WO2022017532A1 - 一种c-Src SH3 RT-loop作为靶点用于抗血栓 - Google Patents

一种c-Src SH3 RT-loop作为靶点用于抗血栓 Download PDF

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WO2022017532A1
WO2022017532A1 PCT/CN2021/108371 CN2021108371W WO2022017532A1 WO 2022017532 A1 WO2022017532 A1 WO 2022017532A1 CN 2021108371 W CN2021108371 W CN 2021108371W WO 2022017532 A1 WO2022017532 A1 WO 2022017532A1
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src
integrin
loop
antagonist
amino acid
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French (fr)
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奚晓东
罗成
毛建华
阮铮
奚闻达
龙章彪
朱孔凯
肖兵
王韵
黄建松
蒋昊
刘静秋
蒋华良
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上海交通大学医学院附属瑞金医院
中国科学院上海药物研究所
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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/82—Translation products from oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis

Definitions

  • the present invention belongs to the field of molecular biology and biomedicine, in particular, the present invention relates to c-Src SH3 RT-loop as a target for antithrombotic.
  • Cardiovascular and cerebrovascular thrombotic diseases such as myocardial infarction and cerebral infarction seriously affect human life and health.
  • the platelet-involved thrombosis plays an important role in the occurrence and development of cardiovascular and cerebrovascular thrombotic diseases.
  • platelets activate and lead to pathology through a series of functions such as adhesion, stretching, and aggregation.
  • Thrombosis can cause ischemic necrosis of the heart and brain tissue in the affected vascular distribution area, which can seriously endanger the patient's life. Therefore, antiplatelet therapy has become the first choice for the treatment of cardiovascular and cerebrovascular thrombotic diseases.
  • Classic antiplatelet drugs include cyclooxygenase (COX) inhibitor aspirin, which is currently the most widely studied and used antiplatelet drug in antiplatelet therapy.
  • COX cyclooxygenase
  • -529 and Ser-516 are irreversibly acetylated, thereby blocking the synthesis of TXA2 and exerting an antiplatelet effect.
  • the common adverse reactions of aspirin are gastrointestinal discomfort and gastrointestinal bleeding, and the risk of bleeding is dose-related. To avoid bleeding side effects, high antithrombotic doses cannot be used.
  • ADP P2Y12 receptor antagonists including thienopyridines, representative drugs are ticlopidine (Ticlopidine), clopidogrel (Clopidogrel) and prasugrel ( Prasugrel).
  • Non-thiophene pyridines representative drugs are ticagrelor (Ticagrelor) and cangrelor (Cangrelor).
  • the second-generation P2Y12 receptor antagonist clopidogrel is widely used, which can irreversibly inhibit the platelet ADP receptor, thereby inhibiting the platelet aggregation induced by the release of ADP from activated platelets, and bleeding is still its main side effect. It can be seen that although the classic antiplatelet drugs can exert a good antithrombotic effect, due to the limitation of bleeding side effects, a sufficient dose cannot be used to exert a deep antithrombotic effect.
  • Integrin ⁇ IIb ⁇ 3 As the final common pathway mediating platelet activation, aggregation and thrombus formation, is the main target of antithrombotic drug research. In fact, important progress has been made in the research on integrin ⁇ IIb ⁇ 3 as an antithrombotic drug target. Currently, it is mainly focused on integrin ⁇ IIb ⁇ 3 receptor antagonist drugs, which have obtained good clinical effects. Currently, there are three integrin ⁇ IIb ⁇ 3 receptor antagonist antiplatelet drugs approved by the U.S.
  • FDA Food and Drug Administration
  • ⁇ IIb ⁇ 3 receptor antagonists specifically exert antithrombotic effects by interfering with the interaction of integrin ⁇ IIb ⁇ 3 and its ligands.
  • ⁇ IIb ⁇ 3 receptor antagonist drugs block bidirectional signal transduction by preventing integrin ⁇ IIb ⁇ 3 from binding to its ligand, that is, it exerts an antithrombotic effect and affects the normal hemostatic function.
  • the choice of clinical antithrombotic drug dose should also take into account the risk of bleeding side effects, which makes it difficult to achieve better antithrombotic effect by increasing the dose of antithrombotic drugs.
  • Integrin ⁇ 3/Src interaction plays an important role in platelet out-in signal transduction.
  • the object of the present invention is to provide the use of a c-Src SH3 RT-loop antagonist (antagonist) in antithrombotic aspects.
  • a first aspect of the present invention provides the use of a c-Src SH3 RT-loop antagonist (antagonist) for the preparation of a composition or formulation for:
  • the c-Src is human (including human) c-Src.
  • the c-Src SH3 RT-loop is a human (including human) c-Src SH3 RT-loop.
  • the "interfering with the interaction between integrin ⁇ 3 and c-Src" is selected from the following group:
  • the integrin ⁇ 3 includes integrin ⁇ IIb ⁇ 3.
  • the antagonist is a c-Src SH3 RT-loop region specific antagonist.
  • the "c-Src SH3 RT-loop region-specific antagonist" refers to that the antagonist antagonizes (or affects) the binding of integrin ⁇ 3 to the RT-loop region of c-Src SH3, However, it does not antagonize (or affect) or substantially does not antagonize the binding of integrin ⁇ 3 to the n-Src loop region of c-Src SH3.
  • the c-Src SH3 RT-loop antagonist does not antagonize (or affect) or substantially does not affect the binding (or interaction) of integrin ⁇ 3 with the c-Src SH3 n-loop region.
  • the KD value of the dissociation constant (referred to as KD R95A ) for the interaction of the antagonist with the R95A mutant c-Src protein is the same as that of the antagonist and the wild-type c-Src protein
  • the ratio (KD R95A /KD wt ) of the dissociation constant Kd values (denoted as KD wt ) at which the interaction occurs, is > 5, preferably > 10, more preferably > 20, most preferably > 40.
  • the KD value of the dissociation constant (referred to as KD E97A ) for the interaction between the antagonist and the E97A mutant c-Src protein is the same as that of the antagonist and the wild-type c-Src protein.
  • the amino acid sequence of the R95A mutant c-Src protein is shown in SEQ ID No: 1, and the R at position 98 is mutated to A.
  • amino acid sequence of the E97A mutant c-Src protein is shown in SEQ ID No: 1, and the E at position 100 is mutated to A.
  • the c-Src SH3 RT-loop antagonist is a dual antagonist of RT-loop region and n-Src loop region.
  • the c-Src SH3 RT-loop antagonist comprises antagonizing the amino acid at position R95 and/or E97.
  • the antagonist is selected from the group consisting of small molecule antagonists, antisense nucleotides, miRNA, siRNA, or a combination thereof.
  • the antagonist includes: DCDBS84 or a pharmaceutically acceptable salt thereof:
  • the antagonist is a structural derivative of DCDBS84, or other small molecule candidate compounds targeting c-Src SH3.
  • the c-Src protein is mammalian c-Src protein, preferably human and rodent c-Src protein, more preferably human or mouse c-Src protein.
  • the RT-loop region of the c-Src SH3 domain is RT-loop region.
  • the RT-loop region of the c-Src SH3 domain or its encoding gene is derived from mammals (including human and mouse).
  • the c-Src protein is selected from the following group:
  • (B) pass the amino acid sequence shown in SEQ ID NO.: 1 through one or several (usually 1-60, preferably 1-30, more preferably 1-20, optimally 1-5 (a) c-Src protein derivatives formed by substitution, deletion or addition of amino acid residues, or active fragments thereof;
  • the c-Src SH3 domain is selected from the following group:
  • (B) pass the amino acid sequence shown in SEQ ID NO.: 2 through one or several (usually 1-10, preferably 1-5, more preferably 1-3, optimally 1-2 (a) c-Src SH3 domain derivatives formed by substitution, deletion or addition of amino acid residues, or active fragments thereof;
  • the c-Src SH3 RT-loop is selected from the following group:
  • (B) pass the amino acid sequence shown in SEQ ID NO.: 3 through one or several (usually 1-5, preferably 1-3, more preferably 1-2, optimally 1) RT-loop region derivatives formed by substitution, deletion or addition of amino acid residues;
  • (C) A polypeptide whose amino acid sequence is shown in SEQ ID NO.: 3 and has a mutation selected from the group consisting of R95A, E97A.
  • the antagonist or composition or formulation does not increase or substantially does not increase the risk of bleeding (or "improve bleeding").
  • the thrombus includes cardiovascular and cerebrovascular disease thrombus; more preferably, the thrombus is a cardiovascular and cerebrovascular disease thrombus selected from the group consisting of myocardial infarction thrombus, cerebral infarction thrombus, ischemic Stroke, atherothrombotic, or a combination thereof.
  • the prevention and/or treatment of thrombus does not affect or improve bleeding while achieving antithrombotic effect.
  • the improving bleeding includes inhibiting bleeding, not increasing bleeding risk, reducing bleeding risk, not causing bleeding side effects and/or not affecting hemostatic function.
  • the hemostatic function includes platelet hemostatic function.
  • the hemostasis includes physiological hemostasis.
  • the inhibiting platelet aggregation comprises inhibiting the two-phase aggregation of platelets.
  • the inhibition of platelet aggregation includes not inhibiting platelet aggregation in one phase.
  • the composition includes a pharmaceutical composition.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and a safe and effective amount of the antagonist.
  • the dosage form of the composition is selected from the following group: solid dosage form, liquid dosage form, and semi-solid dosage form.
  • composition is selected from the group consisting of oral preparations and injections.
  • the dosage form of the composition or preparation is selected from the group consisting of tablets, granules, capsules, injections, infusions, ointments, gels, solutions, microspheres or films.
  • composition or preparation further includes other antithrombotic drugs (eg aspirin).
  • other antithrombotic drugs eg aspirin.
  • the additional antithrombotic drug is selected from the group consisting of aspirin, clopidogrel, eptifibatide, Xuesaitong, Ginkgo biloba, or a combination thereof.
  • the second aspect of the present invention provides the use of a c-Src SH3 RT-loop agonist (agonist) for the preparation of a composition or formulation for use in
  • a third aspect of the present invention provides a method for interfering with the interaction between integrin ⁇ 3 and c-Src protein, comprising the steps of:
  • the method is an in vitro method.
  • step (a) in the presence of c-Src SH3 RT-loop antagonist, cells expressing integrin ⁇ 3 and c-Src protein are cultured, and the binding of integrin ⁇ 3 and c-Src protein is determined condition.
  • the c-Src protein includes wild-type c-Src protein and mutant c-Src protein.
  • the mutant c-Src protein includes: R95A mutant c-Src protein, E97A mutant c-Src protein, or a combination thereof.
  • a fourth aspect of the present invention provides a method for antithrombotic (or inhibiting platelet aggregation and/or adhesion), the method comprising the step of administering a c-Src SH3 RT-loop antagonist to a subject in need thereof.
  • the subject is human and non-human mammals (rodents, rabbits, monkeys, livestock, dogs, cats, etc.).
  • a fifth aspect of the present invention provides a method for screening candidate antithrombotic compounds, the method comprising the steps of:
  • test substance is a candidate compound for antithrombosis .
  • the candidate compound is a c-Src SH3 RT-loop antagonist.
  • the drug to be tested is a compound, a protein drug or a gene drug.
  • step (a) the test is performed in a cell-free system.
  • step (a) the test is performed in a system with cells, wherein the cells express integrin ⁇ 3 and c-Src protein.
  • the cells are platelets.
  • the sixth aspect of the present invention provides a method for screening antithrombotic candidate compounds, the method comprising the steps of:
  • the test substance is a candidate compound for antithrombosis
  • the candidate compound is a c-Src SH3 RT-loop antagonist.
  • the method further includes:
  • the RLP1 polypeptide and the c-Src protein are contacted, and the number of the second complex formed by the RLP1 polypeptide and the c-Src protein in the control group is observed;
  • the candidate compound is a c-Src SH3 RT-loop specific antagonist (ie, with RT- The loop area is mainly used, and basically has no effect with the n-loop area);
  • the candidate compound is a dual antagonist of c-Src SH3 RT-loop and n-loop region ( That is, it works with both RT-loop area and n-loop area).
  • the "significantly lower” refers to the number of complexes or the degree of binding or the number of bindings in the test group (denoted as C1) and the number of complexes or the degree of binding or the number of bindings in the control group ( The ratio (C1/C0) denoted as C0) ⁇ 1/2, preferably ⁇ 1/3, more preferably ⁇ 1/4, most preferably ⁇ 1/5.
  • the "equivalent” refers to the number of complexes or the degree of binding or the number of bindings in the test group (denoted as C1) and the number of complexes or the degree of binding or the amount of binding in the control group (denoted as C1)
  • the ratio (C1/C0) of C0) is 0.8-1.2.
