙坦、替勃龙或其组合。
本发明第十四方面，提供了一种BCAT1E61A突变检测试剂的用途，用于制备以试剂盒，所述试剂盒用于检测患者样本是否存在BCAT1E61A突变。
在另一优选例中，所述试剂盒用于判断患者是否患有BCAT1E61A突变相关的疾病。
在另一优选例中，所述试剂盒含有检测试剂，所述检测试剂用于检测支链氨基酸转移酶1(BCAT1)是否存在E61A突变。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了BCAT1高表达及BCAT1E61A突变在许多癌症中发生。
图2显示了BCAT1E61A突变促进酶活性。
图3显示了BCAT1高表达及BCAT1E61A突变促进细胞生长。
图4显示了BCAT1高表达及BCAT1E61A突变促进细胞迁移和侵袭。
图5显示了BCAT1及BCAT1E61A突变通过RhoC影响细胞迁移和侵袭。
图6显示了BCAT1E61A突变导致肺组织增生及促进体内支链氨基酸分解代谢。
图7显示了BCAT1E61A突变促进肝癌发生和发展。
图8显示了BCAT1E61A突变促进粒细胞白血病发生。
图9显示了BCAT1分解代谢产物支链酮酸结合RhoC促进RhoC活力。
图10显示了BCAT1高表达及BCAT1E61A突变作用机制模型图。
图11显示了坎地沙坦直接结合BCAT1及BCAT1E61A突变酶活位点抑制酶活。
图12显示了坎地沙坦对BCAT1下游关键因子RhoC活力及细胞迁移的抑制。
图13显示了回补BCAT1分解代谢产物支链酮酸有效挽救坎地沙坦处理细胞对RhoC活力的抑制。
图14显示了靶向BCAT1-RhoC代谢信号轴抑制胃癌细胞体内转移。
图15显示了对BCAT1E61A抑制剂的筛选结果。
具体实施方式
本发明人通过广泛而深入的研究，首次意外地发现了BCAT1E61A功能获得性活化突变体蛋白，即BCAT1E61A突变蛋白。研究表明，本发明的BCAT1E61A突变蛋白在突变后，其催化活性显著提高。本发明还开发针对本发明突变蛋白的靶向抑制剂，并验证了这些靶向抑制剂可用于治疗肿瘤等BCAT1E61A突变相关疾病。在此基础上完成了本发明。
具体地，本发明的实验证明，抑制BCAT1E61A突变，能明显缓解BCAT1E61A突变造成的肿瘤等代谢相关疾病。坎地沙坦等BCAT1特异性靶向抑制剂，可直接结合BCAT1E61A并抑制其活性。坎地沙坦等BCAT1特异性靶向抑制剂可以抑制 BCAT1E61A突变造成的肿瘤等疾病。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生、可以有但不是必须有，可以是1个、2个或3个。
缩写
RhoC表示Ras Homolog Family Member C，中文名为Ras同源家族成员C，属于小G蛋白超家族的亚家族成员之一，作为一个分子开关，可以打开或关闭各种细胞内信号通路，还参与许多生理病理过程。
BCAT1及功能获得性活化突变体
支链转氨酶1(branched-chain aminotransferase 1,BCAT1)催化人类支链氨基酸降解的第一步可逆反应,催化支链氨基酸和酮戊二酸通过转氨基反应生成对应的支链酮酸和谷氨酸。
如本文所用，术语“本发明的突变蛋白”、“本发明蛋白”、“本发明的功能获得性活化突变体”、“本发明的功能获得性活化突变蛋白”、或“本发明BCAT1E61A蛋白”可互换使用，指在对应于人支链氨基酸转移酶1的第61位存在E→A的突变并且具有支链氨基酸转移酶活性。应理解，该术语不仅包括在野生型的人BCAT1的氨基酸序列(SEQ ID No:1)基础上具有E61A突变的突变蛋白，还包括可含有额外突变的突变蛋白，只要这些额外突变不影响或基本上不影响BCAT1E61A的转氨酶功 能。
如本文所用，术语“E61A”指在氨基酸序列的第61位上具有从谷氨酸(E)突变为亮氨酸(A)的氨基酸突变。相应地，对应的核苷酸密码子GCG突变为GAG。
本发明所涉及的氨基酸序列如下所示：
野生型BCAT1蛋白的氨基酸序列(SEQ ID No:1)：
BCAT1E61A突变蛋白的氨基酸序列(SEQ ID No:2)：
