A c-Src SH3 RT-loop as a target for anti-thrombosis

Abstract

The present invention relates to using c-Src SH3 RT-loop as a target for anti-thrombosis. Specifically, the present invention provides the use of a c-Src SH3 RT-loop antagonist for preparing a composition or preparation, and the composition or preparation is used for: (a) interfering with the interaction between integrin β3 and c-Src; (b) inhibiting platelet spreading on solid-phase fibrinogen; (c) inhibiting platelet aggregation and / or adhesion; and / or (d) preventing and / or treating thrombosis. The present invention discovers for the first time that a drug combination or preparation targeting the RT-loop region of the c-Src SH3 domain can effectively treat thrombotic diseases without increasing the risk of bleeding.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and biomedicine. Specifically, the present invention relates to using c-Src SH3 RT-loop as a target for anti-thrombosis. Background Art

[0002] Cardiovascular and cerebrovascular thrombotic diseases, such as myocardial infarction and cerebral infarction, seriously affect human life and health. Platelet-mediated thrombosis plays a crucial role in the development and progression of these diseases. When vascular endothelial damage or atherosclerotic plaque rupture occurs, platelets activate and, through a series of processes including adhesion, extension, and aggregation, lead to pathological thrombosis. This can cause ischemic necrosis of heart and brain tissue in the affected vascular distribution, potentially endangering the patient's life. Therefore, antiplatelet therapy has become the preferred treatment for cardiovascular and cerebrovascular thrombotic diseases.

[0003] Classic antiplatelet drugs include the cyclooxygenase (COX) inhibitor aspirin, currently the most widely studied and used antiplatelet agent in antiplatelet therapy. It primarily exerts its antiplatelet effect by inhibiting arachidonic acid cyclooxygenase (COX), leading to irreversible acetylation of Ser-529 and Ser-516, thereby blocking the synthesis of TXA2. Common adverse reactions of aspirin include gastrointestinal discomfort and gastrointestinal bleeding, with the risk of bleeding being dose-dependent. To avoid bleeding side effects, very high antithrombotic doses should be avoided. Another classic class of antiplatelet drugs are adenosine diphosphate (ADP) P2Y12 receptor antagonists, including thienopyridines, with representative drugs including ticlopidine, clopidogrel, and prasugrel. Representative non-thienopyridines include ticagrelor and cangrelor. Among them, the second-generation P2Y12 receptor antagonist clopidogrel is widely used. It can irreversibly inhibit platelet ADP receptors, thereby inhibiting platelet aggregation induced by the release of ADP from activated platelets. Bleeding remains its main side effect. This shows that although classic antiplatelet drugs can exert a good antithrombotic effect, they cannot be used at sufficient doses to achieve a deep antithrombotic effect due to the limitation of bleeding side effects.

[0004] To more specifically target platelet receptors involved in thrombosis, researchers have developed receptor antagonists targeting integrin αIIbβ3. As the final common pathway mediating platelet activation, aggregation, and thrombosis, integrin αIIbβ3 is a major target for antithrombotic drug research. Indeed, significant progress has been made in the study of integrin αIIbβ3 as an antithrombotic drug target, with the current focus on integrin αIIbβ3 receptor antagonists, which have demonstrated excellent clinical efficacy. Currently, three integrin αIIbβ3 receptor antagonist antiplatelet drugs have been approved by the US Food and Drug Administration (FDA) for clinical antithrombotic treatment: abciximab, eptifibatide, and tirofiban. These αIIbβ3 receptor antagonists exert their specific antithrombotic effects by interfering with the interaction between integrin αIIbβ3 and its ligands. However, this strategy still presents significant challenges. αIIbβ3 receptor antagonists block integrin αIIbβ3 binding to its ligand, thereby disrupting bidirectional signaling. This means that while exerting antithrombotic effects, they also impair normal hemostasis. A review of clinical trials shows that approximately 2% of patients treated with integrin αIIbβ3 antagonists experience severe intracranial hemorrhage, approximately 15% experience gastrointestinal bleeding, approximately 5-10% experience peritoneal bleeding, and approximately 60-80% experience significant bleeding at the femoral artery puncture site. Like the classic antithrombotic drugs aspirin and clopidogrel, integrin αIIbβ3 antagonists, while effective in preventing thrombosis, also carry an increased risk of bleeding, the most common and significant side effect of current antithrombotic drugs. Therefore, clinical dosage selection for antithrombotic drugs must balance the risk of bleeding, making it difficult to achieve a better antithrombotic effect by increasing the dose. In studies using death as an endpoint, it is difficult to find an appropriate dose threshold to reduce the mortality rate due to thrombosis and bleeding. Therefore, by developing a new generation of antithrombotic drugs that do not affect normal hemostasis, it will be possible to achieve stronger antithrombotic efficacy at a lower risk, which represents the development direction of antithrombotic drugs.

[0005] Targeting platelet outside-in signaling rather than inhibiting the bidirectional signaling function of the intact integrin αIIbβ3 receptor can distinguish antithrombotic effects from normal hemostatic functions, potentially achieving profound antithrombotic effects without increasing bleeding risk. The integrin β3 / Src interaction plays a crucial role in platelet outside-in signaling. This goal can be achieved by designing peptides or small molecules that specifically dissociate the β3 / Src interaction. Previous studies have shown that a synthetic RGT tripeptide attached to the cytoplasmic tail of integrin β3 can specifically inhibit platelet outside-in signaling and related platelet functions. Knockout mice for the RGT tripeptide attached to the cytoplasmic tail of integrin β3 also inhibit platelet outside-in signaling and related functions due to the disruption of the β3 / Src interaction. Since integrin β3 mimetic peptides can inhibit platelet outside-in signaling, could new small molecule drugs be developed by targeting sequences on c-Src that interact with β3? Existing studies have found that the c-Src SH3 domain can interact with integrin β3, but the specific site of action is still unclear. The present invention uses Co-IP and surface plasmon resonance (SPR) and other technical means to find that the RT loop of c-Src SH3 has a tendency to bind to integrin β3, while the n-Src loop has a tendency to classical binding, which is involved in the kinase activity and other functions of c-Src. Therefore, the design of new antithrombotic drugs with the RT loop of c-Src SH3 as a target is expected to achieve deep antithrombotic effects while having little effect on normal hemostasis and the activity and function of c-Src. Therefore, there is a need in the art to develop a specific target for the prevention or treatment of thrombosis. Summary of the Invention

[0006] The object of the present invention is to provide a c-Src SH3 RT-loop antagonist for use in anti-thrombotic applications.

[0007] In a first aspect, the present invention provides a use of a c-Src SH3 RT-loop antagonist for preparing a composition or formulation for:

[0008] (a) Interfering with the interaction between integrin β3 and c-Src;

[0009] (b) inhibiting platelet extension on solid fibrinogen;

[0010] (c) inhibiting platelet aggregation and / or adhesion; and / or

[0011] (d) Prevention and / or treatment of thrombosis.

[0012] In another preferred embodiment, the c-Src is human (including human) c-Src.

[0013] In another preferred embodiment, the c-Src SH3 RT-loop is a human (including human) c-Src SH3 RT-loop.

[0014] In another preferred embodiment, the “interference with the interaction between integrin β3 and c-Src” is selected from the following group:

[0015] (a1) Reduce the binding between integrin β3 and the RT-loop region of c-Src SH3;

[0016] (a2) Blocks the binding between integrin β3 and the RT-loop region of c-Src SH3.

[0017] In another preferred embodiment, the integrin β3 includes integrin αIIbβ3.

[0018] In another preferred embodiment, the antagonist is a c-Src SH3 RT-loop region specific antagonist.

[0019] In another preferred embodiment, the "c-Src SH3 RT-loop region specific antagonist" refers to the antagonist that antagonizes (or affects) the binding of integrin β3 and the RT-loop region of c-Src SH3, but does not antagonize (or affect) or substantially does not antagonize the binding of integrin β3 and the n-Src loop region of c-Src SH3.

[0020] In another preferred embodiment, the c-Src SH3 RT-loop antagonist does not antagonize (or affect) or substantially does not affect the binding (or interaction) between integrin β3 and the c-Src SH3 n-loop region.

[0021] In another preferred embodiment, the dissociation constant KD value (denoted as KD) of the interaction between the antagonist and the R95A mutant c-Src protein R95A ), and the dissociation constant Kd value of the interaction between the antagonist and wild-type c-Src protein (denoted as KD wt ) ratio (KD R95A / KD wt ), ≥5, preferably ≥10, more preferably ≥20, and most preferably ≥40.