  • a seventh aspect of the present invention provides a c-Src mutein, the mutein has an amino acid mutation at one or more positions selected from the group consisting of positions 95, 96, 97, 98, 99, 100, or Combinations thereof, wherein the numbering of amino acid positions is based on SEQ ID No: 1.
  • the mutein has an amino acid mutation at a position selected from the group consisting of position 95, position 97, or a combination thereof.
  • the c-Src mutein has a mutant amino acid mutation selected from the group consisting of R95A, E97A, or a combination thereof.
  • the c-Src mutein further has an amino acid mutation at a position selected from the group consisting of position 116, position 118, position 131, or a combination thereof.
  • the c-Src mutein has a mutant amino acid mutation selected from the group consisting of G116A, W118A and Y131A.
  • the eighth aspect of the present invention provides a polynucleotide encoding the c-Src mutein according to the seventh aspect of the present invention.
  • the ninth aspect of the present invention provides a vector containing the polynucleotide according to the eighth aspect of the present invention.
  • the tenth aspect of the present invention provides a host cell containing the vector according to the ninth aspect of the present invention, or a combination of the polynucleotides according to the eighth aspect of the present invention in the genome.
  • the eleventh aspect of the present invention provides a detection kit, the kit includes:
  • the detection reagent includes a reagent for detecting the amount of c-Src protein or mRNA.
  • the detection reagent includes a reagent for detecting whether there is an amino acid mutation or nucleotide mutation in the RT-loop region.
  • the detection reagent detects whether there is an amino acid mutation at positions 95, 96, 97, 98, 99 and 100 of the c-Src protein, and/or whether there is a nucleoside corresponding to the amino acid mutation acid mutation.
  • the amino acid mutation includes: R95A, E97A, or a combination thereof
  • a twelfth aspect of the present invention provides the use of the detection kit according to the eleventh aspect of the present invention, for preparing a diagnostic kit for evaluating whether a test object (such as a thrombus patient) is suitable for use c-Src SH3 RT-loop antagonist.
  • the diagnostic kit is also used to assess the risk of thrombus in the test subject.
  • Figure 1 shows a structural mimic diagram of the binding of RGT peptide, NITYRGT peptide, and PXXP domain-containing canonical binding peptide (APPIPPPR) to the c-Src SH3 domain.
  • APPIPPPR PXXP domain-containing canonical binding peptide
  • Figure 2 shows the amino acid sequence of the c-Src SH3 domain and the corresponding secondary structure pattern.
  • Figure 3 shows that c-Src SH3 mutants (R95A, E97A, G116A, W118A and Y131A) were transfected in 293T ⁇ 3 cells (293T cells were transfected with integrin ⁇ 3) using Co-IP to detect the relationship between each mutant and ⁇ 3 differences in interactions.
  • Figure 4 shows the binding of RLP1 polypeptide (containing PXXP domain, binding to c-Src SH3 is considered canonical binding) to c-Src SH3 mutants (R95A, E97A, G116A, W118A and Y131A).
  • Figure 5 shows the structural simulation of the binding of RGT peptide, the designed and synthesized small molecule compound DCDBS84, and the canonical binding peptide containing PXXP domain (APPIPPPR) to the c-Src SH3 domain.
  • APPIPPPR canonical binding peptide containing PXXP domain
  • Figure 6 shows nuclear magnetic resonance experiments to detect the binding site of DCDBS84 in the c-Src SH3 domain.
  • FIG. 7 shows a map of the binding site according to the chemical shift interference (CSP) analysis of amino acid sites based on the results of NMR experiments.
  • CSP chemical shift interference
  • Figure 8 shows the binding constants of DCDBS84 to c-Src SH3 (WT) and each mutant (R95A, E97A, G116A, W118A and Y131A) detected using surface plasmon resonance (SPR).
  • Figure 9 shows the RLP1 peptide (containing PXXP domain, binding to c-Src SH3 is considered to be canonical binding) c-Src SH3 (WT) and each mutant (R95A, E97A, G116A, W118A and Y131A) detected by SPR binding constant.
  • Figure 10 shows a diagram of the overall strategy for gene targeting to construct c-Src E97A transgenic mice.
  • Figure 11 shows the results of genotype identification of c-Src E97A transgenic mice.
  • Figure 12 shows that c-Src E97A transgenic mice can dissociate the interaction between integrin ⁇ 3 and c-Src using Co-IP method.
  • Figure 13 shows that c-Src E97A transgenic mice are able to reduce Thrombin-induced platelet aggregation.
  • Figure 14 shows a statistical graph of the ability of c-Src E97A transgenic mice to reduce Thrombin-induced platelet aggregation.
  • Figure 15 shows that c-Src E97A transgenic mice are able to reduce platelet spread on solid phase fibrinogen.
  • Figure 16 shows the result that c-Src E97A transgenic mice can reduce platelet adhesion on solid phase fibrinogen.
  • Figure 17 shows FeCl 3 model of carotid artery injury induced, compared to control mice and the WT, c-Src E97A transgenic mice could inhibit thrombosis.
  • Figure 18 shows that c-Src E97A transgenic mice did not increase mouse bleeding time compared to WT control mice in a mouse tail clipping experiment.
  • the inventors unexpectedly discovered for the first time that the inhibition of the RT-loop region target of the c-Src SH3 domain can effectively treat thrombosis without increasing the risk of bleeding.
  • the present invention synthesized a PXXP-containing polypeptide RLP1 (RKLPPRPSK), and detected RLP1 and c-Src SH3 wild type respectively.
  • the interaction with each mutant showed that the binding site of RLP1 was inclined to W118, and this region was mainly the region where the n-Src loop of c-Src SH3 was located.
  • the small molecule DCDBS84 targeting c-Src SH3 was screened experimentally, and whether the direct action target of DCDBS84 on c-Src SH3 was differentiated from the classically bound target.
  • the main binding sites of DCDBS84 and c-Src SH3 were R95, E97, W118, W119 and Y131 by nuclear magnetic resonance (NMR) and chemical shift analysis.
  • the present invention also uses SPR to detect the binding constants of the classical binding peptide RLP1 and c-Src SH3 mutants R95A, E97A, G116A, W118A, Y131A and wild type (WT), the results found that the G116A, W118A and Y131A mutations were significantly weakened
  • the binding of RLP1 to c-Src SH3 suggested that G116, W118 and Y131 were mainly involved in the binding of RLP1 to c-Src SH3.
  • This experiment further confirmed that the binding site of small molecule DCDBS84 in c-Src SH3 was mainly located in RT-loop, and E97 was the main target.
  • the structural simulation of the binding site of DCDBS84 and c-Src SH3 also suggested that it is mainly composed of E97 and its surrounding RT-loop amino acids.
  • c-Src E97A transgenic mice were constructed in experiments, genotype identification confirmed WT mice and c-Src E97A mutant mice, platelets were isolated from mice, and Co-IP experiments confirmed that c-Src E97A mutant mice were able to dissociate platelets
  • the interaction of ⁇ 3/c-Src in ⁇ 3/c-Src inhibits platelet aggregation, extension, adhesion and other functions mediated by platelet "outside-in" signal transduction.
  • c-Src E97A mutant mouse model inhibit thrombosis induced by FeCl 3 can be formed thrombus, and does not increase bleeding time compared to wild-type mice in the tail cut experiment bleeding.
  • the results showed that the c-Src SH3 RT-loop and the amino acid sites dominated by E97 can become new antithrombotic targets that do not affect the normal hemostatic function, and provide target information for the research and development of new antithrombotic drugs.
  • the terms “comprising,” “including,” and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • antithrombotic includes the prevention and/or treatment of blood clots.
  • prevention refers to a method of preventing the onset of a disease and/or its attendant symptoms or protecting a subject from acquiring a disease. "Prevention” as used herein also includes delaying the onset of the disease and/or its attendant symptoms and reducing the risk of the disease in a subject.
  • Treatment includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • the compositions or pharmaceutical compositions of the present invention inhibit by inhibiting the levels observed in the absence of the compositions, kits, food kits or nutraceutical kits, combinations of active ingredients Mitochondrial oxidative phosphorylation pathways reduce, inhibit and/or reverse associated diseases (eg, tumors) and their complications, eg, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.
  • the Src kinase family is a protein with protein tyrosine kinase (PTK) activity, of which c-Src is an important part of the Src kinase family.
  • PTK protein tyrosine kinase
  • amino acid sequences are numbered in order from N-terminal to C-terminal.
  • amino acid sequence of human c-Src is shown in SEQ ID NO.: 1:
  • SH3 As used herein, “SH3”, “SH3 domain”, “SH3 domain protein”, “SH3 protein” are used interchangeably.
  • amino acid sequence of a representative wild-type human c-Src SH3 domain is shown in SEQ ID NO.:2:
  • R95A, E97A mutant As used herein, in human c-Src, in the c-Src protein of the R95A, E97A mutant, R95A, E97A are based on the numbering of the following SH3 domain amino acids (SEQ ID NO.: 2), positions 84 to Bit 141 looks like this:
  • R95A refers to the mutation of amino acid residue 95 in amino acid numbering from R amino acid to A amino acid
  • E97A refers to the mutation of amino acid residue 97 in amino acid numbering from E amino acid to A amino acid
  • other sites The amino acid mutations of are as described above.
  • c-Src SH3 RT-loop is used interchangeably with "RT-loop region of the c-Src SH3 domain".
  • the human RT-loop region is located in the human c-Src SH3 domain, and the amino acid sequence is as follows: YDYESRTETDL (SEQ ID No: 3)
  • Integrin ⁇ IIb ⁇ 3 is a transmembrane heterodimer composed of ⁇ IIb and ⁇ 3 subunits through non-covalent bonds. It is mainly expressed on the surface of platelets and megakaryocytes. It is the main membrane receptor on the surface of platelets and can mediate platelet bidirectional signaling Transduction, therefore, plays a key role in platelet activation, maintenance of normal platelet function, and thrombosis. Platelet activators such as thrombin, ADP, etc. interact with the corresponding receptors to cause a configuration change of integrin ⁇ IIb ⁇ 3 and increase the affinity of its ligands, such as soluble fibrinogen. This process is an inside-out signal transduction, a landmark event.
  • Integrin ⁇ IIb ⁇ 3 As the final common pathway mediating platelet activation, aggregation and thrombus formation, is the main target of antithrombotic drug research. In fact, important progress has been made in the research on integrin ⁇ IIb ⁇ 3 as an antithrombotic drug target. Currently, it is mainly focused on integrin ⁇ IIb ⁇ 3 receptor antagonist drugs, which have obtained good clinical effects. Currently, there are three integrin ⁇ IIb ⁇ 3 receptor antagonist antiplatelet drugs approved by the U.S.
  • FDA Food and Drug Administration
  • ⁇ IIb ⁇ 3 receptor antagonists specifically exert antithrombotic effects by interfering with the interaction of integrin ⁇ IIb ⁇ 3 and its ligands.
  • ⁇ IIb ⁇ 3 receptor antagonist drugs block the bidirectional signal transduction by preventing the integrin ⁇ IIb ⁇ 3 from binding to its ligand, that is, it exerts an antithrombotic effect and affects the normal hemostatic function.
  • the choice of clinical antithrombotic drug dose should also take into account the risk of bleeding side effects, which makes it difficult to achieve better antithrombotic effect by increasing the dose of antithrombotic drugs.
  • the integrin ⁇ IIb ⁇ 3 cytoplasmic tail interacts with the cytoplasmic protein c-Src, and has functions related to platelet "outside-in” signaling, such as stable platelet adhesion and extension on solid-phase fibrinogen, biphasic aggregation and fiber. It plays an important role in protein clot retraction, but has little effect on platelet "inside-out” functions such as platelet binding to soluble fibrinogen, initial platelet adhesion, and first-phase aggregation. Studies have shown that the "outside-in” signal of platelets is mainly involved in the process of platelet thrombus formation, while “inside-out” plays a greater role in hemostasis.
  • Src kinase forms a constitutive binding to the three amino acids of RGT at the C-terminus of integrin ⁇ 3.
  • Src kinase interacts with the RGT sequence at the C-terminus of ⁇ 3, and phosphorylation of ⁇ 3 cytoplasmic segments Y747 and Y759 occurs, becoming an important event in outward-in signal transduction.
  • the present invention provides the use of a c-Src SH3 RT-loop antagonist (antagonist), and the c-Src SH3 RT-loop antagonist includes (but is not limited to) one or more uses of the following group:
  • integrin ⁇ 3 includes (but is not limited to) integrin ⁇ IIb ⁇ 3
  • the c-Src SH3 RT-loop antagonist is a c-Src SH3 RT-loop region specific antagonist.