在本发明中，本发明的突变蛋白还包括其保守性变异体，指与本发明突变蛋白的氨基酸序列(SEQ ID No:2)相比，在除了第61位(E61A)之外的其他位置，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1

活性成分
本发明还提供了针对本发明突变蛋白的靶向抑制剂及其应用。本发明的突变蛋白的靶向抑制剂可用于抑制BCAT1E61A突变蛋白酶活功能，进而治疗BCAT1E61A突变相关的疾病。
在本发明中，可将本发明突变蛋白的靶向抑制剂作为活性成分用于制备药物，以治疗相关疾病。
在本发明中，所述靶向抑制剂包括(但并不限于)：小分子、抗体、多肽、寡核苷酸、适体、ADC、或其组合。
本发明的代表性的靶向抑制剂选自下组：坎地沙坦、氯沙坦、替勃龙或其组合。
坎地沙坦(Candesartan)是一种非肽类血管紧张素Ⅱ受体拮抗剂，可选择性的、难以逆转地阻滞ATI受体，而无明显副作用。主要用于治疗高血压。坎地沙坦结构式如式(I)所示：
氯沙坦(Losartan)结构式如式(II)所示：
利维爱/替勃龙片(Tibolone)如式(III)所示：
药物组合物
由于本发明靶向抑制剂(也称为本发明化合物)具有优异的BCAT1E61A突变蛋白的抑制活性，因此本发明靶向抑制剂(包括化合物及其各种晶型，药学上可接受的盐)以及含有本发明靶向抑制剂为主要活性成分的药物组合物可用于预防和/或治疗(稳定、减轻或治愈)BCAT1E61A突变蛋白相关的疾病。
本发明的药物组合物包含安全有效量范围内的本发明靶向抑制剂及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明靶向抑制剂/剂，更佳地，含有10-200mg本发明靶向抑制剂/剂。较佳地， 所述的“一剂”为一个胶囊或药片。
本发明的药物组合物包含安全有效量范围内的本发明靶向抑制剂及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明靶向抑制剂/剂，更佳地，含有10-200mg本发明靶向抑制剂/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明靶向抑制剂以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明靶向抑制剂或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、肠胃外(静脉内、肌肉内或皮下)。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上 述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明靶向抑制剂可以单独给药，或者与其他药学上可接受的化合物联合给药。
联合给药时，所述药物组合物还包括与一种或多种(2种，3种，4种，或更多种)其他药学上可接受的化合物。该其他药学上可接受的化合物中的一种或多种可与本发明的化合物同时、分开或顺序地施用。
使用药物组合物时，是将安全有效量的本发明靶向抑制剂适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选20～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
检测用途和试剂盒
本发明还提供用于检测支链氨基酸转移酶1(BCAT1)是否存在E61A突变的检测试剂和检测试剂盒。
本发明的检测试剂或试剂盒可用于检测患者样本是否存在BCAT1E61A突变。优选地，本发明的检测试剂为核酸检测试剂。
本发明的试剂盒用于判断患者是否患有BCAT1E61A突变相关的疾病。
在本发明中，所述试剂盒还可包括使用说明书。所述说明书描述了所述试剂盒或检测的用法、与BCAT1E61A突变相关的疾病等内容。
本发明的主要优点包括：
1.重组人BCAT1E61A功能获得性活化突变体蛋白，相较于野生型，其酶活力显著增强。
2.坎地沙坦、氯沙坦、替勃龙为已批准临床用药，相较于全新药物，安全性有更强保证，在临床试验等环节有显著优势。