[0022] In another preferred embodiment, the dissociation constant KD value (denoted as KD) of the interaction between the antagonist and the E97A ​​mutant c-Src protein E97A ), and the dissociation constant KD value of the interaction between the antagonist and wild-type c-Src protein (denoted as KDwt ) ratio (KD E97A / KD wt ), ≥5, preferably ≥10, more preferably ≥20, and most preferably ≥40.

[0023] In another preferred embodiment, the amino acid sequence of the R95A mutant c-Src protein is as shown in SEQ ID No: 1, and the R at position 98 is mutated to A.

[0024] In another preferred embodiment, the amino acid sequence of the E97A ​​mutant c-Src protein is as shown in SEQ ID No: 1, and the E at position 100 is mutated to A.

[0025] In another preferred embodiment, the c-Src SH3 RT-loop antagonist is a dual antagonist of the RT-loop region and the n-Src loop region.

[0026] In another preferred embodiment, the c-Src SH3 RT-loop antagonist includes antagonism of amino acids at positions R95 and / or E97.

[0027] In another preferred embodiment, the antagonist is selected from the group consisting of small molecule antagonists, antisense nucleotides, miRNA, siRNA, or a combination thereof.

[0028] In another preferred embodiment, the antagonist includes: DCDBS84 or a pharmaceutically acceptable salt thereof:

[0029]

[0030] In another preferred embodiment, the antagonist is a structural derivative of DCDBS84, or other small molecule candidate compounds targeting c-Src SH3.

[0031] In another preferred embodiment, the c-Src protein is a mammalian c-Src protein, preferably a human or rodent c-Src protein, more preferably a human or mouse c-Src protein.

[0032] In another preferred embodiment, the RT-loop region of the c-Src SH3 domain is the RT-loop region.

[0033] In another preferred embodiment, the RT-loop region of the c-Src SH3 domain or its encoding gene is derived from mammals (including humans and mice).

[0034] In another preferred embodiment, the c-Src protein is selected from the following group:

[0035] (A) a polypeptide with an amino acid sequence as shown in SEQ ID NO.: 1;

[0036] (B) a c-Src protein derivative or an active fragment thereof obtained by substituting, deleting or adding one or more (usually 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-5) amino acid residues of the amino acid sequence of SEQ ID NO.: 1;

[0037] (C) A c-Src protein derivative or an active fragment thereof having a sequence identity of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% to the amino acid sequence of SEQ ID NO.: 1.

[0038] In another preferred embodiment, the c-Src SH3 domain is selected from the following group:

[0039] (A) a polypeptide with an amino acid sequence as shown in SEQ ID NO.: 2;

[0040] (B) a c-Src SH3 domain derivative, or an active fragment thereof, obtained by substituting, deleting, or adding one or more (usually 1-10, preferably 1-5, more preferably 1-3, and most preferably 1-2) amino acid residues of the amino acid sequence of SEQ ID NO.: 2;

[0041] (C) A c-Src SH3 domain derivative or an active fragment thereof having a sequence identity of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% to the amino acid sequence of SEQ ID NO.: 2.

[0042] In another preferred embodiment, the c-Src SH3 RT-loop is selected from the following group:

[0043] (A) a polypeptide with an amino acid sequence as shown in SEQ ID NO.: 3;

[0044] (B) an RT-loop derivative formed by substituting, deleting or adding one or more (usually 1-5, preferably 1-3, more preferably 1-2, and most preferably 1) amino acid residues of the amino acid sequence of SEQ ID NO.: 3;

[0045] (C) A polypeptide having an amino acid sequence as shown in SEQ ID NO.: 3 and having a mutation selected from the group consisting of R95A and E97A.

[0046] In another preferred embodiment, the antagonist, composition or formulation does not increase or substantially increase the risk of bleeding (or referred to as "improving bleeding").

[0047] In another preferred embodiment, the thrombus includes a cardiovascular and cerebrovascular disease thrombus; more preferably, the thrombus is a cardiovascular and cerebrovascular disease thrombus selected from the following group: myocardial infarction thrombus, cerebral infarction thrombus, ischemic stroke, atherosclerotic thrombus, or a combination thereof.

[0048] In another preferred embodiment, the prevention and / or treatment of thrombosis does not affect or improves bleeding while achieving anti-thrombotic effects.

[0049] In another preferred embodiment, the improvement of bleeding includes inhibiting bleeding, not increasing the risk of bleeding, reducing the risk of bleeding, not causing bleeding side effects and / or not affecting the hemostatic function.

[0050] In another preferred embodiment, the hemostatic function includes platelet hemostatic function.

[0051] In another preferred embodiment, the hemostasis includes physiological hemostasis.

[0052] In another preferred embodiment, the inhibiting platelet aggregation includes inhibiting platelet two-phase aggregation.

[0053] In another preferred embodiment, the inhibiting platelet aggregation includes not inhibiting one phase of platelet aggregation.

[0054] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0055] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and a safe and effective amount of the antagonist.

[0056] In another preferred embodiment, the dosage form of the composition is selected from the following group: solid dosage form, liquid preparation, semi-solid preparation.

[0057] In another preferred embodiment, the composition is selected from the following group: oral preparations and injections.

[0058] In another preferred embodiment, the dosage form of the composition or preparation is selected from the following group: tablets, granules, capsules, injections, infusions, ointments, gels, solutions, microspheres or films.

[0059] In another preferred embodiment, the composition or preparation further comprises other antithrombotic drugs (such as aspirin).

[0060] In another preferred embodiment, the additional antithrombotic drug is selected from the group consisting of aspirin, clopidogrel, eptifibatide, Xuesetong, ginkgo leaf tablets, or a combination thereof.

[0061] In a second aspect, the present invention provides a use of a c-Src SH3 RT-loop agonist for preparing a composition or preparation.

[0062] (a) Promotes the interaction between integrin β3 and c-Src;

[0063] (b) promoting the extension of platelets on solid fibrinogen;

[0064] (c) promoting platelet aggregation and adhesion; and / or

[0065] (d) Promote blood coagulation.

[0066] In a third aspect, the present invention provides a method for interfering with the interaction between integrin β3 and c-Src protein, comprising the steps of:

[0067] (a) In the presence of a c-Src SH3 RT-loop antagonist, integrin β3 and c-Src protein are brought into contact, thereby interfering with the interaction between integrin β3 and c-Src protein.

[0068] In another preferred embodiment, the method is an in vitro method.

[0069] In another preferred embodiment, in step (a), a c-Src SH3 RT-loop antagonist is present, cells expressing integrin β3 and c-Src protein are cultured, and the binding between integrin β3 and c-Src protein is measured.

[0070] In another preferred embodiment, the c-Src protein includes wild-type c-Src protein and mutant c-Src protein.

[0071] In another preferred embodiment, the mutant c-Src protein includes: R95A mutant c-Src protein, E97A ​​mutant c-Src protein, or a combination thereof.

[0072] In a fourth aspect, the present invention provides an anti-thrombotic method (or an anti-platelet aggregation and / or adhesion method), comprising the step of administering a c-Src SH3 RT-loop antagonist to a subject in need thereof.

[0073] In another preferred embodiment, the subject is a human or non-human mammal (rodent, rabbit, monkey, livestock, dog, cat, etc.).

[0074] In a fifth aspect, the present invention provides a method for screening candidate antithrombotic compounds, the method comprising the steps of:

[0075] (a) In a test group, integrin β3 and c-Src protein are contacted in the presence of a test substance, and whether integrin β3 in the test group forms a binding with the RT-loop region of the c-Src SH3 domain is observed; in a control group, integrin β3 and c-Src protein are contacted in the absence of the test substance, and whether integrin β3 in the control group forms a binding with the RT-loop region of the c-Src SH3 domain is observed;

[0076] If the degree or quantity of binding between integrin β3 and the RT-loop region of the c-Src SH3 domain in the test group is significantly lower than that in the control group, it indicates that the test substance is a candidate compound for anti-thrombosis.

[0077] Wherein, the candidate compound is a c-Src SH3 RT-loop antagonist.

[0078] In another preferred embodiment, the drug to be tested is a compound, a protein drug or a gene drug.

[0079] In another preferred embodiment, in step (a), the test is performed in a cell-free system.

[0080] In another preferred embodiment, in step (a), the test is performed in a cell-containing system, wherein the cells express integrin β3 and c-Src protein.

[0081] In another preferred embodiment, the cells are platelets.