  • the c-Src SH3 RT-loop antagonist does not antagonize (or affect) or substantially does not affect the binding (or interaction) of integrin ⁇ 3 with the c-Src SH3 n-loop region.
  • c-Src SH3 RT-loop antagonists described in the present invention are not particularly limited, as long as they can have an antagonistic effect on c-Src SH3 RT-loop.
  • the c-Src SH3 RT-loop antagonists can be small molecule antagonists, antisense nucleotides, miRNAs, siRNAs, and the like.
  • the antagonist is the DCDBS84 compound:
  • the c-Src protein is not particularly limited, and is preferably a c-Src protein of mammals such as humans and rodents.
  • the amino sequence of human c-Src protein is the polypeptide shown in SEQ ID NO.:1.
  • positions 87-144 (SEQ ID NO: 2 sequence) are human c-Src SH3 domains.
  • the amino sequence of human c-Src SH3 RT-loop is the polypeptide shown in SEQ ID NO.:3.
  • the c-Src SH3 RT-loop antagonist does not increase or substantially does not increase the risk of bleeding (or "improve bleeding") during antithrombotic treatment.
  • improving bleeding includes inhibiting bleeding, not increasing bleeding risk, reducing bleeding risk, not causing bleeding side effects and/or not affecting hemostatic function.
  • the thrombus includes cardiovascular and cerebrovascular disease thrombus.
  • the thrombus includes (but is not limited to): myocardial infarction thrombus, cerebral infarction thrombus, ischemic stroke, atherosclerotic thrombus, or a combination thereof.
  • compositions or formulations, combinations of active ingredients and kits and methods of administration are provided.
  • the present invention also provides a composition comprising a c-Src SH3 RT-loop antagonist.
  • composition of the present invention is preferably a pharmaceutical composition.
  • compositions of the present invention may include a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid, semi-solid, liquid or gel fillers which are suitable for human or animal use and which must be of sufficient purity and low enough toxicity. "Compatibility” refers to the components in the pharmaceutical composition and the active ingredients of the drug and their intermingling with each other without significantly reducing the efficacy of the drug.
  • the pharmaceutically acceptable carrier is not particularly limited, and materials commonly used in the art can be selected, or prepared by conventional methods, or purchased from the market.
  • pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, skin penetration enhancers, colorants, flavors, stabilizers, antioxidants, preservatives ,
  • cellulose and its derivatives such as
  • the dosage form of the composition is not particularly limited, and can be a solid dosage form, a liquid dosage form, or a semi-solid dosage form.
  • the dosage forms of the compositions and preparations include but are not limited to oral preparations, injection preparations, and external preparations.
  • the dosage forms of the compositions and preparations include, but are not limited to: tablets, granules, capsules, injections, infusions, ointments, gels, solutions, microspheres or films.
  • the injection is an intravenous injection.
  • the pharmaceutical preparation should match the mode of administration, and the preferred mode of administration is oral administration, injection administration (such as intravenous injection), and when used, a therapeutically effective amount of the drug is administered to the desired object (such as human or non-human). mammal).
  • the term "therapeutically effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount” may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs, etc. different.
  • a safe and effective daily dose of the first active ingredient will generally be at least about 0.1 mg, and in most cases will not exceed about 2500 mg.
  • the dose is 1 mg to 500 mg; a safe and effective amount of the second active ingredient is usually at least about 0.01 mg, and in most cases no more than 2500 mg.
  • the dose range is 0.1 mg to 2500 mg.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the present invention finds for the first time that inhibition of the RT-loop region target of the c-Src SH3 domain can effectively treat thrombosis without increasing the risk of bleeding.
  • amino acid sequence of mouse c-Src is shown in SEQ ID NO.: 4:
  • mice c-Src SH3 domain The amino acid sequence of mouse c-Src SH3 domain is shown in SEQ ID NO.:5:
  • mice c-Src SH3 domain containing RT-loop region is shown in SEQ ID NO.: 6:
  • R95, E97 are based on the numbering of the following SH3 domain amino acids (SEQ ID NO.: 5), positions 84 to 147 as follows:
  • RGT peptide crystal structure
  • NITYRGT peptide NMR structure
  • APPIPPPR classical binding peptide containing PXXP domain
  • the classical binding peptides of RGT, NITYRGT and PXXP domains were obtained by computer structural analysis (APPIPPPR) Structural simulation of binding to the c-Src SH3 domain.
  • APPIPPPR computer structural analysis
  • the binding direction of the classical binding peptide (APPIPPPR) to c-Src SH3 is basically perpendicular to the binding direction of NITYRGT to c-Src SH3, and it is more inclined to the n-Src loop. According to the amino acid sequence information and secondary structure information of c-Src SH3, the structural pattern of c-Src SH3 was drawn ( Figure 2).
  • a mutant of integrin ⁇ 3 at the possible binding site of c-Src SH3 was constructed, and co-immunoprecipitation (Co-IP) was used to detect the binding site of ⁇ 3 at c-Src SH3.
  • c-Src SH3 gene point mutation overexpression vectors were constructed, pFlag-CMV4-Src(R95A), pFlag-CMV4-Src(E97A), pFlag-CMV4-Src(G116A), pFlag-CMV4-Src(W118A) and pFlag-CMV4-Src(Y131A).
  • R95 and E97 are located in the RT-loop region
  • G116 and W118 are located in the N-Src loop region
  • Y131 is located in the ⁇ 4 region (as shown in Figure 2).
  • Co-IP co-immunoprecipitation
  • Anti-Flag-tagged antibody M2 (Sigma) 1 ⁇ g or non-specific mouse IgG (sc-2025, Santa Cruz Biotechnology, 1 ⁇ g) was added to the protein supernatant, and the antigen-antibody mixture was incubated overnight at 4°C with rotation. In the next morning, 20 ⁇ l of Protein A+G agarose beads pre-washed with lysis buffer were added, and incubated at 4° C. with rotation for 2 h.
  • Example 3 ELISA method detects the binding of RLP1 peptide to c-Src SH3 mutant
  • Ligand protein molecules that interact with Src kinases in cells generally target the Src-SH3 domain through a canonical (PXXP) motif.
  • the specific experimental procedure is as follows: 50 ⁇ L of coating solution containing Flag antibody M2 (Sigma) (1 ⁇ g/mL, diluted with 0.1M NaHCO3 pH 8.3) was added to each well of a 96-well plate, and incubated at 4°C overnight. On the second day, the cells were washed three times with 1 ⁇ TBST for 5 min each, blocked with 5% BSA for 2 h, and washed three times with 1 ⁇ TBST for use.
  • 293T cells were transfected with pFlag-CMV4-Src(WT) and other 5 Src mutant overexpression vectors pFlag-CMV4-Src(R95A), pFlag-CMV4-Src(E97A), pFlag-CMV4-Src( G116A), pFlag-CMV4-Src (W118A) and pFlag-CMV4-Src (Y131A) were transfected with lipofect 2000 (Invitrogen Life technologies), 48h after transfection, 293T cells were lysed on ice with RIPA for 30min, centrifuged (4°C).
  • the nuclear magnetic resonance spectrum of the amino acid site of the protein (c-Src SH3) before and after the addition of DCDBS84 was determined by nuclear magnetic resonance.
  • the small molecule DCDBS84 targeting c-Src SH3 was screened out by computer simulation screening and analysis based on various information such as the crystal structure of the binding of RGT peptide to c-Src SH3 and the nuclear magnetic resonance structure of NITYRGT and c-Src SH3.
  • the structural simulation of DCDBS84 binding to c-Src SH3 is shown in Figure 5. It can be seen that DCDBS84 mainly binds to the RT-loop of c-Src SH3.
  • NMR experiments were performed on a four-channel Bruker Avance III 600MHz spectrometer.
  • the medium was prepared with 15N instead of 14N to purify the c-Src-SH3 protein labeled with 15N for 2D NMR; when 3D NMR was required, 15N was required to replace 14N and 13C to replace 12C;
  • Figure 6 The two-dimensional 15N-HSQC experiment of the interaction between Src-SH3 and DCDBS84 shown in the left panel), the c-Src-SH3 concentration was 50 ⁇ M, and the DCDBS84 concentration was 20 times that; as shown in Figure 6 (right panel), the The concentration of 15N/13C-labeled c-Src-SH3 protein was adjusted to 1.3 mM to complete the assignment of the amino acid site of the protein.
  • the binding site map was analyzed based on chemical shift interference (CSP) of amino acid sites.
  • CSP chemical shift interference
  • CSP chemical shift perturbation
  • the dissociation constant KD of the classical binding peptide RLP1 and c-Src SH3 protein was detected according to the above method.
  • the dissociation constant of RLP1 with wild-type c-Src SH3 protein is 9.9 ⁇ M
  • the dissociation constant with R95A is 10.9 ⁇ M
  • the dissociation constant with E97A is 22 ⁇ M
  • the dissociation constant with G116A is >80 ⁇ M
  • the dissociation constant with W118A is 49.9 ⁇ M
  • the dissociation constant with Y131A is 85.9 ⁇ M.
  • G116 and W118 located in the n-Src loop and Y131 located in the distal loop are mainly involved in the binding of RLP1 to c-Src SH3.
  • c-Src E97A mutant transgenic mice were constructed by gene targeting.
  • the general strategy for constructing transgenic mice by gene targeting is shown in FIG. 10 .
  • a primer containing the corresponding site mutation (MusE99A) of the mouse gene corresponding to human E97A is designed, and the primer sequence is: c-Src E97A- F:5'-GAACACCTAGTCTGCAGCCC-3',c-Src E97A -R:5'-AGCAGAGAGAAGGAGAGG
  • the amplified fragment has a length of 419 bp (Fig. 11, upper figure).
  • the PCR product was sequenced and analyzed. If the position of the gene corresponding to the 99th amino acid in the mouse is GAG, it is glutamic acid (E), and if it is GCG, it is alanine (A). If there are two peaks of GAG and GCG, is a heterozygote. The results of gene sequencing identification are shown in the lower panel of Figure 11.
  • WT mice and three c-Src E97A transgenic mice were taken, and the mice were anesthetized with phenobarbital according to their body weight.
  • Sodium anticoagulation Sodium anticoagulation.
  • Platelet-rich plasma (PRP) was obtained by centrifugation at 300 ⁇ g for 7 min, 1/4 volume of ACD was added to the PRP seed for anticoagulation, centrifuged at 500 ⁇ g for 10 min, and the supernatant was discarded.
  • PRP Platelet-rich plasma
  • Platelets were washed with CGS wash solution (13mM sodium citrate, 120mM sodium chloride, 30mM glucose, pH 6.5) followed by Tyrode's buffer (0.1% bovine serum albumin, 5mM 4-hydroxyethylpiperazineethanesulfonic acid, Platelets were resuspended in 5.5 mM glucose, 137 mM sodium chloride, 2 mM potassium chloride, 12 mM sodium bicarbonate, 0.3 mM sodium dihydrogen phosphate, 1 mM calcium chloride, 1 mM magnesium chloride, pH 7.4). The platelets were counted by a small animal blood routine detector (PoCH-100iV Diff), the platelet density was adjusted to 3 ⁇ 10 8 /ml, and the cells were allowed to stand for 1 h at room temperature.
  • CGS wash solution 13mM sodium citrate, 120mM sodium chloride, 30mM glucose, pH 6.5
  • Tyrode's buffer 0.1% bovine serum albumin, 5mM 4-
  • the Co-IP method was used to detect the effect of DCDBS84, a small molecule compound, on the interaction between ⁇ 3 of integrin ⁇ IIb ⁇ 3 and c-Src.
  • IP buffer 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.2% NP-40
  • the supernatant was aspirated, and BCA protein was quantified.
  • Example 10 Effect of c-Src E97A transgenic mice on platelet aggregation.
  • Platelets were isolated from 3 each of wild-type (WT) mice and 3 c-Src E97A transgenic mice as described in Example 9. PRP was obtained by centrifugation at 300 ⁇ g for 7 min, and then centrifuged at 500 ⁇ g for 10 min to obtain platelet poor plasma (PPP). The platelet concentration in PRP was adjusted to 2 ⁇ 10 8 / ml with PPP. An additional 200 ⁇ l of PPP was taken to correct the zero point of the instrument Chrono-Log.
  • each group of reagents was incubated with PRP at 37°C for 60min, 200 ⁇ l/tube was placed on the aggregator (37°C, stirring at 1000rpm), 0.1U/ml thrombin (Thrombin) was added to start the reaction after zero point correction, and the aggregation curve was recorded.
  • Thrombin 0.1U/ml thrombin
  • Example 11 Effects of c-Src E97A transgenic mice on platelet extension and adhesion.