3.坎地沙坦、氯沙坦、替勃龙，可以强烈结合，并特异性抑制支链氨基酸转氨酶1(BCAT1)及其功能获得性活化突变蛋白。
4.坎地沙坦、氯沙坦、替勃龙，靶向支链氨基酸转氨酶1(BCAT1)及其功能获得性活化突变，在治疗肿瘤和枫糖尿病等代谢性疾病方面，有巨大应用潜力。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1 BCAT1高表达或者BCAT1E61A突变在数据库及临床肿瘤样本中普遍存在
1.1 BCAT1高表达在数据库的临床肿瘤样本中普遍存在
通过cbioportal(https://www.cbioportal.org/)数据库的结果可以看到，BCAT1在如胃癌、肺癌、胰腺癌和卵巢癌等多种肿瘤中高表达(图1a)。而且,从Protein Atlas数据库中的生存数据可以看到，BCAT1高表达与生存率呈现负相关，即高表达BCAT1提示较差的预后结果(图1b)。
1.2 BCAT1E61A在数据库的临床肿瘤样本中普遍存在
通过CCLE数据库可以看到BCAT1E61A在白血病、胃癌、食管癌、肺癌细胞系中均有检测到(图1c-d)。而且取自临床的106例肿瘤样本中发现了8例在癌组织中发生突变的样本(图1e-f)。以上结果说明BCAT1E61A在肿瘤发生发展 过程中普遍存在。
实施例2 BCAT1E61A突变促进其酶活
2.1原核及真核蛋白表达
以293T细胞cDNA为模板，用PCR克隆出带有相应保护碱基和酶切位点的BCAT1的CDS产物。酶切后的PCR产物分别连接于原核表达载体pET28a和真核表达载体pCDH-puro,其中的真核表达载体在加了用于纯化的flag标签。将连接产物转化DH5α克隆菌株，然后鉴定，测序及抽提质粒。同时，相应的需要以质粒为模板进行PCR点突变，然后同样进行转化、鉴定、测序及抽提质粒。
原核表达则将对应pET28a质粒转化至用于原核表达感受态细胞BL21(DE3)。然后取单克隆，小摇10ml，后按1:1000接种在1L LB培养基中进行扩大培养。在OD值约0.6时，转至16℃，加入0.5mM终浓度IPTG诱导表达过夜，次日离心收集细菌，使用纯化缓冲液(20mM Tris pH 8.0，150mM NaCl)重悬，250W功率的超声破碎，3秒间隔破碎细菌，超速离心收集上清。将上清经过5ml的镍离子交换柱吸附蛋白，咪唑洗脱，(洗脱缓冲液成分：25mM Tris，150mM NaCl，25mM Imidazole，pH7.5)，竞争下洗脱含相似结构的组氨酸标签的目的蛋白。采用GE的AKTA蛋白纯化系统进一步纯化后收集浓缩蛋白。先启动AKTA系统，待有蛋白质经过UV检测到峰值时，收集蛋白。
真核表达则将对应pCDH-puro质粒转染至293T细胞，于48小时后收取细胞。使用含0.5％NP-40的PBS缓冲液裂解，高速离心收集上清，加入flag-beads富集蛋白并过夜，然后离心清洗flag-beads，加入flag肽段洗脱蛋白，收集上清即是纯化好的真核蛋白。
2.2酶活力检测
1μL纯化的原核5mg/ml BCAT1蛋白或者适量的真核蛋白溶于100μL BCAT1酶活反应体系(5μM PLP,50mM(NH4)2SO4,0.05mM NADH,5mM DTT,5mM酮戊二酸，10mM亮氨酸，1U亮氨酸脱氢酶，100mM K2HPO4，pH 7.4)，检测340nm的吸光值变化，取直线区间斜率作为统计结果，与不添加BCAT1蛋白及不加亮氨酸脱氢酶的对照组，相对酶活以百分比方式表示(反应机制如图2a所示)。
2.3 BCAT1E61A突变促进其酶活
使用原核表达的BCAT1和BCAT1E61A突变蛋白，进行酶动力实验。此外，为了排除是否真核修饰影响酶活，使用真核表达的BCAT1和BCAT1E61A突变蛋白，进行酶活力实验。
结果表明：原核表达的BCAT1E61A突变显著促进蛋白催化活力(如图2b所示)。同样地，真核表达的BCAT1E61A突变也显著促进蛋白催化活力(如图2c所示)。
2.4突变细胞系代谢水平整体升高
通过基于LC-MS的非靶向代谢组分析BCAT1突变型细胞系(KATO III和TE1)及野生型细胞系(AGS和KYSE180)，发现BCAT1突变细胞系对比野生型细胞系的总体代谢水平都发生上调(如图2d-e所示)。