[0082] In a sixth aspect, the present invention provides a method for screening candidate antithrombotic compounds, the method comprising the steps of:

[0083] (a) in a first test group, contacting a NITYRGT peptide and a c-Src protein in the presence of a test substance, and observing the amount of a first complex formed between the NITYRGT peptide and the c-Src protein in the test group;

[0084] In a first control group, NITYRGT peptide and c-Src protein are contacted in the absence of the test substance, and the amount of the first complex formed between NITYRGT peptide and c-Src protein in the control group is observed;

[0085] If the amount of the first complex in the first test group is significantly lower than the amount of the first complex in the first control group, it indicates that the test substance is a candidate compound for antithrombotic activity.

[0086] Wherein, the candidate compound is a c-Src SH3 RT-loop antagonist.

[0087] In another preferred embodiment, the method further comprises:

[0088] (b) in a second test group, contacting the RLP1 polypeptide and the c-Src protein in the presence of the test substance, and observing the amount of the second complex formed between the RLP1 polypeptide and the c-Src protein in the test group;

[0089] In a second control group, the RLP1 polypeptide and the c-Src protein are contacted in the absence of the test substance, and the amount of the second complex formed between the RLP1 polypeptide and the c-Src protein in the control group is observed;

[0090] If the amount of the second complex in the second test group is comparable to the amount of the second complex in the second control group, it indicates that the candidate compound is a c-Src SH3 RT-loop specific antagonist (i.e., it mainly acts on the RT-loop region and has little effect on the n-loop region);

[0091] If the amount of the second complex in the second test group is significantly lower than the amount of the second complex in the second control group, it indicates that the candidate compound is a dual antagonist of the c-Src SH3 RT-loop and n-loop regions (i.e., it has effects on both the RT-loop region and the n-loop region).

[0092] In another preferred embodiment, the "significantly lower than" refers to the ratio of the number of complexes or the degree of binding or the amount of binding in the test group (denoted as C1) to the number of complexes or the degree of binding or the amount of binding in the control group (denoted as C0) (C1 / C0) ≤ 1 / 2, preferably ≤ 1 / 3, more preferably ≤ 1 / 4, and most preferably ≤ 1 / 5.

[0093] In another preferred embodiment, the "equivalent" refers to the ratio (C1 / C0) of the number of complexes or the degree of binding or the amount of binding in the test group (denoted as C1) to the number of complexes or the degree of binding or the amount of binding in the control group (denoted as C0) is 0.8-1.2.

[0094] In a seventh aspect, the present invention provides a c-Src mutant protein, wherein the mutant protein has an amino acid mutation at one or more sites selected from the group consisting of: position 95, 96, 97, 98, 99, 100, or a combination thereof, wherein the numbering of the amino acid positions is based on SEQ ID No: 1.

[0095] In another preferred embodiment, the mutant protein has an amino acid mutation at a site selected from the group consisting of: position 95, position 97, or a combination thereof.

[0096] In another preferred embodiment, the c-Src mutant protein has an amino acid mutation selected from the group consisting of R95A, E97A, or a combination thereof.

[0097] In another preferred embodiment, the c-Src mutant protein 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.

[0098] In another preferred embodiment, the c-Src mutant protein has an amino acid mutation selected from the group consisting of G116A, W118A and Y131A.

[0099] In an eighth aspect, the present invention provides a polynucleotide encoding the c-Src mutant protein as described in the seventh aspect of the present invention.

[0100] The ninth aspect of the present invention provides a vector comprising the polynucleotide as described in the eighth aspect of the present invention.

[0101] In a tenth aspect, the present invention provides a host cell, wherein the host cell contains the vector as described in the ninth aspect of the present invention, or the genome thereof contains the polynucleotide as described in the eighth aspect of the present invention.

[0102] In an eleventh aspect of the present invention, a detection kit is provided, comprising:

[0103] (i) Detection reagent for detecting the RT-loop region of the c-Src SH3 domain and its encoding gene.

[0104] In another preferred embodiment, the detection reagent includes a reagent for detecting the amount of c-Src protein or mRNA.

[0105] In another preferred embodiment, the detection reagent includes a reagent for detecting whether there is an amino acid mutation or a nucleotide mutation in the RT-loop region.

[0106] In another preferred embodiment, the detection reagent detects whether there are amino acid mutations at positions 95, 96, 97, 98, 99, and 100 of the c-Src protein, and / or whether there are nucleotide mutations corresponding to the amino acid mutations.

[0107] In another preferred embodiment, the amino acid mutation includes: R95A, E97A, or a combination thereof

[0108] In a twelfth aspect, the present invention provides a use of the detection kit according to the eleventh aspect of the present invention for preparing a diagnostic kit for evaluating whether a test subject (such as a thrombosis patient) is suitable for treatment with a c-SrcSH3 RT-loop antagonist.

[0109] In another preferred embodiment, the diagnostic kit is also used to assess the risk of a test subject suffering from thrombosis.

[0110] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0112] Figure 1 Structural simulations showing the binding of RGT peptide, NITYRGT peptide, and the classical binding peptide containing the PXXP domain (APPIPPPR) to the c-SrcSH3 domain.

[0113] Figure 2 The amino acid sequence of the c-Src SH3 domain and the corresponding secondary structure diagram are shown.

[0114] Figure 3 The results show that after transfection of c-Src SH3 mutants (R95A, E97A, G116A, W118A, and Y131A) into 293Tβ3 cells (293T cells were transfected with integrin β3), Co-IP was used to detect the differences in the interaction between each mutant and β3.

[0115] Figure 4 The binding of RLP1 peptide (containing the PXXP domain, whose binding to c-Src SH3 is considered to be canonical) to c-Src SH3 mutants (R95A, E97A, G116A, W118A, and Y131A) is shown.

[0116] Figure 5 Structural simulation diagram showing the binding of RGT peptide, designed and synthesized small molecule compound DCDBS84, and classic binding peptide containing PXXP domain (APPIPPPR) to c-Src SH3 domain.

[0117] Figure 6 The results show that the binding site of DCDBS84 in the c-Src SH3 domain was detected by NMR experiments.

[0118] Figure 7 The binding site map is displayed based on the results of nuclear magnetic resonance experiments and the chemical shift interference (CSP) of the amino acid sites.

[0119] Figure 8Shown are the binding constants of DCDBS84 to c-Src SH3 (WT) and various mutants (R95A, E97A, G116A, W118A, and Y131A) detected by surface plasmon resonance (SPR).

[0120] Figure 9 Figure 3 shows the binding constants of RLP1 peptide (containing the PXXP domain, whose binding to c-Src SH3 is considered to be canonical) to c-Src SH3 (WT) and its mutants (R95A, E97A, G116A, W118A, and Y131A) detected by SPR.

[0121] Figure 10 Display gene targeting construct c-Src E97A Diagram of the overall strategy for transgenic mice.

[0122] Figure 11 Display c-Src E97A Genotyping results of transgenic mice.

[0123] Figure 12 The application of Co-IP method to detect c-Src E97A Transgenic mice are able to dissociate the interaction between integrin β3 and c-Src.

[0124] Figure 13 Display c-Src E97A Transgenic mice have reduced thrombin-induced platelet aggregation.

[0125] Figure 14 Display c-Src E97A Statistical graph of transgenic mice with reduced thrombin-induced platelet aggregation.

[0126] Figure 15 Display c-Src E97A Transgenic mice have reduced platelet spreading on solid fibrinogen.

[0127] Figure 16 Display c-Src E97A Transgenic mice have reduced platelet adhesion to solid fibrinogen.

[0128] Figure 17 showed that in the FeCl3-induced carotid artery injury model, c-Src E97A Transgenic mice can significantly inhibit thrombosis.

[0129] Figure 18 In the tail-snipping experiment, c-Src E97ATransgenic mice do not have increased bleeding time. DETAILED DESCRIPTION

[0130] After extensive and in-depth research, the present inventors unexpectedly discovered 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.