  • Platelets were isolated from 3 each of wild-type (WT) mice and 3 c-Src E97A transgenic mice as described in Example 9. Platelet concentration was adjusted to 2 ⁇ 10 8 / ml.
  • the platelet membrane was then punched with 0.5% Triton X-100, and then stained with 0.5 ⁇ g/ml phalloidin-rhodamine for 60 min at 37°C, and washed 3 times with 1 ⁇ PBS (10 min each time). . After washing, the fluorescence color was observed with a fluorescence microscope (Leica). Results As shown in Fig. 15, the platelets of c-Src E97A transgenic mice were significantly weaker than WT mice to stretch on solid-phase fibrinogen.
  • Example 12 c-Src E97A transgenic mice with thrombotic occlusion of the carotid arteries induced by FeCl 3.
  • WT and c-Src E97A transgenic mice aged 6-8 weeks were selected as experimental subjects.
  • the mouse carotid artery was stimulated with FeCl 3 , which resulted in endothelial damage, and then started the process of thrombus formation.
  • the blood flow was detected with a Doppler ultrasound probe at the distal end of the FeCl 3 stimulation. When the upstream thrombus formed When the blood vessels are blocked, the blood flow decreases.
  • Example 13 Effects of c-Src E97A transgenic mice on bleeding time detected by tail clipping.
  • the tail clipping experiment is a process in which platelets maintain normal hemostatic function and block the vascular wound after the blood vessel is damaged.
  • the mouse tail was quickly cut off with a sharp blade at a distance of 5mm from the tip of the tail (tail-cut), and then every 15 sec, filter paper was used to dip the blood exuded from the mouse tail, and do not touch the mouse tail to avoid new damage. Stop bleeding and do not recur within 15sec as the timing standard.
  • the c-Src SH3 RT-loop antagonist described in the present application can reduce, inhibit or interfere with the interaction between integrin ⁇ 3 and c-Src, thereby significantly exerting antithrombotic effect without affecting the normal physiological hemostatic effect.

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Abstract

本发明涉及一种c-Src SH3 RT-loop作为靶点用于抗血栓。具体地，本发明提供一种c-Src SH3 RT-loop拮抗剂的用途，用于制备组合物或制剂，所述组合物或制剂用于：(a)干扰整合素β3和c-Src的相互作用；(b)抑制血小板在固相纤维蛋白原上的伸展；(c)抑制血小板的聚集和/或粘附；和/或(d)预防和/或治疗血栓。

Description

一种c-Src SH3 RT-loop作为靶点用于抗血栓 技术领域
本发明属于分子生物学和生物医药领域，具体地，本发明涉及c-Src SH3 RT-loop作为靶点用于抗血栓。
背景技术
心脑血管血栓性疾病如心肌梗塞和脑梗塞等严重影响着人类的生命和健康。而血小板参与的血栓形成在心脑血管血栓性疾病的发生发展中发挥重要作用，当血管内皮损伤或动脉粥样硬化斑块破裂时，血小板活化并通过黏附、伸展、聚集等一系列功能导致病理性血栓形成，引起受累血管分布区域的心、脑组织缺血性坏死，严重可危及患者的生命。因此，抗血小板治疗成为心脑血管血栓性疾病治疗的首选。
经典的抗血小板药物包括环氧化酶(COX)抑制剂阿司匹林，是目前在抗血小板治疗中研究和应用最广泛的抗血小板药物，主要通过抑制花生四烯酸环氧酶(COX)，使Ser-529和Ser-516不可逆的乙酰化，从而阻断TXA2的合成，发挥抗血小板的作用。阿司匹林常见的不良反应是胃肠道不适和消化道出血，出血风险与剂量相关。为避免出血副作用，不能使用很高的抗血栓剂量。另一类经典的抗血小板药物是二磷酸腺苷(ADP)P2Y12受体拮抗剂，包括噻吩吡啶类，代表性药物有噻氯匹定(Ticlopidine)，氯吡格雷(Clopidogrel)和普拉格雷(Prasugrel)。非噻吩吡啶类，代表性药物有替卡格雷(Ticagrelor)和坎格雷洛(Cangrelor)。其中，第二代P2Y12受体拮抗剂氯吡格雷使用较广泛，能够不可逆地抑制血小板ADP受体，从而抑制活化血小板释放ADP所诱导的血小板聚集，出血仍然是其主要的副作用。由此可见，经典的抗血小板药物虽然能够发挥较好的抗血栓作用，但由于出血副作用的限制，不能使用足够的剂量发挥深度抗血栓作用。
为了能够更加特异性的靶向血小板参与血栓形成的受体，人们研发了靶向整合素αIIbβ3的受体拮抗剂。整合素αIIbβ3作为介导血小板活化聚集、血栓形成的最终共同通路，是抗血栓药物研究的主要靶点。而事实上，以整合素αIIbβ3为抗血栓药物靶点的研究取得了重要的进展，目前主要集中于整合素αIIbβ3受体拮抗剂类药物，已获得很好的临床疗效。目前，经美国食品药物监督管理局 (FDA)批准用于临床抗血栓治疗的整合素αIIbβ3受体拮抗剂类抗血小板药物有三种，即阿昔单抗、埃替巴肽、替罗非班。此类αIIbβ3受体拮抗剂通过干扰整合素αIIbβ3与其配体的相互作用而特异性地发挥抗血栓作用。但这一策略仍存在明显问题，αIIbβ3受体拮抗剂药物通过阻止整合素αIIbβ3结合其配体从而阻断双向信号转导，也就是在发挥抗血栓作用的同时影响了正常的止血功能。临床实验回顾统计显示，经整合素αIIbβ3拮抗剂类药物治疗的病人中约有2％发生严重的颅内出血，约有15％发生胃肠道出血，约有5-10％出现腹膜出血，另大约有60-80％受试患者于股动脉穿刺点出现明显出血。与经典的抗血栓药物阿司匹林、氯吡格雷一样，整合素αIIbβ3拮抗剂在发挥有效抗血栓作用的同时也导致患者出血风险的增加，这是目前抗血栓药物最常见也是最重要的副作用。所以，临床上抗血栓药物剂量的选择还要兼顾其出血副作用的风险，致使目前难以通过增加抗血栓药物剂量来达到更好的抗血栓效果。在以死亡作为终点事件的研究中，难以找到一个合适的剂量阈值来减少由于血栓死亡和由于出血死亡的死亡率。因此，通过开发新一代不影响正常止血功能的抗血栓药物，将有可能在低风险的前提下获得更强的抗血栓疗效，这代表了抗血栓药物的发展方向。
靶向血小板外向内信号而非抑制整合素αIIbβ3完整受体的双向信号转导功能，将抗血栓作用及正常止血功能加以区分，将有可能实现在深度抗血栓的同时不增加出血风险。整合素β3/Src相互作用在血小板外向内信号转导中发挥重要作用。通过设计能够靶向β3/Src相互作用的多肽或者小分子，特异性解离β3/Src相互作用，能够实现上述目的。既往研究发现合成的整合素β3胞浆尾端的RGT三肽能够特异性抑制血小板外向内信号及相关的血小板功能；整合素β3胞浆尾端的RGT三肽基因敲除小鼠也能因破坏了β3/Src的相互作用而发挥对血小板外向内信号及相关功能的抑制作用。既然可以通过整合素β3的模拟肽实现对血小板外向内信号的抑制，那么，能否通过靶向c-Src上能够与β3发生相互作用的序列而实现新型小分子药物的研发呢？已有研究发现c-Src SH3结构域能够与整合素β3发生相互作用，但是具体的作用位点尚不清楚。本发明通过Co-IP以及表面等离子共振(SPR)等技术手段，发现c-Src SH3的RT loop具有结合整合素β3的倾向性，而n-Src loop对经典结合具有倾向性，经典结合参与c-Src的激酶活性和其他功能。因此，以c-Src SH3的RT loop为靶点设计新型抗血栓药物，有望实现深度抗血栓的同时几乎不影响正常止血功能以及 c-Src的活性和功能。因此，本领域需要开发一种特异性的靶点用于血栓的预防或治疗。
发明内容
本发明的目的在于提供一种c-Src SH3 RT-loop拮抗剂(antagonist)在抗血栓方面中的用途。
本发明第一方面，提供一种c-Src SH3 RT-loop拮抗剂(antagonist)的用途，用于制备组合物或制剂，所述组合物或制剂用于：
(a)干扰整合素β3和c-Src的相互作用；
(b)抑制血小板在固相纤维蛋白原上的伸展；
(c)抑制血小板的聚集和/或粘附；和/或
(d)预防和/或治疗血栓。
在另一优选例中，所述的c-Src为人源(包括人)c-Src。
在另一优选例中，所述的c-Src SH3 RT-loop为人源(包括人)c-Src SH3 RT-loop。
在另一优选例中，所述的“干扰整合素β3和c-Src的相互作用”选自下组：
(a1)减少整合素β3和c-Src SH3的RT-loop区发生结合；
(a2)阻断整合素β3和c-Src SH3的RT-loop区发生结合。
在另一优选例中，所述的整合素β3包括整合素αIIbβ3。
在另一优选例中，所述的拮抗剂为c-Src SH3 RT-loop区特异性拮抗剂。
在另一优选例中，所述的“c-Src SH3 RT-loop区特异性拮抗剂”指所述拮抗剂拮抗(或影响)整合素β3和c-Src SH3的RT-loop区的结合，但是不拮抗(或影响)或基本上不拮抗整合素β3和c-Src SH3的n-Src loop区的结合。
在另一优选例中，所述的c-Src SH3 RT-loop拮抗剂不拮抗(或影响)或基本不影响整合素β3与c-Src SH3 n-loop区的结合(或相互作用)。
在另一优选例中，所述的拮抗剂与R95A突变型的c-Src蛋白发生相互作用的解离常数KD值(记为KD R95A)，与所述的拮抗剂与野生型c-Src蛋白发生相互作用的解离常数Kd值(记为KD wt)的比值(KD R95A/KD wt)，≥5，较佳地≥10，更佳地≥20，最佳地≥40。
在另一优选例中，所述的拮抗剂与E97A突变型的c-Src蛋白发生相互作用的 解离常数KD值(记为KD E97A)，与所述的拮抗剂与野生型c-Src蛋白发生相互作用的解离常数KD值(记为KD wt)的比值(KD E97A/KD wt)，≥5，较佳地≥10，更佳地≥20，最佳地≥40。
在另一优选例中，所述的R95A突变型的c-Src蛋白的氨基酸序列如SEQ ID No:1所示，并且第98位的R突变为A。
在另一优选例中，所述的E97A突变型的c-Src蛋白的氨基酸序列如SEQ ID No:1所示，并且第100位的E突变为A。
在另一优选例中，所述的c-Src SH3 RT-loop拮抗剂为RT-loop区和n-Src loop区双重拮抗剂。
在另一优选例中，所述的c-Src SH3 RT-loop拮抗剂包括对R95和/或E97位氨基酸进行拮抗。
在另一优选例中，所述的拮抗剂选自下组：小分子拮抗剂、反义核苷酸、miRNA、siRNA、或其组合。
在另一优选例中，所述的拮抗剂包括：DCDBS84或其药学上可接受的盐：