实施例3 BCAT1及BCAT1E61A促进癌症发展的机制研究
3.1 BCAT1及其BCAT1E61A促进细胞生长
在多种肿瘤细胞系中过表达BCAT1及其BCAT1E61A促进细胞生长(如图3a所示)。在多种肿瘤细胞系中敲除BCAT1及其BCAT1E61A降低细胞克隆生成如图3b所示)。
综上说明，BCAT1高表达或者BCAT1E61A突变可促进肿瘤生长。
3.2 BCAT1及其BCAT1E61A促进细胞迁移和侵袭
在多种肿瘤细胞系中敲除BCAT1及其BCAT1E61A降低细胞迁移(如图4a所示)。在多种肿瘤细胞系中过表达BCAT1及其BCAT1E61A促进细胞迁移(如图4b所示)。在肿瘤细胞系中敲除BCAT1及其BCAT1E61A导致细胞侵袭变弱(如图4c所示)。在肿瘤细胞系中过表达BCAT1及其BCAT1E61A促进细胞侵袭(如图4d所示)。
综上说明，BCAT1高表达或者BCAT1E61A突变可以促进肿瘤迁移以及侵袭。
3.3 BCAT1及其BCAT1E61A可与细胞迁移关键因子RhoC互作
通过IP-MS鉴定到BCAT1与RhoC直接互作(如图5a所示)，通过CoIP验证RhoC与BCAT1互作(如图5b所示)。通过IHC染色分析发现临床样本中BCAT1及其BCAT1E61A均高表达(如图5c-d所示)，而且RhoC活力在癌组织中显著升高(如图5e所示)。通过裸鼠腹膜转移模型发现高表达BCAT1及其BCAT1E61A均促进转移(如图5f-g所示)。
综上说明，BCAT1通过增强RhoC活力以促进肿瘤迁移及侵袭。
3.4 BCAT1E61A突变增强肺部细胞增生
通过CRISPR-Cas9技术，构建了敲入型的Bcat1E61A点突变C57小鼠。并对小鼠的病理进行分型研究。
结果表明，与野生型相比，Bcat1E61A点突变小鼠的肺部支气管增生及肺泡细胞增生(如图6a所示)，这提示BCAT1E61A点突变可能在炎症和癌症发生过程起到重要的作用。
3.5 BCAT1E61A突变促进其酶活并促进BCAA分解代谢
通过分离培养转基因小鼠的表皮成纤维细胞，同时在低BCAA培养基中添加缬氨酸-13C5或者亮氨酸-13C6,15N1以追踪支链氨基酸的代谢流。
在标记36小时后，发现标记的支链氨基酸显著减少，而标记的支链酮酸显著增加。这说明BCAT1E61A突变促进其酶活，增强BCAA分解代谢(如图6b-d所示)。
3.6 BCAT1E61A突变促进肝癌发生和发展
野生型和Bcat1E61A点突变杂合和纯合小鼠在出生第14天，腹腔注射二乙基亚硝胺(DEN)2mg/kg，并在给药4周后，腹腔注射四氯化碳(CCl4)1mL/kg，每周2次,共9周，进行肝癌造模(如图7a所示)。造模结束记录小鼠外形、脾、肺等重要脏器、体重和进食量、肝脏/体重比和血清白蛋白水平均无明显变化(如图7b-g所示)。但Bcat1E61A点突变杂合和纯合小鼠谷草转氨酶(AST)显著升高(如图7h所示)，纯合小鼠谷丙转氨酶(ALT)降低(如图7i所示)。解剖发现Bcat1E61A点突变杂合和纯合小鼠肝脏表面均有肿瘤发生(如图7j所示)。病理切片检查发现野生型小鼠肝组织恶性病变程度低，而Bcat1E61A点突变杂合和纯合小鼠肝组织细胞恶性程度高，细胞增殖标记物Ki67染色阳性高于野生型小鼠(如图7k所示)。统计显示Bcat1E61A点突变杂合和纯合小鼠的肝脏肿瘤发生率、肝脏肿瘤数目和最大瘤直径均显著高于相同造模的野生型小鼠(如图7l-n所示)。
3.7 BCAT1E61A突变促进粒细胞白血病的发生和发展
Bcat1E61A突变引起小鼠死亡，有巨大的脾、淋巴结和肝脏(如图8a-b所示)。脾脏和肝脏通过标记物染色发现MPO+CD34+(如图8c所示)；CD3-CD4-CD19-CD20-CD30-CD79a-PAX5-，同时增殖标记物Ki67染色高阳性和Phospho-Histone H3(PH3)水平明显增高(如图8d所示)，这些数据表明BCAT1E61A突变促进粒细胞白血病的发生和发展。
3.8 BCAT1及BCAT1E61A通过下游代谢物BCKA促进RhoC活力