[0131] Specifically, the experiments of the present invention determined whether the c-Src SH3 binding regions of NITYRGT, RGT peptides, and the β3 cytoplasmic tail of integrin αIIbβ3 differ from the c-Src SH3 binding regions of classical binding peptides (containing the PXXP domain). Classical binding of PXXP domain-containing peptides to c-Src SH3 involves c-Src kinase activity (to which the n-Src loop, primarily W118, is known to contribute significantly) and related signal transduction pathways involved in various cellular functions. However, the β3 / c-Src interaction is considered a relatively weaker non-classical binding mechanism, primarily involved in platelet "outside-in" signaling and associated thrombosis. Structural modeling of the binding of NITYRGT, RGT, and a PXXP domain-containing peptide (APPIPPPR) to the c-Src SH3 domain revealed that NITYRGT prefers to bind to the RT-loop of c-Src SH3, while the PXXP domain-containing peptide (APPIPPPR) prefers to bind to the n-Src loop. Furthermore, by cloning c-Src SH3 mutants R95A, E97A, G116A, W118A, and Y131A, and using Co-IP assays to examine the interaction of c-Src WT and these mutants with αIIbβ3, we found that R95A, E97A, G116A, W118A, and Y131A indeed weakened the β3 / c-Src interaction, with E97A ​​showing the most significant reduction, as E97 is located in the RT-loop of c-Src SH3. Since c-SrcSH3 can interact with proteins containing the PXXP domain, this binding is called classical binding. The present invention synthesized the PXXP-containing polypeptide RLP1 (RKLPPRPSK) and tested the interaction of RLP1 with the wild-type and various mutants of c-Src SH3. The results showed that the binding site of RLP1 tends to be W118, which is mainly the region where the n-Src loop of c-Src SH3 is located.

[0132] The small molecule DCDBS84, which targets c-Src SH3, was screened and tested to determine whether DCDBS84 differentiates between its direct and canonical c-Src SH3 targets. Nuclear magnetic resonance (NMR) and chemical shift shift analysis revealed that the binding sites of DCDBS84 to c-Src SH3 primarily include R95, E97, W118, W119, and Y131. Furthermore, surface plasmon resonance (SPR) assays were used to determine the binding constants of DCDBS84 to c-Src SH3 mutants R95A, E97A, G116A, W118A, and Y131A, as well as the wild-type (WT) c-Src SH3. R95A and E97A ​​significantly weakened the binding of DCDBS84 to c-Src SH3, suggesting that R95 and E97 are primarily involved in the binding of DCDBS84 to c-Src SH3. The present invention also used SPR to detect the binding constants of the classic binding peptide RLP1 with c-Src SH3 mutants R95A, E97A, G116A, W118A, Y131A, and the wild type (WT). The results showed that the G116A, W118A, and Y131A mutations significantly weakened the binding of RLP1 to c-Src SH3, suggesting that G116, W118, and Y131 are primarily involved in the binding of RLP1 to c-Src SH3. This experiment further confirmed that the binding site of the small molecule DCDBS84 on c-Src SH3 is primarily located in the RT-loop, with E97 as the primary target. Simultaneously, structural simulations of the DCDBS84-c-Src SH3 binding site also suggested that it is primarily composed of E97 and the surrounding RT-loop amino acids.

[0133] In addition, c-Src E97A Transgenic mice, genotype identification confirmed WT mice and c-Src E97A Mutant mice, platelets isolated from mice, Co-IP experiments confirmed c-Src E97A Mutant mice can dissociate the β3 / c-Src interaction in platelets, inhibiting platelet aggregation, extension, adhesion and other platelet "outside-in" signal transduction-mediated functions. More importantly, c-Src E97A The mutant mice were able to inhibit thrombosis in an FeCl3-induced thrombosis model and did not experience increased bleeding time compared to wild-type mice in a tail-clip bleeding test. These results suggest that the c-Src SH3 RT-loop and amino acid sites, primarily E97, could serve as novel antithrombotic targets without affecting normal hemostasis, providing target information for the development of novel antithrombotic drugs.

[0134] the term

[0135] As used herein, the terms "comprise," "include," and "contain" 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."

[0136] As used herein, the term "antithrombotic" includes the prevention and / or treatment of thrombosis.

[0137] In the present invention, the term "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. As used herein, "prevention" also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0138] The "treatment" of the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination, and reversal. In some embodiments, the composition or pharmaceutical composition of the present invention reduces, inhibits, and / or reverses the relevant disease (such as tumor) and its complications by inhibiting the mitochondrial oxidative phosphorylation pathway, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the levels observed in the absence of the composition, medicine kit, food box, or health product box, or active ingredient combination of the present invention.

[0139] Src and c-Src

[0140] Src was first discovered as an oncogene protein in Rous sarcoma retrovirus (retrovirus roussarcoma virus), and then it was found that v-Src, which is highly conserved and homologous to Src, is ubiquitous in cells.

[0141] The Src kinase family is a group of proteins with protein tyrosine kinase (PTK) activity, of which c-Src is an important component of the Src kinase family.

[0142] Unless otherwise indicated herein, amino acid sequences are numbered from N-terminus to C-terminus.

[0143] The amino acid sequence of human c-Src is shown in SEQ ID NO.: 1:

[0144] SEQ ID NO.: 1:

[0145] (SEQ ID No: 1, the underlined SH3 domain and the italicized RT-loop region)

[0146] SH3 domain and RT-loop region

[0147] As used herein, "SH3," "SH3 domain," "SH3 domain protein," and "SH3 protein" are used interchangeably.

[0148] The amino acid sequence of a representative wild-type human c-Src SH3 domain is shown in SEQ ID NO.: 2:

[0149] (SEQ ID No: 2, corresponding to positions 87-144 in SEQ ID No: 1).

[0150] As used herein, in human c-Src, R95, E97, T98, L100, D117, W118, W119, A138 are numbered based on the amino acids of the following SH3 domain (SEQ ID NO.: 2), and positions 84 to 141 are as follows:

[0151]

[0152] As used herein, in human c-Src, in the R95A and E97A ​​mutant c-Src proteins, R95A and E97A ​​are numbered based on the amino acids of the following SH3 domain (SEQ ID NO.: 2), and positions 84 to 141 are as follows:

[0153]

[0154] As described herein, R95A refers to the mutation of the amino acid residue at position 95 from an R amino acid to an A amino acid, and E97A ​​refers to the mutation of the amino acid residue at position 97 from an E amino acid to an A amino acid. Amino acid mutations at other positions are the same as described above.

[0155] As used herein, the terms "c-Src SH3 RT-loop" and "RT-loop region of the c-Src SH3 domain" are used interchangeably. The human RT-loop region is located within the human c-Src SH3 domain and has the following amino acid sequence: YDYESRTETDL (SEQ ID No: 3)

[0156] Integrin and integrin β3 / Src interactions

[0157] Integrin αIIbβ3 is a transmembrane heterodimer composed of two subunits, αIIb and β3, formed by non-covalent bonds. It is primarily expressed on the surfaces of platelets and megakaryocytes and serves as the primary membrane receptor on platelets, mediating bidirectional platelet signaling. Therefore, it plays a key role in platelet activation, maintaining normal platelet function, and thrombosis. Platelet activators such as thrombin and ADP, upon interaction with their corresponding receptors, induce conformational changes in integrin αIIbβ3, increasing its affinity for its ligands, such as soluble fibrinogen. This process is inside-out signaling, with hallmark events including unstable platelet adhesion, free fibrinogen binding, and reversible aggregation. Activated integrin αIIbβ3 and ligand binding activate outside-in signaling, with hallmark events including stable platelet adhesion, extension, irreversible aggregation, and fibrin clot retraction. Ultimately, platelets aggregate and form a relatively stable thrombus, completing the physiological and pathological processes of hemostasis and thrombosis. The current consensus is that the realization of hemostasis and thrombosis requires the joint participation of inside-out and outside-in signal transduction, and the pathological process of thrombosis requires the increase of platelet plugs under conditions of high blood flow impact. Therefore, thrombosis is relatively more dependent on outside-in signal transduction.