Figure PCTCN2021108371-appb-000001
在另一优选例中，所述的拮抗剂为DCDBS84的结构衍生物，或者其他以c-Src SH3为靶点的小分子候选化合物。
在另一优选例中，所述的c-Src蛋白为哺乳动物的c-Src蛋白，较佳地人和啮齿动物的c-Src蛋白，更佳地为人或小鼠的c-Src蛋白。
在另一优选例中，所述c-Src SH3结构域的RT-loop区域RT-loop区域。
在另一优选例中，所述c-Src SH3结构域的RT-loop区域或其编码基因来源于哺乳动物(包括人、鼠)。
在另一优选例中，所述的c-Src蛋白选自下组：
(A)氨基酸序列如SEQ ID NO.:1所示的多肽；
(B)将SEQ ID NO.:1所示的氨基酸序列经过一个或几个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-5个)氨基酸残基的取代、缺失或添加而形 成的c-Src蛋白衍生物，或其活性片段；
(C)序列与SEQ ID NO.:1所示的氨基酸序列相比，同源性≥90％，较佳地≥95％，更佳地≥98％，最佳地≥99％的c-Src蛋白衍生物，或其活性片段。
在另一优选例中，所述的c-Src SH3结构域选自下组：
(A)氨基酸序列如SEQ ID NO.:2所示的多肽；
(B)将SEQ ID NO.:2所示的氨基酸序列经过一个或几个(通常为1-10个，较佳地1-5个，更佳地1-3个，最佳地1-2个)氨基酸残基的取代、缺失或添加而形成的c-Src SH3结构域衍生物，或其活性片段；
(C)序列与SEQ ID NO.:2所示的氨基酸序列相比，同源性≥90％，较佳地≥95％，更佳地≥98％，最佳地≥99％的c-Src SH3结构域衍生物，或其活性片段。
在另一优选例中，所述的c-Src SH3 RT-loop选自下组：
(A)氨基酸序列如SEQ ID NO.:3所示的多肽；
(B)将SEQ ID NO.:3所示的氨基酸序列经过一个或几个(通常为1-5个，较佳地1-3个，更佳地1-2个，最佳地1个)氨基酸残基的取代、缺失或添加而形成的RT-loop区衍生物；
(C)氨基酸序列如SEQ ID NO.:3所示且具有选自下组突变的多肽：R95A、E97A。
在另一优选例中，所述的拮抗剂或组合物或制剂不增加或基本上不增加出血风险(或称为“改善出血”)。
在另一优选例中，所述的血栓包括心脑血管性疾病血栓；更佳地，所述的血栓为心脑血管性疾病血栓选自下组：心肌梗塞血栓、脑梗塞血栓、缺血性卒中、动脉粥样硬化血栓，或其组合。
在另一优选例中，所述的预防和/或治疗血栓在实现抗血栓的同时，不影响出血或改善出血。
在另一优选例中，所述的改善出血包括抑制出血、不增加出血风险、降低出血风险、不会引起出血副作用和/或不影响止血功能。
在另一优选例中，所述的止血功能包括血小板止血功能。
在另一优选例中，所述的止血包括生理性止血。
在另一优选例中，所述抑制血小板的聚集包括抑制血小板的二相聚集。
在另一优选例中，所述抑制血小板的聚集包括不抑制血小板的一相聚集。
在另一优选例中，所述的组合物包括药物组合物。
在另一优选例中，所述的药物组合物还包括药学上可接受的载体和安全有效量的所述拮抗剂。
在另一优选例中，所述的组合物的剂型选自下组：固体剂型、液态制剂、半固态制剂。
在另一优选例中，所述的组合物选自下组：口服制剂、注射剂。
在另一优选例中，所述的组合物或制剂的剂型选自下组：片剂、颗粒剂、胶囊剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
在另一优选例中，所述的组合物或制剂还包括其它抗血栓药物(如阿司匹林)。
在另一优选例中，所述的额外的抗血栓的药物选自下组：阿司匹林、氯吡格雷、依替巴肽、血塞通、银杏叶片，或其组合。
本发明第二方面，提供一种c-Src SH3 RT-loop激动剂(agonist)的用途，用于制备组合物或制剂，所述组合物或制剂用于
(a)促进整合素β3和c-Src相互作用；
(b)促进血小板在固相纤维蛋白原上的伸展；
(c)促进血小板的聚集和粘附；和/或
(d)促进凝血。
本发明第三方面，提供一种干扰整合素β3和c-Src蛋白的相互作用的方法，包括步骤：
(a)在c-Src SH3 RT-loop拮抗剂存在下，使得整合素β3和c-Src蛋白接触，从而干扰整合素β3和c-Src蛋白的相互作用。
在另一优选例中，所述的方法为体外方法。
在另一优选例中，在步骤(a)中，c-Src SH3 RT-loop拮抗剂存在，培养表达整合素β3和c-Src蛋白的细胞，并测定整合素β3和c-Src蛋白的结合情况。
在另一优选例中，所述的c-Src蛋白包括野生型c-Src蛋白、突变型c-Src蛋白。
在另一优选例中，所述的突变型c-Src蛋白包括：R95A突变型的c-Src蛋白、E97A突变型的c-Src蛋白、或其组合。
本发明第四方面，提供一种抗血栓(或抑制血小板的聚集和/或粘附)的方法，所述方法包括步骤：给需要的对象施用c-Src SH3 RT-loop拮抗剂。
在另一优选例中，所述对象为人和非人哺乳动物(啮齿动物、兔、猴、家畜、狗、猫等)。
本发明第五方面，提供一种筛选抗血栓的候选化合物的方法，所述方法包括步骤：
(a)测试组中，在测试物存在下，使整合素β3和c-Src蛋白接触，并观察所述测试组中整合素β3是否与c-Src SH3结构域的RT-loop区形成结合；在对照组中，在所述测试物不存在下，使整合素β3和c-Src蛋白接触，并观察所述对照组中整合素β3是否与c-Src SH3结构域的RT-loop区形成结合；
其中，如果测试组中整合素β3是否与c-Src SH3结构域的RT-loop区形成结合程度或数量显著低于对照组中的结合程度或数量，就表明该测试物是对抗血栓的候选化合物，
其中，所述的候选化合物为c-Src SH3 RT-loop拮抗剂。
在另一优选例中，所述的待测药物为化合物、蛋白药物或基因药物。
在另一优选例中，在步骤(a)中，在无细胞的体系中进行测试。
在另一优选例中，在步骤(a)中，在有细胞的体系中进行测试，其中所述的细胞表达整合素β3和c-Src蛋白。
在另一优选例中，所述的细胞为血小板。
本发明第六方面，提供一种筛选抗血栓的候选化合物的方法，所述方法包括步骤：
(a)在第一测试组中，在测试物存在下，使NITYRGT肽和c-Src蛋白接触，并观察所述测试组中NITYRGT肽与c-Src蛋白形成的第一复合物的数量；
在第一对照组中，在所述测试物不存在下，使NITYRGT肽和c-Src蛋白接触，并观察所述对照组中NITYRGT肽与c-Src蛋白形成的第一复合物的数量；
其中，如果第一测试组中第一复合物的数量显著低于第一对照组中第一复合物的数量，就表明该测试物是对抗血栓的候选化合物，
其中，所述的候选化合物为c-Src SH3 RT-loop拮抗剂。
在另一优选例中，所述方法还包括：
(b)在第二测试组中，在所述测试物存在下，使RLP1多肽和c-Src蛋白接触，并观察所述测试组中RLP1多肽与c-Src蛋白形成的第二复合物的数量；
在第二对照组中，在所述测试物不存在下，使RLP1多肽和c-Src蛋白接触，并观察所述对照组中RLP1多肽与c-Src蛋白形成的第二复合物的数量；
如果在第二测试组中第二复合物的数量与第二对照组中二复合物的数量的相当，则提示所述候选化合物为c-Src SH3 RT-loop特异性拮抗剂(即与RT-loop区作用为主，而与n-loop区基本无作用)；
如果在第二测试组中第二复合物的数量显著低于第二对照组中二复合物的数量，则提示所述候选化合物为c-Src SH3 RT-loop和n-loop区双重拮抗剂(即与RT-loop区和n-loop区均有作用)。
在另一优选例中，所述的“显著低于”指在测试组中的复合物数量或结合程度或结合数量(记为C1)与对照组中的复合物数量或结合程度或结合数量(记为C0)之比(C1/C0)≤1/2，较佳地≤1/3，更佳地≤1/4，最佳地≤1/5。
在另一优选例中，所述的“相当”指在测试组中的复合物数量或结合程度或结合数量(记为C1)与对照组中的复合物数量或结合程度或结合数量(记为C0)之比(C1/C0)为0.8-1.2。
本发明第七方面，提供一种c-Src突变蛋白，所述的突变蛋白在选自下组一个或多个位点具有氨基酸突变：第95、96、97、98、99、100位，或其组合，其中，氨基酸位置的编号基于SEQ ID No:1。
在另一优选例中，所述的突变蛋白在选自下组位点具有氨基酸突变：第95位、第97位、或其组合。
在另一优选例中，所述的c-Src突变蛋白具有选自下组的突变氨基酸突变：R95A、E97A、或其组合。
在另一优选例中，所述的c-Src突变蛋白还在选自下组位点具有氨基酸突变：116位、118位、131位、或其组合。
在另一优选例中，所述的c-Src突变蛋白具有选自下组的突变氨基酸突变：G116A、W118A和Y131A。
本发明第八方面，提供一种多核苷酸，所述多核苷酸编码如本发明第七方面所述的c-Src突变蛋白。
本发明第九方面，提供一种载体，所述载体含有如本发明第八方面所述的多核苷酸。
本发明第十方面，提供一种宿主细胞，所述的宿主细胞含有如本发明第九方面所述的载体，或基因组中组合含有如本发明第八方面所述的多核苷酸。
本发明第十一方面，提供一种检测试剂盒，所述的试剂盒包括：
(i)用于检测c-Src SH3结构域的RT-loop区域其编码基因的检测试剂。
在另一优选例中，所述检测试剂包括检测c-Src蛋白或mRNA数量的试剂。
在另一优选例中，所述检测试剂包括检测所述RT-loop区域是否存在氨基酸突变或核苷酸突变的试剂。
在另一优选例中，所述检测试剂检测在c-Src蛋白的第95、96、97、98、99、100位是否存在氨基酸突变，和/或是否存在对应于所述氨基酸突变的核苷酸突变。
在另一优选例中，所述的氨基酸突变包括：R95A、E97A、或其组合
本发明第十二方面，提供如本发明第十一方面所述的检测试剂盒的用途，用于制备一诊断试剂盒，所述诊断试剂盒用于评估测试对象(如血栓患者)是否适合采用c-Src SH3 RT-loop拮抗剂进行治疗。
在另一优选例中，所述的诊断试剂盒还用于评估测试对象患血栓的风险。
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
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示RGT肽、NITYRGT肽以及含PXXP结构域的经典结合肽(APPIPPPR)与c-Src SH3结构域结合的结构模拟图。
图2显示c-Src SH3结构域的氨基酸序列以及对应的二级结构模式图。
图3显示在293T β3细胞(293T细胞中转染了整合素β3)中转染c-Src SH3突变体(R95A、E97A、G116A、W118A和Y131A)后应用Co-IP检测各突变体与β3的相互作用的差异。
图4显示RLP1多肽(含PXXP domain,与c-Src SH3的结合被认为是经典结合)与c-Src SH3突变体(R95A、E97A、G116A、W118A和Y131A)的结合。
图5显示RGT肽、设计合成的小分子化合物DCDBS84、以及含PXXP结构域的经典结合肽(APPIPPPR)与c-Src SH3结构域结合的结构模拟图。
图6显示核磁共振实验检测DCDBS84在c-Src SH3结构域的结合位点。
图7显示基于核磁共振实验结果，根据氨基酸位点的化学位移干扰(CSP)分析出结合位点图。
图8显示应用表面等离子共振(SPR)检测的DCDBS84与c-Src SH3(WT)以及各突变体(R95A、E97A、G116A、W118A和Y131A)的结合常数。
图9显示应用SPR检测的RLP1肽(含PXXP domain,与c-Src SH3的结合被认为是经典结合)c-Src SH3(WT)以及各突变体(R95A、E97A、G116A、W118A和Y131A)的结合常数。
图10显示基因打靶构建c-Src E97A转基因小鼠的总体策略图。
图11显示c-Src E97A转基因小鼠的基因型鉴定结果。
图12显示应用Co-IP方法检测c-Src E97A转基因小鼠能够解离整合素β3与c-Src的相互作用。
图13显示c-Src E97A转基因小鼠能够减少Thrombin诱导的血小板聚集。
图14显示c-Src E97A转基因小鼠能够减少Thrombin诱导的血小板聚集的统计图。
图15显示c-Src E97A转基因小鼠能够降低血小板在固相纤维蛋白原上的伸展图。
图16显示c-Src E97A转基因小鼠能够降低血小板在固相纤维蛋白原上的黏附结果。
图17显示在FeCl 3诱导的颈动脉损伤模型中，与WT对照小鼠相比，c-Src E97A转基因小鼠能够明显抑制血栓形成。
图18显示在小鼠剪尾实验中，与WT对照小鼠相比，c-Src E97A转基因小鼠不会增加小鼠出血时间。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，对c-Src SH3结构域的RT-loop区域靶点的抑制能够有效地治疗血栓的同时不增加出血风险。