通过使用计算机分子模拟，发现BCKA可以直接结合RhoC，而且等温热滴定(ITC)实验验证了该结果(如图9a所示)。在体外鸟感受交换实验也证实了BCKA可 以促进RhoC活力(如图9b所示)。
综上所有结果，分析得出一个模型：BCAT1及其BCAT1E61A促进BCKA产生增加区室化浓度以促进RhoC活力，进一步促进肿瘤的迁移和侵袭(如图10所示)。
实施例4 BCAT1抑制剂筛选
4.1通过酶活力筛选BCAT1E61A抑制剂
使用FDA已批准药物库(FDA approved drug library)，终浓度为10μM加入上述实施例2.2的检测BCAT1E61A酶活的反应体系内的吸光值变化。每组三个重复，对照组不加抑制剂，作为活力值的100％。
此外，对于筛选出的具有一定抑制活性的化合物，进一步通过ΔOD值随时间的变化曲线来测量化合物的抑制活性。
结果：
如图15a所示，在六百多种化合物中，绝大多数化合物对于BCAT1E61A酶活没有抑制作用，然而，少数几种对BCAT1E61A酶活有一定的抑制作用，包括坎地沙坦、氯沙坦、替勃龙。
如图15b所示，坎地沙坦、氯沙坦和替勃龙对BCAT1E61A酶活有显著的靶向抑制活性。按对BCAT1E61A酶活的抑制作用大小排序如下：坎地沙坦>氯沙坦>替勃龙。
4.2测定坎地沙坦与BCAT1或BCAT1E61A结合实验
通过改变坎地沙坦的浓度，测定坎地沙坦对野生型和突变型BCAT1E61A的抑制活性。
通过改变底物亮氨酸的浓度，从0.1mM到5mM梯度。等热滴定实验使用纯化的BCAT1或BCAT1E61A蛋白和坎地沙坦。
结果：
如图11a-b所示，坎地沙坦能够以浓度依赖的方式抑制BCAT1或BCAT1E61A的酶活。
如图11c中所示，坎地沙坦能够直接结合BCAT1或BCAT1E61A蛋白，并且，坎地沙坦更强烈地结合BCAT1E61A蛋白。
4.3使用计算机分子模拟BCAT1或BCAT1E61A和坎地沙坦的结合
通过BCAT1或BCAT1E61A结构模型和坎地沙坦三维结构进行软件拟合分析。
结果表明，坎地沙坦能够结合在BCAT1或BCAT1E61A蛋白酶活中心，封闭了 BCAT1或BCAT1E61A蛋白与正常底物的结合以抑制其活性(如图11d表示坎地沙坦与BCAT1或BCAT1E61A结构的表面图；图11e为表示坎地沙坦与BCAT1E61A结合位点的局部图)。
实施例5坎地沙坦在活体水平抑制BCAT1或BCAT1E61A活性来降低细胞迁移
5.1坎地沙坦在活体水平抑制过表达BCAT1或BCAT1E61A导致的RhoC活力增加
对过表达BCAT1或BCAT1E61A的细胞系计数后进行处理和不处理坎地沙坦(150μM终浓度)，在18小时后裂解细胞，分析其RhoC活力水平。
RhoC活力结果表明，未处理坎地沙坦对照组中BCAT1或BCAT1E61A过表达都明显增加RhoC活力，处理坎地沙坦实验组中BCAT1或BCAT1E61A过表达都明显抑制RhoC活力(如图12a所示)。
5.2坎地沙坦在活体水平抑制BCAT1E61A突变导致的RhoC活力增加
对从转基因小鼠中分离的野生型，杂合Bcat1E61A和纯合Bcat1E61A的成纤维细胞计数后进行处理和不处理坎地沙坦(150μM终浓度)。在18小时后裂解细胞，分析其RhoC活力水平。
RhoC活力结果表明，未处理坎地沙坦对照组中杂合Bcat1E61A和纯合Bcat1E61A都明显增加RhoC活力，而处理过坎地沙坦的实验组中杂合Bcat1E61A和纯合Bcat1E61A的RhoC活力都被明显抑制(如图12b所示)。
5.3坎地沙坦以浓度依赖方式抑制RhoC活力
对肿瘤细胞系TE1和MGC803加入50-250μM终浓度坎地沙坦处理细胞。在18小时后裂解细胞，分析其RhoC活力水平。
RhoC活力结果表明，坎地沙坦在细胞水平以浓度依赖方式抑制RhoC活力(如图12c所示)。
5.4坎地沙坦在活体水平以浓度依赖方式抑制细胞迁移
对肿瘤细胞系TE1和MGC803加入50-250μM终浓度坎地沙坦处理细胞，在18小时后计数细胞约10万/小室细胞进行Transwell实验，分析坎地沙坦对细胞迁移能力的影响。
迁移实验结果表明，坎地沙坦在细胞水平以浓度依赖方式抑制细胞迁移(如图12d所示)。