[0158] To more specifically target platelet receptors involved in thrombosis, receptor antagonists targeting integrin αIIbβ3 have been developed. As the final common pathway mediating platelet activation, aggregation, and thrombosis, integrin αIIbβ3 is a major target for antithrombotic drug research. Indeed, significant progress has been made in the study of integrin αIIbβ3 as an antithrombotic drug target, with the current focus on integrin αIIbβ3 receptor antagonists, which have demonstrated excellent clinical efficacy. Currently, three integrin αIIbβ3 receptor antagonist antiplatelet drugs have been approved by the US Food and Drug Administration (FDA) for clinical antithrombotic treatment: abciximab, eptifibatide, and tirofiban. These αIIbβ3 receptor antagonists exert their specific antithrombotic effects by interfering with the interaction between integrin αIIbβ3 and its ligands. However, this strategy still presents significant challenges. αIIbβ3 receptor antagonists block integrin αIIbβ3 binding to its ligand, thereby disrupting bidirectional signaling. This means that while exerting antithrombotic effects, they also impair normal hemostasis. Clinical trials have shown that approximately 2% of patients treated with integrin αIIbβ3 antagonists experience severe intracranial hemorrhage, approximately 15% experience gastrointestinal bleeding, approximately 5-10% experience peritoneal bleeding, and approximately 60-80% experience significant bleeding at the femoral artery puncture site. Like the classic antithrombotic drugs aspirin and clopidogrel, integrin αIIbβ3 antagonists, while effective in preventing thrombosis, also carry an increased risk of bleeding, the most common and significant side effect of current antithrombotic drugs. Therefore, clinically, the choice of antithrombotic drug dosage must also consider the risk of bleeding side effects, making it difficult to achieve a better antithrombotic effect by increasing the antithrombotic drug dose. In studies using death as an endpoint, it is difficult to find an appropriate dose threshold to reduce the mortality rate due to thrombosis and bleeding. Therefore, by developing a new generation of antithrombotic drugs that do not affect normal hemostasis, it will be possible to achieve stronger antithrombotic efficacy at a lower risk, which represents the development direction of antithrombotic drugs.

[0159] Studies have found that the cytoplasmic tail of integrin αIIbβ3 plays an important role in platelet "outside-in" signaling, such as stable platelet adhesion and extension on solid fibrinogen, two-phase aggregation, and fibrin clot retraction, through interaction with the cytoplasmic protein c-Src. However, it has little effect on platelet "inside-out" signaling, such as platelet binding to soluble fibrinogen, initial platelet adhesion, and one-phase aggregation. Studies have shown that platelet "outside-in" signaling primarily participates in platelet thrombosis, while "inside-out" signaling plays a greater role in hemostasis.

[0160] Studies have shown that the SH3 domain of Src kinase constitutively binds to the three amino acids of RGT at the C-terminus of integrin β3. During outside-in signaling following integrin activation, Src kinase interacts with the RGT sequence at the C-terminus of β3 and phosphorylates Y747 and Y759 of the cytoplasmic segment of β3, a key event in outside-in signaling. In vivo experiments have demonstrated that RGT knockout mice are protected from FeCl3-induced carotid artery thrombosis. In tail-cut bleeding experiments, some mice exhibited prolonged bleeding time, but did not exhibit spontaneous bleeding, postoperative hemorrhage, bloody stools, hematuria, or anemia. Given the crucial role of the β3RGT / c-Src interaction in platelet "outside-in" signaling, it is reasonable to suggest that synthetic RGT tripeptides could exert antithrombotic effects by competing with endogenous β3RGT / c-Src interactions.

[0161] use

[0162] The present invention provides a use of a c-Src SH3 RT-loop antagonist, which includes (but is not limited to) one or more uses of the following groups:

[0163] (a) Interfering with the interaction between integrin β3 and c-Src;

[0164] (b) inhibiting platelet extension on solid fibrinogen;

[0165] (c) inhibiting platelet aggregation and / or adhesion; and

[0166] (d) Prevention and / or treatment of thrombosis.

[0167] In a preferred embodiment, integrin β3 includes (but is not limited to) integrin αIIbβ3

[0168] In another preferred embodiment of the present invention, the c-Src SH3 RT-loop antagonist is a c-Src SH3 RT-loop region-specific antagonist. Typically, the c-Src SH3 RT-loop antagonist does not antagonize (or affect) or substantially does not affect the binding (or interaction) between integrin β3 and the c-Src SH3 n-loop region.

[0169] The specific type of c-Src SH3 RT-loop antagonist described in the present invention is not particularly limited, as long as it can have an antagonistic effect on the c-Src SH3 RT-loop. For example, the c-Src SH3 RT-loop antagonist can be a small molecule antagonist, antisense nucleotide, miRNA, and siRNA. Preferably, the antagonist is the DCDBS84 compound:

[0170]

[0171] In the present invention, the c-Src protein is not particularly limited, but is preferably a mammalian c-Src protein, such as a human or rodent c-Src protein. Representatively, the amino acid sequence of the human c-Src protein is the polypeptide set forth in SEQ ID NO.:1. Positions 87-144 (SEQ ID NO:2) of the polypeptide set forth in SEQ ID NO.:1 represent the human c-Src SH3 domain. The amino acid sequence of the human c-Src SH3 RT-loop is the polypeptide set forth in SEQ ID NO.:3.

[0172] In a preferred embodiment of the present invention, the c-Src SH3 RT-loop antagonist does not increase or substantially increase the risk of bleeding during antithrombotic treatment (or referred to as "improving bleeding"). In the present invention, improving bleeding includes inhibiting bleeding, not increasing the risk of bleeding, reducing the risk of bleeding, not causing bleeding side effects, and / or not affecting hemostasis.

[0173] In the present invention, the thrombus includes thrombus caused by cardiovascular and cerebrovascular diseases. Preferably, the thrombus includes (but is not limited to): myocardial infarction thrombus, cerebral infarction thrombus, ischemic stroke, atherosclerotic thrombus, or a combination thereof.

[0174] Compositions or formulations, combinations of active ingredients and kits and methods of administration

[0175] The present invention also provides a composition comprising a c-Src SH3 RT-loop antagonist.

[0176] The composition of the present invention is preferably a pharmaceutical composition. The composition of the present invention may include a pharmaceutically acceptable carrier.

[0177] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, liquid, or gel fillers suitable for human or animal use and of sufficient purity and low toxicity. "Compatibility" refers to the ability of the components of a pharmaceutical composition to function with the active ingredient of the drug, and for them to coexist with each other, without significantly reducing the efficacy of the drug.

[0178] It should be understood that in the present invention, the pharmaceutically acceptable carrier is not particularly limited and can be selected from commonly used materials in the art, or prepared by conventional methods, or purchased from the market. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, 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 regulators, transdermal enhancers, colorants, flavorings, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0179] In the present invention, the dosage form of the composition is not particularly limited, and can be a solid dosage form, a liquid preparation, or a semi-solid preparation.

[0180] In the present invention, the dosage forms of the compositions and preparations include but are not limited to oral preparations, injection preparations, and external preparations.

[0181] Typically, the dosage forms of the compositions and preparations include, but are not limited to, tablets, granules, capsules, injections, infusions, pastes, gels, solutions, microspheres, or films.

[0182] Typically, the injection is an intravenous injection.

[0183] The pharmaceutical preparation should be compatible with the mode of administration. The preferred modes of administration are oral administration and injection (e.g., intravenous injection). When used, a therapeutically effective amount of the drug is administered to the desired subject (e.g., a human or non-human mammal). As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will 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.

[0184] In one administration regimen, a safe and effective daily dose of the first active ingredient is generally at least about 0.1 mg and, in most cases, does not exceed about 2500 mg. Preferably, this dose is 1 mg to 500 mg. A safe and effective amount of the second active ingredient is generally at least about 0.01 mg and, in most cases, does not exceed 2500 mg. Preferably, this dose ranges from 0.1 mg to 2500 mg. Of course, the specific dose will also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0185] The main advantages of the present invention are:

[0186] The present invention discovered 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.

[0187] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, parts and percentages are by weight.

[0188] Example

[0189] The amino acid sequence of mouse c-Src is shown in SEQ ID NO.: 4:

[0190] SEQ ID NO.: 4

[0191] MGSNKSKPKDASQRRRSLEPSENVHGAGGAFPASQTPSKPASADGHRGPSAAFVPPAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTRKVDVREGDWWLAHSLSTG QTGYIPSNYVAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEI PRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKGETGKYLRLPQLVDMSAQIASGMAYVERMNYVHRDLRAANILVGEN LVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL

[0192] The amino acid sequence of the mouse c-Src SH3 domain is shown in SEQ ID NO.: 5:

[0193] SEQ ID NO.: 5

[0194] TTFVALYDYESRTETDLSFKKGERLQIVNNTRKVDVREGDWWLAHSLSTGQTGYIPSNYVAPSD

[0195] The amino acid sequence of the mouse c-Src SH3 domain containing the RT-loop region is shown in SEQ ID NO.: 6:

[0196] SEQ ID NO.: 6

[0197] YDYESRTETDL

[0198] The structural formula of compound DCDBS84 is as follows:

[0199]

[0200] In the Examples, in human c-Src, R95, E97, T98, L100, D117, W118, W119, and A138 are numbered based on the amino acids of the following SH3 domain (SEQ ID NO.: 2), and positions 84 to 141 are as follows.