具体地，在本发明的实验中，明确NITYRGT，RGT肽以及整合素αIIbβ3的β3胞浆尾段在c-Src SH3的结合区域与经典结合肽(含PXXP结构域)在c-Src SH3的结合区域是否有所区分。含PXXP结构域多肽与c-Src SH3的经典结合涉及到c-Src激酶活性(已知以W118为主的n-Src loop对此贡献较大)以及相关信号转导参与细胞的多种功能，而β3/c-Src的相互作用被认为是结合相对较弱的非经典结合，结合后的主要功能是参与血小板“外向内”信号转导及相关的血栓形成。通过结构模拟分析NITYRGT，RGT以及含PXXP结构域的多肽(APPIPPPR)与c-Src SH3结构域结合，发现NITYRGT在c-Src SH3的结合倾向于RT-loop，而PXXP结构域的多肽(APPIPPPR)在c-Src SH3的结合倾向于n-Src loop。进一步地，通过克隆c-Src SH3的突变体R95A、E97A、G116A、W118A、Y131A，应用Co-IP实验检测了c-Src WT及各突变体与αIIbβ3的相互作用，发现R95A、E97A、G116A、W118A、Y131A的确能够减弱β3/c-Src的相互作用，减弱程度最明显的是E97A，E97所在的区域是c-Src SH3的RT-loop。由于c-Src SH3可以与包含PXXP结构域的蛋白发生相互作用，这种结合被称为经典结合，本发明合成了含PXXP的多肽RLP1(RKLPPRPSK)，检测了RLP1分别与c-Src SH3野生型和各突变体的相互作用，结果发现，RLP1的结合位点倾向于W118，而这个区域主要是c-Src SH3的n-Src loop所在的区域。
实验筛选出靶向c-Src SH3的小分子DCDBS84，检测了DCDBS84在c-Src SH3的直接作用靶点与经典结合的靶点是否有所区分。应用核磁共振实验(NMR)以及化学位移迁移分析了DCDBS84与c-Src SH3的结合位点主要有R95、E97、W118、W119和Y131等。进一步，通过应用表面等离子共振(SPR)实验，检测了DCDBS84与c-Src SH3的突变体R95A、E97A、G116A、W118A、Y131A以及野生型(WT)的结合常数，发现R95A和E97A明显减弱了DCDBS84与c-Src SH3的结合，提示，R95和E97主要参与了DCDBS84与c-Src SH3的结合。同时，本发明也应用SPR检测了经典结合肽RLP1与c-Src SH3的突变体R95A、E97A、G116A、W118A、Y131A以及野生型(WT)的结合常数，结果发现G116A、W118A和Y131A突变明显减弱了RLP1与c-Src SH3的结合，提示，G116、 W118和Y131主要参与了RLP1与c-Src SH3的结合。该实验进一步证实了小分子DCDBS84在c-Src SH3的结合位点主要位于RT-loop，以E97为主要靶点。同时，对DCDBS84与c-Src SH3结合位点的结构模拟也提示其主要由E97及其周围的RT-loop氨基酸组成。
此外，实验构建了c-Src E97A转基因小鼠，基因型鉴定确认WT小鼠和c-Src E97A突变小鼠，从小鼠分离血小板，Co-IP实验证实c-Src E97A突变小鼠能够解离血小板中β3/c-Src的相互作用，抑制血小板的聚集、伸展、黏附等血小板“外向内”信号转导介导的功能。尤为重要的是，c-Src E97A突变小鼠能够在FeCl 3诱导的血栓形成模型中抑制血栓形成，并且在剪尾流血实验中与野生型小鼠相比不增加流血时间。结果表明：c-Src SH3 RT-loop以及以E97为主的氨基酸位点能够成为不影响正常止血功能的新型抗血栓靶点，为新型抗血栓药物的研发提供靶点信息。
术语
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“抗血栓”包括预防和/或治疗血栓。
在本发明中，术语“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。本文中使用的"预防"还包括延迟疾病和/或它的附随症状的发作和降低对象的得病的风险。
本发明所述的“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述的组合物、药盒、食品盒或保健品盒、活性成分组合时观察到的水平相比，本发明所述组合物或药物组合物通过抑制线粒体氧化磷酸化通路将相关疾病(如肿瘤)及其并发症减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％。
Src和c-Src
Src作为一个癌基因蛋白起初发现于Rous肉瘤逆转录病毒(retrovirus rous sarcoma virus)，随后发现在细胞中普遍存在高度保守并与其同源的v-Src。
Src激酶家族是具有酪氨酸蛋白激酶(protein tyrosine kinase，PTK)活性的蛋白质，其中c-Src是Src激酶家族的一个重要组成部分。
在本文中，除非另有说明，否则氨基酸序列为从N端到C端进行排序编号。
人c-Src的氨基酸序列如SEQ ID NO.:1所示:
SEQ ID NO.:1:
Figure PCTCN2021108371-appb-000002
Figure PCTCN2021108371-appb-000003
(SEQ ID No:1，下划线为SH3结构域，斜体为RT-loop区)
SH3结构域和RT-loop区
如本文所用，“SH3”、“SH3结构域”、“SH3结构域蛋白”、“SH3蛋白”可互换使用。
代表性的野生型的人c-Src SH3结构域的氨基酸序列如SEQ ID NO.:2所示:
Figure PCTCN2021108371-appb-000004
Figure PCTCN2021108371-appb-000005
(SEQ ID No:2，对应于SEQ ID No:1中第87-144位)。
如本文所用，在人c-Src中，R95、E97、T98、L100、D117、W118、W119、A138是基于对以下SH3结构域的氨基酸(SEQ ID NO.:2)进行编号，第84位至第141位如下所示：
Figure PCTCN2021108371-appb-000006
如本文所用，在人c-Src中，R95A、E97A突变型的c-Src蛋白中，R95A、E97A是基于对以下SH3结构域的氨基酸(SEQ ID NO.:2)进行编号，第84位至第141位如下所示：
Figure PCTCN2021108371-appb-000007
如本文所述，R95A是指氨基酸编号的第95位氨基酸残基由R氨基酸突变成A氨基酸，E97A是指氨基酸编号的第97位氨基酸残基由E氨基酸突变成A氨基酸，其它位点的氨基酸突变同上所述。
如本文所用，术语“c-Src SH3 RT-loop”与“c-Src SH3结构域的RT-loop区域”可互换使用。人RT-loop区域位于人c-Src SH3结构域，氨基酸序列如下所示:YDYESRTETDL(SEQ ID No:3)
整合素和整合素β3/Src相互作用
整合素αIIbβ3是由αIIb和β3两个亚基通过非共价键组成的跨膜异二聚体，主要表达于血小板及巨核细胞表面，是血小板表面主要的膜受体，可介导血小板双向信号转导，因此在血小板活化、维持血小板正常功能以及血栓形成中起关键作用。血小板激活剂如凝血酶、ADP等与相应受体作用后引起整合素αIIbβ3的构型改变并导致与其配体即可溶性纤维蛋白原等的亲和力增高，此过程为内向外信号转导，标志性事件是血小板的非稳定粘附、游离纤维蛋白原结合和可逆聚集等；整合素αIIbβ3被活化并结合配体，激活外向内信号转导，标志性事件是血小板的稳定粘附、伸展、不可逆聚集、纤维蛋白凝块回缩等，最终促使血小板相互聚集并形成较为稳定的血栓而完成止血和血栓形成的生理或病理过程。目前的共识是，止血和血栓形成的实现需要内向外和外向内信号转导共同参与，而血栓形成的病理过程需要在对抗高血流冲击力条件下增大血小板栓子，因此，血栓形成对外向内信号转导的依赖性相对更大。
为了能够更加特异性的靶向血小板参与血栓形成的受体，研发了靶向整合素αIIbβ3的受体拮抗剂。整合素αIIbβ3作为介导血小板活化聚集、血栓形成的最终共同通路，是抗血栓药物研究的主要靶点。而事实上，以整合素αIIbβ3为抗血栓药物靶点的研究取得了重要的进展，目前主要集中于整合素αIIbβ3受体拮抗剂类药物，已获得很好的临床疗效。目前，经美国食品药物监督管理局(FDA)批准用于临床抗血栓治疗的整合素αIIbβ3受体拮抗剂类抗血小板药物有三种，即阿昔单抗、埃替巴肽、替罗非班。此类αIIbβ3受体拮抗剂通过干扰整合素αIIbβ3与其配体的相互作用而特异性地发挥抗血栓作用。但这一策略仍存在明显问题，αIIbβ3受体拮抗剂药物通过阻止整合素αIIbβ3结合其配体从而阻断双 向信号转导，也就是在发挥抗血栓作用的同时影响了正常的止血功能。临床实验显示，经整合素αIIbβ3拮抗剂类药物治疗的病人中约有2％发生严重的颅内出血，约有15％发生胃肠道出血，约有5-10％出现腹膜出血，另大约有60-80％受试患者于股动脉穿刺点出现明显出血。与经典的抗血栓药物阿司匹林、氯吡格雷一样，整合素αIIbβ3拮抗剂在发挥有效抗血栓作用的同时也导致患者出血风险的增加，这是目前抗血栓药物最常见也是最重要的副作用。所以，临床上抗血栓药物剂量的选择还要兼顾其出血副作用的风险，致使目前难以通过增加抗血栓药物剂量来达到更好的抗血栓效果。在以死亡作为终点事件的研究中，难以找到一个合适的剂量阈值来减少由于血栓死亡和由于出血死亡的死亡率。因此，通过开发新一代不影响正常止血功能的抗血栓药物，将有可能在低风险的前提下获得更强的抗血栓疗效，这代表了抗血栓药物的发展方向。
研究发现整合素αIIbβ3胞浆尾段通过与胞浆蛋白c-Src相互作用，在血小板“外向内”信号相关的功能如血小板在固相纤维蛋白原上的稳定黏附和伸展，二相聚集和纤维蛋白凝块回缩中发挥重要作用，而对血小板“内向外”功能如血小板结合可溶性纤维蛋白原、血小板初步黏附以及一相聚集影响不大。研究表明，血小板的“外向内”信号主要参与了血小板血栓形成过程，而“内向外”在发挥止血功能中的作用较大。
研究显示，Src激酶的SH3结构域与整合素β3C末端的RGT三个氨基酸形成组成性结合。在整合素活化后的外向内信号转导中，Src激酶与β3C末端的RGT序列相互作用，并且发生β3胞浆段Y747和Y759的磷酸化，成为外向内信号转导的重要事件。体内实验证实，RGT敲除小鼠可避免FeCl3刺激颈动脉导致的血栓形成；在切尾流血实验中，部分小鼠出血时间延长，但是没有出现自发性出血、手术后大出血、血便、血尿、贫血等情况。既然，β3 RGT/c-Src的相互作用在血小板“外向内”信号转导中发挥如此重要的作用，合成RGT三肽通过竞争内源性β3 RGT/c-Src的相互作用能够发挥抗血栓作用。
用途
本发明提供一种c-Src SH3 RT-loop拮抗剂(antagonist)的用途，c-Src SH3 RT-loop拮抗剂包括(但不限于)下组的一种或多种用途：
(a)干扰整合素β3和c-Src的相互作用；
(b)抑制血小板在固相纤维蛋白原上的伸展；
(c)抑制血小板的聚集和/或粘附；和
(d)预防和/或治疗血栓。
在一个优选例中，整合素β3包括(但不限于)整合素αIIbβ3
在本发明的另一优选例中，所述的c-Src SH3 RT-loop拮抗剂是为c-Src SH3 RT-loop区特异性拮抗剂。代表性地，所述的c-Src SH3 RT-loop拮抗剂不拮抗(或影响)或基本不影响整合素β3与c-Src SH3 n-loop区的结合(或相互作用)。
本发明所述的c-Src SH3 RT-loop拮抗剂具体种类并没有特别的限制，只要能够具有对c-Src SH3 RT-loop具有拮抗作用即可。例如，所述的c-Src SH3 RT-loop拮抗剂可以为小分子拮抗剂、反义核苷酸、miRNA和siRNA等。优选地，所述的拮抗剂为DCDBS84化合物：
Figure PCTCN2021108371-appb-000008
在本发明中，c-Src蛋白并没有特别的限定，优选为哺乳动物如人和啮齿动物的c-Src蛋白。代表性地，人c-Src蛋白的氨基序列如SEQ ID NO.:1所示的多肽。在SEQ ID NO.:1所示的多肽中第87-144位(SEQ ID No:2序列)为人c-Src SH3结构域。人c-Src SH3 RT-loop的氨基序列如SEQ ID NO.:3所示的多肽。
在本发明的一个优选例中，c-Src SH3 RT-loop拮抗剂在抗血栓的治疗过程中不增加或基本上不增加出血风险(或称为“改善出血”)。在本发明中，所述的改善出血包括抑制出血、不增加出血风险、降低出血风险、不会引起出血副作用和/或不影响止血功能。
在发明中，所述的血栓包括心脑血管性疾病血栓。优选地，所述的血栓包括(但不限于)：心肌梗塞血栓、脑梗塞血栓、缺血性卒中、动脉粥样硬化血栓，或其组合。
组合物或制剂、活性成分的组合和药盒和施用方法
本发明还提供一种组合物，所述的组合物包含c-Src SH3 RT-loop拮抗剂。
本发明所述的组合物优选为药物组合物。本发明所述的组合物可以包括药学上可接受的载体。