5.5回补BCAT1分解代谢产物支链酮酸有效挽救坎地沙坦处理细胞对RhoC活力的抑制
对肿瘤细胞系TE1和MGC803加入50-250μM终浓度坎地沙坦处理细胞。同时分别加入BCAA和α-KG，或BCKA和Glu。在18小时后裂解细胞，分析其RhoC活力水平。
RhoC活力结果表明，回补BCKA和Glu能明显挽救坎地沙坦对RhoC活力的抑制(如图13所示)。
5.6靶向BCAT1-RhoC代谢信号轴抑制胃癌细胞体内转移
稳定过表达对照空载质粒，BCAT1野生型质粒或BCAT1E61A突变体的胃癌细胞株SGC7901接种于5周龄裸鼠腹膜内。分别以坎地沙坦，1/5含量支链氨基酸的饮食，或在上述稳定细胞株分别敲减RhoC或ARHGEF1接种于裸鼠腹膜内。接种后，观察记录小鼠外形和体重，5周后，各处理组小鼠麻醉处死后检测腹膜成瘤，并计算腹膜肿瘤指数(PCI)。结果显示接种过表达野生型BCAT1和BCAT1E61A突变体的胃癌细胞的小鼠腹水明显(如图14a所示)且均显著促进胃癌细胞在腹腔内的播散转移(如图14b所示)和接种瘤的重量(如图14c所示)。与对照组相比，坎地沙坦，1/5含量支链氨基酸的饮食以及敲减RhoC和显著降低了过表达野生型BCAT1和BCAT1E61A突变体胃癌细胞的PCI，即能显著抑制胃癌细胞在腹腔内的播散转移(如图14b所示)和接种瘤重量(如图14c所示)。此外，接种空载质粒、BCAT1野生型质粒或BCAT1E61A突变体的胃癌细胞株的各组小鼠之间体重无明显差异，各处理组间小鼠体重也无显著差异(如图14d所示)。
以上结果证明，靶向BCAT1-RhoC代谢信号轴抑制胃癌细胞体内转移。
讨论
本发明首次报道了BCAT1E61A在肿瘤发生发展过程中的重要功能，是癌症驱动基因突变。发现肿瘤临床样本中存在BCAT1E61A突变位点,该突变可以显著提升BCAT1的酶活功能，导致代谢物BCKA显著增加，从而促进下游RhoC的活力。
进一步在小鼠模型中，本发明人也验证了BCAT1突变在癌症发生及发展中的关键作用,尤其是该突变可导致白血病,以及促进化学诱导肝癌的发生发展。
此外，本发明对多种不同化合物进行了通过酶活抑制筛选实验，筛选到了坎地沙坦等数个显著靶向抑制BCAT1突变蛋白的酶活的抑制剂，这些靶向抑制剂可用于抑制BCAT1突变型相关的肿瘤。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (23)

  1. 一种支链氨基酸转移酶1(BCAT1)突变蛋白，其特征在于，所述的突变蛋白在对应于人支链氨基酸转移酶1的第61位存在E→A的突变，并且具有支链氨基酸转移酶活性。
  2. 如权利要求1所述的突变蛋白，其特征在于，所述突变蛋白的转氨酶活性A1与野生型BCAT1蛋白的转氨酶活性A0之比(A1/A0)为约1.5，更佳地为约1.47。
  3. 如权利要求1所述的突变蛋白，其特征在于，所述突变蛋白的氨基酸序列如SEQ ID No:2所示。
  4. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1所述的BCAT1E61A突变蛋白。
  5. 一种载体，其特征在于，所述载体含有如权利要求4所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求5所述的载体，或所述宿主细胞的核酸中含有如权利要求4所述的分离的多核苷酸。
  7. 如权利要求6所述的宿主细胞，其特征在于，所述宿主细胞表达BCAT1E61A突变蛋白。
  8. 一种制备如权利要求1所述的BCAT1E61A突变蛋白的方法，其特征在于，包括步骤：
    在适合表达的条件下，培养如权利要求6所述的宿主细胞，从而表达如权利要求1所述的BCAT1E61A突变蛋白；和
    分离表达产物，从而获得如权利要求1所述的BCAT1E61A突变蛋白。
  9. 一种如权利要求1所述的突变蛋白的靶向抑制剂的用途，其特征在于，用于制备一药物组合物或制剂，所述药物组合物或制剂用于治疗BCAT1E61A突变相关的疾病。
  10. 如权利要求9所述的用途，其特征在于，所述的BCAT1E61A突变相关的疾病选自下组：代谢疾病、癌症或其组合。