[0201]

[0202] In mice, R95 and E97 are numbered based on the amino acids of the following SH3 domain (SEQ ID NO.: 5), with positions 84 to 147 as shown below:

[0203] 84 TTFVALY DYESRTETDL SFKKGERLQI 110

[0204] 111 VNNTRKVDVR EGDWWLAHSL STGQTGYIPS NYVAPSD 147

[0205] Example 1 Structural simulation of binding peptide to c-Src SH3 domain

[0206] Based on the RGT peptide (crystal structure), NITYRGT peptide (NMR structure) and the classical binding peptide containing the PXXP domain (APPIPPPR) (NMR structure), the structural simulation diagram of the binding of RGT, NITYRGT and the classical binding peptide containing the PXXP domain (APPIPPPR) to the c-Src SH3 domain was obtained through computer structural analysis. Figure 1 As shown in the figure, the binding of the YRGT tetrapeptide from the solid phase crystal structure to c-Src SH3 is biased towards the n-Src loop, while the binding of the NITYRGT heptapeptide from the liquid phase NMR structure to c-Src SH3 is more biased towards the RT-loop. The binding direction of the classical binding peptide (APPIPPPR) to c-SrcSH3 is basically perpendicular to the binding direction of NITYRGT to c-Src SH3, and is more biased towards the n-Src loop. Based on the amino acid sequence information and secondary structure information of c-Src SH3, the structural model diagram of c-Src SH3 was drawn (as shown in the figure). Figure 2 ).

[0207] Example 2: Detection of binding sites by co-immunoprecipitation (Co-IP)

[0208] In this example, mutants of the possible binding site of integrin β3 at c-Src SH3 were constructed, and the binding site of β3 at c-Src SH3 was detected by co-immunoprecipitation (Co-IP).

[0209] Five c-Src SH3 gene point mutation overexpression vectors were constructed, namely 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, and Y131 is located in the β4 region (e.g. Figure 2 shown).

[0210] The co-immunoprecipitation technique (Co-IP) was used to detect the binding of c-Src SH3 mutant to integrin β3 subunit. First, 0.4 ml of platelets (concentration 3×10 8 / 100 μg of protein was lysed in 50 μl of Protein A+G agarose beads pre-washed with lysis buffer. The beads were incubated with rotation at 4°C for 2 h to remove nonspecific contaminants and reduce background. After incubation, the beads were centrifuged at 1000 × g for 5 min at 4°C, and the supernatant was transferred to a fresh centrifuge tube. 1 μg of anti-Flag tag antibody M2 (Sigma) or 1 μg of nonspecific mouse IgG (sc-2025, Santa Cruz Biotechnology) was added to the supernatant, and the antigen-antibody mixture was incubated with rotation at 4°C overnight. The following morning, 20 μl of Protein A+G agarose beads pre-washed with lysis buffer were added, and the beads were incubated with rotation at 4°C for 2 h. The beads-antigen-antibody complex was then centrifuged at 1000 × g for 5 min at 4°C to collect the agarose bead-antigen-antibody complex. The supernatant was removed and the beads were washed five times with pre-chilled RIPA lysis buffer, using 800 μl each wash, and then allowed to stand on ice for 10 min. The agarose beads-antigen-antibody complex was then resuspended in 1× SDS-PAGE sample buffer (100 mM Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 4% SDS, 20% glycerol, 0.1% Bromophenol Blue) and boiled at 100°C for 8 min to denature the protein. The immunoprecipitates were detected by Western blot. Figure 3 As shown, R95 and E97 mutations significantly reduced β3 / c-Src binding.

[0211] Example 3 ELISA method was used to detect the binding of RLP1 peptide to c-Src SH3 mutant

[0212] Ligand proteins that interact with Src kinase in cells typically target the Src-SH3 domain through a canonical (PXXP) motif. The RLP1 peptide (containing a canonical PXXP motif) mimics the common PXXP motif sequence found in various ligand proteins that interact with the Src-SH3 domain in cells. The specific experimental procedure is as follows: 50 μL of coating buffer containing Flag antibody M2 (Sigma) (1 μg / mL, diluted in 0.1 M NaHCO₃, pH 8.3) was added to each well of a 96-well plate and incubated overnight at 4°C. The next day, the plate was washed three times with 1× TBST (5 minutes each time), blocked with 5% BSA for 2 hours, and then washed three times with 1× TBST before use. In the early stage, 293T cells were transfected with pFlag-CMV4-Src (WT) and five other 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). Lipofect 2000 (Invitrogen Life technologies) was used for transfection. 48 hours after transfection, 293T cells were lysed on ice with RIPA for 30 minutes and the supernatant was collected by centrifugation (4°C, 18000 rpm). 50 μL of the supernatant was added to each well of a 96-well plate coated with Flag antibody and incubated overnight at 4°C. A portion of the supernatant from each experimental group was retained for relative quantification by Western blotting. On the third day, wash three times with TBST, add 50 μL of biotin-labeled RLP1 and control (RLA) peptide (1 μg / mL) to each well, and incubate at 37°C for 1 hour. Then wash three times with 1×TBST, 5 minutes each time, add 50 μL of HRP-labeled avidin to each well, and incubate at 37°C for 1 hour. Wash three times with 1×TBST, add 100 μL of TMB display solution to each well, incubate at room temperature for 15 minutes, and then add 100 μL of sulfuric acid (1M) to terminate the reaction. Use an enzyme reader to detect the absorbance of each well at a wavelength of 450 nm. The relative quantitative results of the absorbance value and the grayscale value of the Western blot band are shown as follows. Figure 4 The results showed that W118 and Y131 mutations significantly reduced the binding of RLP1 peptide to c-Src.

[0213] Example 4 Nuclear Magnetic Resonance Determination of Amino Acid Binding Sites

[0214] In this example, the nuclear magnetic resonance spectra of the amino acid sites of the protein (c-Src SH3) before and after the addition of DCDBS84 were determined by nuclear magnetic resonance.

[0215] Based on the crystal structure of RGT peptide bound to c-Src SH3, the nuclear magnetic resonance structure of NITYRGT bound to c-Src SH3, and other information, a small molecule targeting c-Src SH3, DCDBS84, was selected through computer simulation screening and analysis. The structural simulation of DCDBS84 binding to c-Src SH3 is shown in the figure below. Figure 5 As shown, DCDBS84 mainly binds to the RT-loop of c-Src SH3.

[0216] NMR experiments were performed on a four-channel Bruker Avance III 600 MHz spectrometer. Culture media were prepared with 15N instead of 14N to purify 15N-labeled c-Src-SH3 protein for 2D NMR. For 3D NMR, 15N replaced 14N and 13C replaced 12C. Figure 6 (Left) shows the two-dimensional 15N-HSQC experiment of the interaction between Src-SH3 and DCDBS84, with the concentration of c-Src-SH3 at 50 μM and the concentration of DCDBS84 at 20 times that of c-Src-SH3; Figure 6 As shown in the right figure, the concentration of 15N / 13C labeled c-Src-SH3 protein was adjusted to 1.3 mM to complete the assignment of the amino acid sites of the protein.

[0217] Example 5 Chemical Shift Interference (CSP) Analysis of Binding Sites

[0218] In the NMR experiment, the binding site map was analyzed based on the chemical shift interference (CSP) of the amino acid sites.

[0219] Calculate chemical shift perturbation (CSP) according to the formula: The CSP mean + standard error is used as the baseline, and values ​​greater than this value are considered binding sites. Figure 7 The results shown showed that R95, E97, T98, L100, D117, W118, W119, and A138 may be the binding sites of SH3 and the compound.

[0220] Example 6: Binding properties of antagonist DCDBS84 to wild-type and mutant c-Src SH3

[0221] In this example, the dissociation constants of DCDBS84 with c-Src SH3 (WT) and mutants (R95A, E97A, G116A, W118A, Y131A) were determined by surface plasmon resonance experiments.

[0222] Surface plasmon resonance testing was performed on a BIACORE T200 instrument (GE). Purified c-Src SH3 protein (concentration 2 mg / ml) was diluted to 0.1 mg / ml with 10 mM CH3COONa (pH 4.2), and then the SH3 protein was coupled to a CM5 chip using a standard amino coupling method. DCDBS84 was diluted with a buffer solution (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), and the sample was continuously injected for 60 seconds at a flow rate of 20 μl / s and dissociated for 120 seconds. The changes in the response value over time were recorded, and the dissociation constant KD of DCDBS84 and c-SrcSH3 protein (WT and mutant) was obtained by analysis using the BIA Evaluation Software (GE Healthcare) program. Figure 8 As shown, the dissociation constants of DCDBS84 with wild-type c-Src SH3 protein were 0.83 μM, 48 μM with R95A, 35 μM with E97A, 8.1 μM with G116A, 6.24 μM with W118A, and 1.52 μM with Y131, suggesting that R95 and E97, located in the RT-loop, are primarily involved in the binding of DCDBS84 to c-Src SH3.