如本文所用“药学上可接受的载体”是指一种或多种相容性固体、半固体、液体或凝胶填料，它们适合于人体或动物使用，而且必须有足够的纯度和足够低的毒性。“相容性”是指药物组合物中的各组分和药物的活性成分以及它们之间相互掺和，而不明显降低药效。
应理解，在本发明中，所述的药学上可接受的载体没有特别的限制，可选用本领域常用材料，或用常规方法制得，或从市场购买得到。药学可接受的载体部分例子有纤维素及其衍生物(如甲基纤维素、乙基纤维素、羟丙甲基纤维素、羧甲基纤维素钠等)、明胶、滑石粉、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)、缓冲剂、螯合剂、增稠剂、pH调节剂、透皮促进剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、抑菌剂、无热原水等。
在本发明中，所述的组合物的剂型并没有特别限制，可以为固体剂型、液态制剂、半固态制剂。
在本发明中，组合物和制剂的剂型包括但不限于口服制剂、注射制剂、外用制剂。
代表性地，组合物和制剂的剂型包括但不限于：片剂、颗粒剂、胶囊剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
典型地，所述的注射剂为静脉注射剂。
药物制剂应与给药方式相匹配，优选的给药方式为口服给药、注射给药(如静脉注射)，使用时，是将治疗有效量的药物施用于所需对象(如人或非人哺乳动物)。如本文所用，术语“治疗有效量”，是指对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。本领域的普通技术人员应该理解，所述的“治疗有效量”可随着药物组合物的形式、给药途径、所用药物的辅料、疾病的严重程度以及与其他药物联合用药等情况的不同而有所不同。
在一个施用方式中，第一活性成分的安全有效日使用剂量通常至少约0.1mg，而且在大多数情况下不超过约2500mg。较佳地，该剂量是1mg-500mg； 第二活性成分的安全有效量通常至少约0.01mg，而且在大多数情况下不超过2500mg。较佳地，该剂量范围是0.1mg至2500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是在熟练医师技能范围之内的。
本发明的主要优点在于：
本发明首次发现c-Src SH3结构域的RT-loop区域靶点的抑制能够有效地治疗血栓的且不增加出血风险。
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，否则份数和百分比按重量计。
实施例
小鼠c-Src的氨基酸序列如SEQ ID NO.:4所示:
SEQ ID NO.:4
Figure PCTCN2021108371-appb-000009
小鼠c-Src SH3结构域的氨基酸序列如SEQ ID NO.:5所示:
SEQ ID NO.:5
Figure PCTCN2021108371-appb-000010
小鼠c-Src SH3结构域含RT-loop区域的氨基酸序列如SEQ ID NO.:6所示:
SEQ ID NO.:6
Figure PCTCN2021108371-appb-000011
化合物DCDBS84的结构式如下：
Figure PCTCN2021108371-appb-000012
实施例中，在人c-Src中，R95、E97、T98、L100、D117、W118、W119、A138是基于对以下SH3结构域的氨基酸(SEQ ID NO.:2)进行编号，第84位至第141位如下所示。
Figure PCTCN2021108371-appb-000013
在鼠中，R95、E97是基于对以下SH3结构域的氨基酸(SEQ ID NO.:5)进行编号，第84位至第147位如下所示：
Figure PCTCN2021108371-appb-000014
实施例1 结合肽与c-Src SH3结构域结合的结构模拟
根据RGT肽(晶体结构)、NITYRGT肽(核磁共振结构)以及含PXXP结构域的经典结合肽(APPIPPPR)(核磁共振结构)，通过计算机结构分析获得了RGT、NITYRGT以及PXXP结构域的经典结合肽(APPIPPPR)与c-Src SH3结构域结合的结构模拟图。如图1所示，来自于固相的晶体结构的YRGT四肽与c-Src SH3的结 合偏向于n-Src loop,而来自于液相核磁共振结构的NITYRGT七肽与c-Src SH3的结合更偏向于RT-loop。经典结合肽(APPIPPPR)与c-Src SH3的结合方向基本与NITYRGT在c-Src SH3的结合方向垂直，更偏向于n-Src loop。根据c-Src SH3的氨基酸序列信息以及二级结构信息，绘制了c-Src SH3的结构模式图(如图2)。
实施例2：免疫共沉淀(Co-IP)检测结合位点
本实施例通过构建整合素β3在c-Src SH3可能结合位点的突变体，免疫共沉淀(Co-IP)检测β3在c-Src SH3的结合位点。
构建5个c-Src SH3基因点突变过表达载体，pFlag-CMV4-Src(R95A),pFlag-CMV4-Src(E97A),pFlag-CMV4-Src(G116A),pFlag-CMV4-Src(W118A)和pFlag-CMV4-Src(Y131A)。其中R95和E97位于RT-loop区域，G116、W118位于N-Src loop区域，Y131位于β4区域(如图2所示)。
应用免疫共沉淀实验技术(Co-IP)检测了c-Src SH3突变体与整合素β3亚基的结合情况。首先取0.4ml血小板(浓度3×10 8/ml)裂解蛋白(蛋白含量500μg)，加入50μl预先经裂解缓冲液洗涤过的Protein A+G琼脂糖珠子，4℃旋转孵育2h以去除非特异性杂蛋白，降低背景。孵育后4℃1000×g离心5min，将上清转移到新的离心管中。在蛋白上清中加入抗Flag标签的抗体M2(Sigma)1μg或非特异性的鼠IgG(sc-2025，Santa Cruz Biotechnology，1μg)，于4℃旋转孵育抗原抗体混合物过夜。次晨在其中加入20μl预先经裂解缓冲液洗涤的Protein A+G琼脂糖珠子，于4℃旋转孵育2h。随后4℃1000×g离心5min，收集琼脂糖珠子-抗原抗体复合物，去上清，用预冷的RIPA裂解缓冲液反复洗涤5遍，每次用800μl洗涤，冰上静置10分钟。随后以1×SDS-PAGE样品缓冲液(100mM Tris-HCl，pH 6.8，5％β-mercaptoethanol，4％SDS，20％glycerol，0.1％Bromophenol Blue)重悬琼脂糖珠子-抗原抗体复合物，100℃煮沸8min以变性蛋白。通过Western blot检测免疫共沉淀物。如图3所示，R95和E97突变明显减低了β3/c-Src的结合。
实施例3 ELISA的方法检测了RLP1肽与c-Src SH3突变体的结合
细胞中与Src激酶存在相互作用的配体蛋白分子一般通过经典的(PXXP)基序靶向Src-SH3结构域，RLP1多肽(含经典的PXXP基序)是模拟细胞中与Src-SH3结构域相互作用多种配体蛋白分子共同的PXXP基序序列。具体的实验流程如下，96孔板每孔加入50μL含Flag抗体M2(Sigma)的包被液(1μg/mL,用0.1M  NaHCO3 PH 8.3稀释)，4℃孵育过夜。第2天用1×TBST洗3次，每次5min，加5％BSA封闭2h,再用1×TBST洗3次，备用。前期在293T细胞中分别转染pFlag-CMV4-Src(WT)及另外5种Src突变型过表达载体pFlag-CMV4-Src(R95A),pFlag-CMV4-Src(E97A),pFlag-CMV4-Src(G116A),pFlag-CMV4-Src(W118A)和pFlag-CMV4-Src(Y131A),应用lipofect 2000(Invitrogen Life technologies)进行转染，转染48h后用RIPA冰上裂解293T细胞30min，离心(4℃,18000rpm)收集裂解液上清，在包被Flag抗体的96孔板中每孔添加50μL裂解液上清，4℃孵育过夜，各实验组留部分上清液做Western blot相对定量。第3天，用TBST洗3次，每孔添加50μL生物素标记的RLP1及对照(RLA)多肽(1μg/mL)，37℃孵育1h。然后用1×TBST洗3次，每次5min，每孔添加50μL HRP标记的亲和素，37℃孵育1h。再用1×TBST洗涤3次，每孔添加TMB显示液100μL，室温孵育15min,再添加100μL硫酸(1M)终止反应，用酶标仪在450nm波长下检测各孔吸光值。吸光值的数值与Western blot条带灰度值的相对定量结果如图4，结果可见W118和Y131突变明显减低了RLP1肽与c-Src的结合。
实施例4 核磁共振测定氨基酸结合位点
本实施例通过核磁共振实测定加上DCDBS84前后蛋白(c-Src SH3)氨基酸位点的核磁共振谱图。
综合RGT肽与c-Src SH3结合的晶体结构，NITYRGT与c-Src SH3的核磁共振结构等各种信息，经计算机模拟筛选和分析，筛选出了靶向c-Src SH3的小分子DCDBS84。DCDBS84与c-Src SH3结合的结构模拟图如图5所示。可见DCDBS84主要结合在c-Src SH3的RT-loop。
核磁共振实验在四通道Bruker Avance III 600MHz频谱仪上进行。用15N取代14N配制培养基，以纯化出带有15N标记的c-Src-SH3蛋白，从而进行二维核磁共振；当需要行三维核磁时，则需要15N取代14N，13C取代12C；图6(左图)所示Src-SH3和DCDBS84之间的相互作用的二维15N-HSQC实验，c-Src-SH3浓度在50μM，DCDBS84浓度为其20倍；如图6(右图)所示，将15N/13C标记的c-Src-SH3蛋白浓度调至1.3mM，完成对该蛋白氨基酸位点的归属。
实施例5 化学位移干扰(CSP)分析出结合位点
在核磁共振实验中，根据氨基酸位点的化学位移干扰(CSP)分析出结合位点 图。
计算化学位移干扰(Chemical shift perturbation，CSP),按照公式：
Figure PCTCN2021108371-appb-000015
以CSP平均值+标准误作为基线，大于此值作为结合位点。根据如图7所示的结果，发现R95、E97、T98、L100、D117、W118、W119、A138可能是SH3与化合物的结合位点。
实施例6：拮抗剂DCDBS84与野生型和突变型c-Src SH3的结合性能
在本实施例中，通过表面等离子共振实验，测定DCDBS84与c-Src SH3(WT)以及突变体(R95A,E97A,G116A,W118A,Y131A)的解离常数。
表面等离子共振测试在BIACORE T200仪器(GE公司)上进行。用10mM CH3COONa(pH 4.2)将纯化的c-Src SH3蛋白(浓度2mg/ml)稀释至0.1mg/ml，然后通过标准氨基偶联方法将SH3蛋白偶联至CM5芯片上。采用缓冲溶液(20mM Tris-HCl,PH8.0，100mM NaCl)将DCDBS84稀释后，以20μl/s的流速连续进样60秒钟，解离120秒钟。记录随时间增长响应值的变化情况，经BIA Evaluation Software(GE Healthcare)程序分析获得DCDBS84与c-Src SH3蛋白(WT和突变体)的解离常数KD。如图8所示，DCDBS84与野生型c-Src SH3蛋白的解离常数为0.83μM，与R95A的解离常数为48μM，与E97A的解离常数为35μM，与G116A的解离常数为8.1μM，与W118A的解离常数为6.24μM，与Y131的解离常数为1.52μM。提示，位于RT-loop的R95和E97主要参与了DCDBS84与c-Src SH3的结合。
进一步的，按照上述方法检测了经典结合肽RLP1与c-Src SH3蛋白(WT和突变体)的解离常数KD。如图9所示，RLP1与野生型c-Src SH3蛋白的解离常数为9.9μM，与R95A的解离常数为10.9μM，与E97A的解离常数为22μM，与G116A的解离常数>80μM，与W118A的解离常数为49.9μM，与Y131A的解离常数为85.9μM。提示，位于n-Src loop的G116和W118以及位于远端loop的Y131主要参与了RLP1与c-Src SH3的结合。
上述结果提示：小分子化合物DCDBS84在c-Src SH3的主要结合位点倾向于以RT-loop的R95和E97为主，而含PXXP结构域的经典结合肽(RLP1)c-Src SH3的主要结合位点倾向于以n-Src loop的G116和W118以及远端loop的Y131为主。表明，非经典结合与经典结合在c-Src SH3上的结合确实有所区分。
实施例7 基因打靶构建转基因小鼠
本实施例通过基因打靶构建c-Src E97A突变转基因小鼠，基因打靶构建转基因小鼠的总体策略如图10所示。
实施例8：c-Src E97A转基因小鼠的基因型鉴定