  11. 如权利要求9所述的用途，其特征在于，所述疾病是BCAT1E61A阳性的疾病。
  12. 如权利要求9所述的用途，其特征在于，所述抑制剂选自下组：小分子、抗体、多肽、寡核苷酸、适体、或其组合。
  13. 如权利要求9所述的用途，其特征在于，所述的靶向抑制剂选自下组：坎 地沙坦、氯沙坦、替勃龙或其组合。
  14. 一种试剂盒，其特征在于，所述的试剂盒包括：
    (a)第一活性成分或含有所述第一活性成分的第一药物组合物，所述第一活性成分为针对如权利要求1所述的突变蛋白的靶向抑制剂；和
    (b)检测试剂，所述检测试剂用于检测支链氨基酸转移酶1(BCAT1)是否存在E61A突变。
  15. 一种BCAT1靶向抑制剂的用途，其特征在于，用于制备一药物组合物或制剂，所述药物组合物或制剂用于抑制RhoC的活性。
  16. 一种活性成分的组合，其特征在于，所述组合包括：
    (i)第一活性成分或含有所述第一活性成分的药物组合物，所述第一活性成分为针对如权利要求1所述的突变蛋白的靶向抑制剂；和
    (ii)第二活性成分，所述的第二活性成分用于降低RhoC的表达和/或活性。
  17. 一种如权利要求16所述的活性成分的组合的用途，其特征在于，用于制备一药物，所述药物用于治疗BCAT1高表达或突变型BCAT1相关的疾病。
  18. 一种体外抑制RhoC活性的方法，其特征在于，包括步骤：
    (a)在BCAT1靶向抑制剂存在下，培养表达BCAT1蛋白和RhoC蛋白的细胞，从而抑制所述所述细胞中的RhoC活性。
  19. 如权利要求18所述的方法，其特征在于，所述的BCAT1靶向抑制剂选自下组：坎地沙坦、氯沙坦、替勃龙、抗BCAT1的抗体或其组合。
  20. 一种筛选候选治疗药物的方法，其特征在于，包括步骤：
    (a)在测试组中，在测试物存在下，测试BCAT1E61A突变蛋白的活性T1；并且在空白对照组中，在测试物不存在下，测试BCAT1E61A突变蛋白的活性T0；其中，除了测试物之外，所述测试组和空白对照组的其他测试条件相同；
    (b)将测试组的活性T1与空白对照组的活性T0进行比较；
    其中，如果所述的T1显著低于T0，则提示所述测试物为潜在的候选治疗药物。
  21. 如权利要求20所述的方法，其特征在于，所述的“显著低于”指T1/T0≤0.5，较佳地≤0.25，更佳地≤0.1。
  22. 一种治疗疾病的方法，其特征在于，包括步骤：向需要治疗的受试者施用治疗有效量的BCAT1E61A靶向抑制剂。
  23. 一种BCAT1E61A突变检测试剂的用途，其特征在于，用于制备以试剂盒，所述试剂盒用于检测患者样本是否存在BCAT1E61A突变。
PCT/CN2023/086648 2022-04-25 2023-04-06 支链氨基酸转氨酶1的功能获得性突变体及其应用 WO2023207545A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0613687A1 (en) * 1993-03-05 1994-09-07 Akzo Nobel N.V. Use of a pregnane derivatives for the treatment of tumours
US20100029560A1 (en) * 2007-01-16 2010-02-04 Phigenix, Inc Compositions and methods for diagnosing, treating, and preventing prostate conditions
US20130072397A1 (en) * 2010-05-14 2013-03-21 Deutsches Krebsforschungszentrum Methods for the diagnosis and prognosis of a tumor using bcat1 protein