[0223] Furthermore, the dissociation constant KD of the classical binding peptide RLP1 and c-Src SH3 protein (WT and mutant) was detected according to the above method. Figure 9 As shown, the dissociation constants of RLP1 with wild-type c-Src SH3 protein were 9.9 μM, 10.9 μM with R95A, 22 μM with E97A, >80 μM with G116A, 49.9 μM with W118A, and 85.9 μM with Y131A, suggesting that G116 and W118 in the n-Src loop and Y131 in the distal loop are primarily involved in the binding of RLP1 to c-Src SH3.

[0224] These results suggest that the primary binding sites of the small molecule DCDBS84 on c-Src SH3 tend to be R95 and E97 of the RT-loop, while the primary binding sites of the canonical PXXP domain-containing peptide (RLP1) on c-Src SH3 tend to be G116 and W118 of the n-Src loop and Y131 of the distal loop. This suggests that there is indeed a distinction between non-canonical and canonical binding on c-Src SH3.

[0225] Example 7 Gene Targeting to Construct Transgenic Mice

[0226] In this example, c-Src was constructed by gene targeting E97A Mutant transgenic mice, the overall strategy for constructing transgenic mice by gene targeting is as follows Figure 10 shown.

[0227] Example 8: c-Src E97A Genotyping of transgenic mice

[0228] In this example, primers containing the mouse gene mutation site (MusE99A) corresponding to human E97A ​​were designed based on the mouse Src gene (NC_000068.7) sequence. The primer sequences are: c-Src E97A -F:5'-GAACACCTAGTCTGCAGCCC-3',c-Src E97A -R:5'-AGCAGAGAGAAGGAGAGG

[0229] CT-3', amplified fragment length 419 bp (such as Figure 11 The PCR product was sequenced and analyzed. If the 99th amino acid in the mouse gene is GAG, it is glutamic acid (E), and if it is GCG, it is alanine (A). If two peaks of GAG and GCG appear, it is a heterozygote. The results of gene sequencing are as follows Figure 11 As shown in the figure below.

[0230] Example 9: Co-immunoprecipitation

[0231] In this example, c-Src was detected by co-immunoprecipitation (Co-IP). E97A β3 / c-Src interaction in transgenic mouse platelets.

[0232] Wild-type (WT) mice and c-Src E97AThree transgenic mice were anesthetized with phenobarbital according to their body weight. Cardiac blood was collected and anticoagulated with 0.38% sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation at 300×g for 7 minutes. One-quarter volume of ACD was added to the PRP for anticoagulation, and the platelets were centrifuged at 500×g for 10 minutes. The supernatant was discarded. Platelets were washed with CGS washing solution (13 mM sodium citrate, 120 mM sodium chloride, 30 mM glucose, pH 6.5) and resuspended in Tyrode's buffer (0.1% bovine serum albumin, 5 mM 4-hydroxyethylpiperazineethanesulfonic acid, 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). Platelet counts were performed using a small animal blood routine tester (PoCH-100iV Diff), and the platelet density was adjusted to 3×10 8 / ml, and let it stand at room temperature for 1 hour.

[0233] The Co-IP method was used to detect the effect of the small molecule compound DCDBS84 on the interaction between integrin αIIbβ3β3 and c-Src. 400 μl of the solution was used to prepare 3×10 8Platelets at a concentration of 100 μg / ml were lysed on ice in IP buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.2% NP-40) for 30 minutes. Centrifuge (4°C, 12,000 rpm, 15 minutes) and remove the supernatant for BCA protein quantification. Add 50 μl of Protein A / G agarose beads pre-washed with IP buffer and incubate at 4°C with rotation for 2 hours. Centrifuge (4°C, 1,000 g, 5 minutes) and transfer the supernatant to a fresh centrifuge tube. Anti-integrin mouse β3 antibody SZ-21 (1 μg) or non-specific mouse IgG (sc-2025, Santa Cruz Biotechnology, 1 μg), or rabbit monoclonal antibody c-Src antibody (36D10, #2109, Cell signaling Technology, 1 μg) and non-specific rabbit IgG (#2729, Cell signaling Technology, 1 μg) were added to the protein supernatant and the antigen-antibody mixture was incubated overnight at 4°C with rotation. The next morning, 20 μl of Protein A / G agarose beads pre-washed with IP buffer were added to the mixture and incubated at 4°C with rotation for 2 hours. The mixture was centrifuged (4°C, 1000g, 5 minutes) to collect the agarose bead-antigen-antibody complex and washed three times with pre-cooled 1× PBS buffer. Finally, the agarose bead-antigen-antibody complex was resuspended with 1× SDS loading buffer and boiled at 100°C for 10 minutes. The immunoprecipitate was detected by Western blot. The results are shown in Figure 2. Figure 12 As shown, whether β3 antibody IP or c-Src antibody IP was used, c-Src E97A The β3 / c-Src interaction was significantly weakened in mouse platelets.

[0234] Example 10: c-Src E97A Effects of transgenic mice on platelet aggregation.

[0235] The method described in Example 9 was used to extract the WT and c-Src E97A Platelets were isolated from three transgenic mice. PRP was obtained by centrifugation at 300 × g for 7 min, and then platelet-poor plasma (PPP) was obtained by centrifugation at 500 × g for 10 min. The platelet concentration in PRP was adjusted to 2 × 10 8 / ml. Then take 200 μl of PPP to calibrate the zero point of the instrument light transmission aggregometer (Chrono-Log). Then, each group of reagents was incubated with PRP at 37°C for 60 minutes, and 200 μl / tube was placed on the aggregometer (37°C, 1000 rpm stirring). After calibrating the zero point, 0.1 U / ml thrombin was added to start the reaction and the aggregation curve was recorded. Figure 13 As shown, compared with WT mice, c-Src E97A Transgenic mice significantly inhibited platelet aggregation in the second phase without affecting the first phase, indicating that c-Src E97A The transgenic mice significantly inhibited thrombosis without affecting normal physiological hemostasis. Figure 13 The statistical chart is as follows Figure 14 shown.

[0236] Example 11: c-Src E97A Effects of transgenic mice on platelet extension and adhesion.

[0237] The method described in Example 9 was used to extract the WT and c-Src E97A Platelets were isolated from three transgenic mice. The platelet concentration was adjusted to 2 × 10 8 / ml.

[0238] Add 50 μl of fibrinogen (0.1 M, pH 8.3, diluted in sodium bicarbonate, 20 μg / ml) to a 96-well plate and coat overnight at 4°C. The next morning, wash three times with 1× PBS and block with bovine serum albumin (BSA 20 mg / ml) at 37°C for 60 min. Take 50 μl of the washed platelet suspension (concentration 2×10 8 Platelets were added to a 96-well plate and allowed to adhere in a 37°C incubator for 60 minutes. Non-adherent platelets were removed by washing with PBS three times, and the adhered platelets were fixed with 4% paraformaldehyde and washed with PBS three times. The platelet membrane was then perforated with 0.5% Triton X-100, and the platelets were stained with 0.5 μg / ml phalloidin-rhodamine at 37°C for 60 minutes and washed three times with 1× PBS (10 minutes each time). After washing, the fluorescence was observed using a fluorescence microscope (Leica). The results are shown in Figure 2. Figure 15 As shown, c-Src E97A The platelets of transgenic mice extended significantly less on solid fibrinogen than those of WT mice.

[0239] The coating and blocking methods of fibrinogen in the adhesion experiment are the same as those in the stretching experiment. 50 μl of platelets are added to a 96-well plate and allowed to adhere in a 37°C incubator for 60 minutes. After adhesion is completed, wash with PBS 5 times to remove unadhered and unstable adhered platelets. Add 10 μl / well of CCK-8 to the wells where platelets adhere and incubate in a 37°C incubator for 2 hours. Finally, read the OD value at a wavelength of 405 nm using an enzyme reader. Use the wells without platelets as blank controls to calculate the amount of adhered platelets. Set up 3 replicate wells for each sample and take the average of the results. Figure 16 As shown, compared with WT mice, c-Src E97A Transgenic mice can significantly inhibit the adhesion function of platelets on solid fibrinogen (p<0.01), thereby inhibiting thrombosis.