在本实施例中，根据小鼠Src基因(NC_000068.7)序列设计包含与人E97A相对应的小鼠基因相应位点突变(MusE99A)引物，引物序列为：c-Src E97A-F:5'-GAACACCTAGTCTGCAGCCC-3',c-Src E97A-R:5'-AGCAGAGAGAAGGAGAGG
CT-3'，扩增片段长度419bp(如图11上图)。PCR产物进行测序分析，在小鼠第99位氨基酸对应基因的位置若为GAG则为谷氨酸(E)，如为GCG则为丙氨酸(A)，若出现GAG和GCG两个峰，则为杂合子。基因测序鉴定结果如图11下图所示。
实施例9：免疫共沉淀
在本实施例中，通过免疫共沉淀(Co-IP)检测c-Src E97A转基因小鼠血小板中β3/c-Src的相互作用。
取野生型(WT)小鼠和c-Src E97A转基因小鼠各3只，根据小鼠体重应用苯巴比妥对小鼠进行麻醉，然后对小鼠进行心脏采血，应用0.38％的枸橼酸钠抗凝。300×g离心7min得到富血小板血浆(PRP)，在PRP种加入1/4体积的ACD抗凝，以500×g离心10min，弃上清。用CGS洗涤液(13mM枸橼酸钠，120mM氯化钠，30mM葡萄糖，pH 6.5)洗涤血小板，再用Tyrode's缓冲液(0.1％牛血清白蛋白，5mM 4-羟乙基哌嗪乙磺酸，5.5mM葡萄糖，137mM氯化钠，2mM氯化钾，12mM碳酸氢钠，0.3mM磷酸二氢钠，1mM氯化钙，1mM氯化镁，pH 7.4)重悬血小板。应用小动物血常规检测仪(PoCH-100iV Diff)对血小板进行计数，将血小板密度调整为3×10 8个/ml，室温静置1h备用。
应用Co-IP方法检测小分子化合物DCDBS84对整合素αIIbβ3的β3与c-Src相互作用的影响。取400μl浓度为3×10 8个/ml的血小板，用IP buffer(50mM Tris-HCl,pH7.4,50mM NaCl,0.2％NP-40)冰上裂解血小板30min，离心(4℃,12000rpm,15min)吸取上清，BCA蛋白定量。加入50μl预先经IP buffer洗涤过的Protein A/G琼脂糖珠子，4℃旋转孵育2h，离心(4℃,1000g,5min),再将上清转移到新的离心管中。在蛋白上清中加入抗整合素小鼠β3抗体SZ-21(1μg)或非特异性的鼠IgG(sc-2025，Santa Cruz Biotechnology，1μg)，或者兔单克隆抗体c-Src抗体(36D10， #2109，Cell signaling Technology,1μg)以及非特异性的兔IgG(#2729,Cell signaling Technology,1μg)于4℃旋转孵育抗原抗体混合物过夜。次晨向混合物中加入20μl预先经IP buffer洗涤的Protein A/G琼脂糖珠子，于4℃旋转孵育2h,离心(4℃,1000g,5min)，收集琼脂糖珠子-抗原抗体复合物，用预冷的1×PBS缓冲液洗涤3遍。最后用1×SDS loading buffer重悬琼脂糖珠子-抗原抗体复合物，100℃煮沸10min。通过Western blot检测免疫共沉淀物。结果如图12所示，无论是应用β3抗体IP还是应用c-Src抗体IP，与WT小鼠相比，c-Src E97A小鼠血小板中β3/c-Src的相互作用明显减弱。
实施例10：c-Src E97A转基因小鼠对血小板聚集的影响。
按照实施例9所述的方法从野生型(WT)小鼠和c-Src E97A转基因小鼠各3只中分离血小板。300×g离心7min得到PRP，再以500×g离心10min得到乏血小板血浆(Platelet poor plasma,PPP)，用PPP调整PRP中血小板浓度至2×10 8/ml。再取200μl PPP以校正仪器透光聚集仪(Chrono-Log)零点。随后各组试剂分别与PRP于37℃孵育60min后，以200μl/管上聚集仪(37℃，1000rpm搅拌)，校正零点后加入0.1U/ml凝血酶(Thrombin)启动反应，记录聚集曲线。如图13所示，与WT小鼠相比，c-Src E97A转基因小鼠明显抑制血小板二相聚集，而不影响一相聚集，表明c-Src E97A转基因小鼠明显抑制血栓形成且不影响正常的生理性止血作用。图13的统计图如图14所示。
实施例11：c-Src E97A转基因小鼠对血小板伸展、黏附的影响。
按照实施例9所述的方法从野生型(WT)小鼠和c-Src E97A转基因小鼠各3只中分离血小板。血小板浓度调整至2×10 8/ml。
在96孔板中加50μl纤维蛋白原(0.1M，pH 8.3的碳酸氢钠稀释，20μg/ml)，4℃包被过夜。次晨经1×PBS洗涤3次后以牛血清白蛋白(BSA 20mg/ml)37℃封闭60min。取洗涤血小板悬液50μl(浓度2×10 8个/ml)，加到96孔板中，于37℃温箱中黏附60min。通过PBS洗涤3次去除未黏附的血小板，以4％多聚甲醛固定稳定黏附的血小板，PBS洗涤3次。随后以0.5％曲拉通X-100对血小板膜打孔，再用0.5μg/ml鬼笔环肽-罗丹明在37℃下染血小板60min，并以1×PBS洗涤3次(每次10min)。洗涤结束后，用荧光显微镜(Leica)观察其荧光显色。结果如图15所示，c-Src E97A转基因小鼠的血小板在固相纤维蛋白原上的伸展明显弱于WT小鼠。
黏附实验中纤维蛋白原的包被与封闭方法同伸展实验，取50μl血小板加到96 孔板中，于37℃温箱中黏附60min。黏附完成后，PBS洗涤5次以去除未黏附以及不稳定黏附的血小板。将CCK-8 10μl/孔加到血小板黏附的孔中，置于37℃温箱孵育2h。最后酶标仪405nm波长下读OD值，以未加血小板孔作为空白对照，计算黏附的血小板量。每个样品设3个复孔，结果取均值。如图16所示，与WT小鼠相比，c-Src E97A转基因小鼠能够显著抑制血小板在固相纤维蛋白原上的黏附功能(p<0.01)，从而抑制血栓形成。
实施例12：c-Src E97A转基因小鼠对FeCl 3诱导的颈动脉阻塞血栓形成的影响。
选取6-8周龄的WT和c-Src E97A转基因小鼠作为实验对象。按照文献报道的实验方法，应用FeCl 3刺激小鼠颈动脉，从而导致内皮的损伤，继而开始血栓形成过程，在FeCl 3刺激的远心端以多普勒超声探头检测血流，当上游血栓形成并堵塞血管时，则血流下降。
从结果(图17)可以看出，相比较于WT小鼠，c-Src E97A转基因小鼠在FeCl 3刺激下颈动脉血栓形成时间明显延长。表明，c-Src E97A转基因小鼠对FeCl 3诱导的颈动脉血栓形成起明显抑制作用，具有抗血栓疗效。
实施例13：c-Src E97A转基因小鼠通过剪尾检测对出血时间的影响。
为评估c-Src E97A转基因小鼠对止血功能的影响，选取6-8周龄的WT和c-Src E97A转基因小鼠进行研究。剪尾实验是在血管破损后，血小板维持正常的止血功能，堵塞血管伤口的过程。在距离尾尖5mm处以锋利的刀片快速切断小鼠尾巴(tail-cut)，然后以每15sec用滤纸来蘸小鼠尾巴渗出的血液，切勿触碰小鼠尾巴以免造成新的损伤。以出血停止而且15sec不复发作为计时标准。
从结果(图18)可以看出，WT小鼠约在7.4min时停止出血，而相比较于WT组，c-Src E97A转基因小鼠其出血时间未有明显延长，约8.9min,与WT相比无统计学差异。表明c-Src E97A转基因小鼠不会延长剪尾出血时间，基本不影响正常的生理性止血作用。
讨论
本申请所述的c-Src SH3 RT-loop拮抗剂能够降低、抑制或干扰整合素β3和c-Src的相互作用，从而显著发挥抗血栓作用，且不影响正常的生理性止血作用。
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 (15)

  1. 一种c-Src SH3 RT-loop拮抗剂的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于：
    (a)干扰整合素β3和c-Src的相互作用；
    (b)抑制血小板在固相纤维蛋白原上的伸展；
    (c)抑制血小板的聚集和/或粘附；和/或
    (d)预防和/或治疗血栓。
  2. 如权利要求1所述的用途，其特征在于，所述的拮抗剂为c-Src SH3 RT-loop区特异性拮抗剂。
  3. 如权利要求1所述的用途，其特征在于，所述的c-Src SH3 RT-loop拮抗剂不拮抗(或影响)或基本不影响整合素β3与c-Src SH3 n-loop区的结合(或相互作用)。
  4. 如权利要求1所述的用途，其特征在于，所述的拮抗剂选自下组：小分子拮抗剂、反义核苷酸、miRNA、siRNA、或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述的“干扰整合素β3和c-Src的相互作用”选自下组：
    (a1)减少整合素β3和c-Src SH3的RT-loop区发生结合；
    (a2)阻断整合素β3和c-Src SH3的RT-loop区发生结合。
  6. 如权利要求1所述的用途，其特征在于，所述的拮抗剂与R95A突变型的c-Src蛋白发生相互作用的解离常数KD值(记为KD R95A)，与所述的拮抗剂与野生型c-Src蛋白发生相互作用的解离常数Kd值(记为KD wt)的比值(KD R95A/KD wt)，≥5，较佳地≥10，更佳地≥20，最佳地≥40；
    所述的R95A突变型的c-Src蛋白的氨基酸序列如SEQ ID No:1所示，并且第98位的R突变为A。
  7. 如权利要求1所述的用途，其特征在于，所述的拮抗剂与E97A突变型的c-Src蛋白发生相互作用的解离常数KD值(记为KD E97A)，与所述的拮抗剂与野生型c-Src蛋白发生相互作用的解离常数KD值(记为KD wt)的比值(KD E97A/KD wt)，≥5，较佳地≥10，更佳地≥20，最佳地≥40；
    所述的E97A突变型的c-Src蛋白的氨基酸序列如SEQ ID No:1所示，并且第100位的E突变为A。
  8. 如权利要求1所述的用途，其特征在于，所述的拮抗剂包括：DCDBS84 或其药学上可接受的盐：
    Figure PCTCN2021108371-appb-100001
  9. 如权利要求1所述的用途，其特征在于，所述的c-Src SH3 RT-loop选自下组：
    (A)氨基酸序列如SEQ ID NO.:3所示的多肽；
    (B)将SEQ ID NO.:3所示的氨基酸序列经过一个或几个(通常为1-5个，较佳地1-3个，更佳地1-2个，最佳地1个)氨基酸残基的取代、缺失或添加而形成的RT-loop区衍生物；
    (C)氨基酸序列如SEQ ID NO.:3所示且具有选自下组突变的多肽：R95A、E97A。
  10. 如权利要求1所述的用途，其特征在于，所述的c-Src SH3 RT-loop选自下组：
    (A)氨基酸序列如SEQ ID NO.:3所示的多肽；
    (B)将SEQ ID NO.:3所示的氨基酸序列经过一个或几个(通常为1-5个，较佳地1-3个，更佳地1-2个，最佳地1个)氨基酸残基的取代、缺失或添加而形成的RT-loop区衍生物；
    (C)氨基酸序列如SEQ ID NO.:3所示且具有选自下组突变的多肽：R95A、E97A。
  11. 一种c-Src SH3 RT-loop激动剂(agonist)的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于
    (a)促进整合素β3和c-Src相互作用；
    (b)促进血小板在固相纤维蛋白原上的伸展；
    (c)促进血小板的聚集和粘附；和/或
    (d)促进凝血。
  12. 一种干扰整合素β3和c-Src蛋白的相互作用的方法，其特征在于，包括步骤：
    (a)在c-Src SH3 RT-loop拮抗剂存在下，使得整合素β3和c-Src蛋白接触，从而干扰整合素β3和c-Src蛋白的相互作用。
  13. 一种抗血栓(或抑制血小板的聚集和/或粘附)的方法，其特征在于，所述方法包括步骤：给需要的对象施用c-Src SH3 RT-loop拮抗剂。
  14. 一种筛选抗血栓的候选化合物的方法，其特征在于，所述方法包括步骤：
    (a)测试组中，在测试物存在下，使整合素β3和c-Src蛋白接触，并观察所述测试组中整合素β3是否与c-Src SH3结构域的RT-loop区形成结合；在对照组中，在所述测试物不存在下，使整合素β3和c-Src蛋白接触，并观察所述对照组中整合素β3是否与c-Src SH3结构域的RT-loop区形成结合；
    其中，如果测试组中整合素β3是否与c-Src SH3结构域的RT-loop区形成结合程度或数量显著低于对照组中的结合程度或数量，就表明该测试物是对抗血栓的候选化合物，
    其中，所述的候选化合物为c-Src SH3 RT-loop拮抗剂。
  15. 一种c-Src突变蛋白，其特征在于，所述的突变蛋白在选自下组一个或多个位点具有氨基酸突变：第95、96、97、98、99、100位，或其组合，其中，氨基酸位置的编号基于SEQ ID No:1。
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