US20130310545A1 (en) * 2011-01-28 2013-11-21 Deutsches Krebsforschungszentrum Inhibitors of Branched-Chain-Aminotransferase-1 (BCAT1) for the Treatment of Brain Tumors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0613687A1 (en) * 1993-03-05 1994-09-07 Akzo Nobel N.V. Use of a pregnane derivatives for the treatment of tumours
US20100029560A1 (en) * 2007-01-16 2010-02-04 Phigenix, Inc Compositions and methods for diagnosing, treating, and preventing prostate conditions
US20130072397A1 (en) * 2010-05-14 2013-03-21 Deutsches Krebsforschungszentrum Methods for the diagnosis and prognosis of a tumor using bcat1 protein
US20130310545A1 (en) * 2011-01-28 2013-11-21 Deutsches Krebsforschungszentrum Inhibitors of Branched-Chain-Aminotransferase-1 (BCAT1) for the Treatment of Brain Tumors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU YUPING; HE WENWEN; WANG WENYAN; ZHU DAN; DENG MINGFEN: "Suppression Effects of Candesartan on Proliferation, Migration and Invasion of Ovarian Cancer Caov-3 Cells and Its Mechanism", TUMOR, SHANGHAI, CN, vol. 38, no. 11, 30 November 2018 (2018-11-30), CN , pages 1001 - 1010, XP009550017, ISSN: 1000-7431 *
RONG WU; JUN-TAO ZHANG; WEI-BIN LI; KUN WANG; ZHI-ZHEN LIU: "Losartan Attenuates Angiogenesis in Hepatocellular Carcinoma via Down-Regulation of AT1R/VEGF Signaling Pathway", CHINA JOURNAL OF MODERN MEDICINE, ZHONGNAN DAXUE, CN, vol. 28, no. 26, 30 September 2018 (2018-09-30), CN , pages 7 - 13, XP009550014, ISSN: 1005-8982 *
SHAFEI MAI AHMED, FORSHAW THOMAS, DAVIS JASMINE, FLEMBAN ARWA, QUALTROUGH DAVID, DEAN SARAH, PERKS CLAIRE, DONG MING, NEWMAN ROBER: "BCATc modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer", ONCOTARGET, vol. 11, no. 21, 26 May 2020 (2020-05-26), pages 1971 - 1987, XP093104474, DOI: 10.18632/oncotarget.27607 *

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