[0240] Example 12: c-Src E97A Effects of transgenic mice on FeCl3-induced carotid artery occlusion and thrombosis.

[0241] WT and c-Src cells aged 6-8 weeks were selected E97A Transgenic mice were used as experimental subjects. Following a reported experimental method, FeCl₃ was used to stimulate the mouse carotid artery, causing endothelial damage and subsequently initiating thrombosis. Blood flow was monitored with a Doppler ultrasound probe distal to the FeCl₃ stimulation site. When upstream thrombosis formed and blocked the vessel, blood flow decreased.

[0242] From the results ( Figure 17 ) It can be seen that compared with WT mice, c-Src E97A The carotid artery thrombosis time of transgenic mice was significantly prolonged under FeCl3 stimulation. E97A The transgenic mice showed a significant inhibitory effect on FeCl3-induced carotid artery thrombosis and had anti-thrombotic efficacy.

[0243] Example 13: c-Src E97A The effect of tail snip on bleeding time in transgenic mice was examined.

[0244] To evaluate c-Src E97A Effects of transgenic mice on hemostasis function, WT and c-Src mice aged 6-8 weeks were selected E97A The study was conducted on transgenic mice. The tail-cutting experiment is a process in which platelets maintain normal hemostasis and plug the wound after a blood vessel ruptures. The mouse's tail is quickly cut with a sharp blade 5 mm from the tip. Blood oozing from the tail is then blotted with filter paper every 15 seconds. Avoid touching the tail to avoid causing new damage. The bleeding stops and does not recur within 15 seconds as the standard.

[0245] From the results ( Figure 18 ) It can be seen that the WT mice stopped bleeding at about 7.4 min, while compared with the WT group, c-Src E97A The bleeding time of transgenic mice was not significantly prolonged, about 8.9 minutes, which was not statistically different from that of WT. E97A Transgenic mice do not prolong the bleeding time after tail clipping and have little effect on normal physiological hemostasis.

[0246] discuss

[0247] The c-Src SH3 RT-loop antagonist described in the present application can reduce, inhibit or interfere with the interaction between integrin β3 and c-Src, thereby significantly exerting an anti-thrombotic effect without affecting normal physiological hemostasis.

[0248] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto. Sequence Listing <110> Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine Shanghai Institute of Materia Medica, Chinese Academy of Sciences <120> A c-Src SH3 RT-loop as a target for antithrombotic therapy <130> P2020-0745 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 536 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Ser Asn Lys Ser Lys Pro Lys Asp Ala Ser Gln Arg Arg Arg 1 5 10 15 Ser Leu Glu Pro Ala Glu Asn Val His Gly Ala Gly Gly Gly Ala Phe 20 25 30 Pro Ala Ser Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp Gly His Arg 35 40 45 Gly Pro Ser Ala Ala Phe Ala Pro Ala Ala Ala Glu Pro Lys Leu Phe 50 55 60 Gly Gly Phe Asn Ser Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly 65 70 75 80 Pro Leu Ala Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu 85 90 95 Ser Arg Thr Glu Thr Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln 100 105 110 Ile Val Asn Asn Thr Glu Gly Asp Trp Trp Leu Ala His Ser Leu Ser 115 120 125 Thr Gly Gln Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp 130 135 140 Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys Ile Thr Arg Arg Glu 145 150 155 160 Ser Glu Arg Leu Leu Leu Asn Ala Glu Asn Pro Arg Gly Thr Phe Leu 165 170 175 Val Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Cys Leu Ser Val Ser 180 185 190 Asp Phe Asp Asn Ala Lys Gly Leu Asn Val Lys His Tyr Lys Ile Arg 195 200 205 Lys Leu Asp Ser Gly Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe Asn 210 215 220 Ser Leu Gln Gln Leu Val Ala Tyr Tyr Ser Lys His Ala Asp Gly Leu 225 230 235 240 Cys His Arg Leu Thr Thr Val Cys Pro Thr Ser Lys Pro Gln Thr Gln 245 250 255 Gly Leu Ala Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser Leu Arg Leu 260 265 270 Glu Val Lys Leu Gly Gln Gly Cys Phe Gly Glu Val Trp Met Gly Thr 275 280 285 Trp Asn Gly Thr Thr Arg Val Ala Ile Lys Thr Leu Lys Pro Gly Thr 290 295 300 Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Gln Val Met Lys Lys Leu 305 310 315 320 Arg His Glu Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu Pro 325 330 335 Ile Tyr Ile Val Thr Glu Tyr Met Ser Lys Gly Ser Leu Leu Asp Phe 340 345 350 Leu Lys Gly Glu Thr Gly Lys Tyr Leu Arg Leu Pro Gln Leu Val Asp 355 360 365 Met Ala Ala Gln Ile Ala Ser Gly Met Ala Tyr Val Glu Arg Met Asn 370 375 380 Tyr Val His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val Gly Glu Asn 385 390 395 400 Leu Val Cys Lys Val Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu Asp 405 410 415 Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp Thr 420 425 430 Ala Pro Glu Ala Ala Leu Tyr Gly Arg Phe Thr Ile Lys Ser Asp Val 435 440 445 Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Thr Thr Lys Gly Arg Val 450 455 460 Pro Tyr Pro Gly Met Val Asn Arg Glu Val Leu Asp Gln Val Glu Arg 465 470 475 480 Gly Tyr Arg Met Pro Cys Pro Pro Glu Cys Pro Glu Ser Leu His Asp 485 490 495 Leu Met Cys Gln Cys Trp Arg Lys Glu Pro Glu Glu Arg Pro Thr Phe 500 505 510 Glu Tyr Leu Gln Ala Phe Leu Glu Asp Tyr Phe Thr Ser Thr Glu Pro 515 520 525 Gln Tyr Gln Pro Gly Glu Asn Leu 530 535 <210> 2 <211> 58 <212> PRT <213> Artifical Sequence <400> 2 Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu Ser Arg Thr Glu Thr Asp 1 5 10 15 Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln Ile Val Asn Asn Thr Glu 20 25 30 Gly Asp Trp Trp Leu Ala His Ser Leu Ser Thr Gly Gln Thr Gly Tyr 35 40 45 Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp 50 55 <210> 3 <211> 11 <212> PRT <213> Artifical Sequence <400> 3 Tyr Asp Tyr Glu Ser Arg Thr Glu Thr Asp Leu 1 5 10 <210> 4 <211> 541 <212> PRT <213> Artifical Sequence <400> 4 Met Gly Ser Asn Lys Ser Lys Pro Lys Asp Ala Ser Gln Arg Arg Arg 1 5 10 15 Ser Leu Glu Pro Ser Glu Asn Val His Gly Ala Gly Gly Ala Phe Pro 20 25 30 Ala Ser Gln Thr Pro Ser Lys Pro Ala Ser Ala Asp Gly His Arg Gly 35 40 45 Pro Ser Ala Ala Phe Val Pro Pro Ala Ala Glu Pro Lys Leu Phe Gly 50 55 60 Gly Phe Asn Ser Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly Pro 65 70 75 80 Leu Ala Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu Ser 85 90 95 Arg Thr Glu Thr Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln Ile 100 105 110 Val Asn Asn Thr Arg Lys Val Asp Val Arg Glu Gly Asp Trp Trp Leu 115 120 125 Ala His Ser Leu Ser Thr Gly Gln Thr Gly Tyr Ile Pro Ser Asn Tyr 130 135 140 Val Ala Pro Ser Asp Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys 145 150 155 160 Ile Thr Arg Arg Glu Ser Glu Arg Leu Leu Leu Asn Ala Glu Asn Pro 165 170 175 Arg Gly Thr Phe Leu Val Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr 180 185 190 Cys Leu Ser Val Ser Asp Phe Asp Asn Ala Lys Gly Leu Asn Val Lys 195 200 205 His Tyr Lys Ile Arg Lys Leu Asp Ser Gly Gly Phe Tyr Ile Thr Ser 210 215 220 Arg Thr Gln Phe Asn Ser Leu Gln Gln Leu Val Ala Tyr Tyr Ser Lys 225 230 235 240 His Ala Asp Gly Leu Cys His Arg Leu Thr Thr Val Cys Pro Thr Ser 245 250 255 Lys Pro Gln Thr Gln Gly Leu Ala Lys Asp Ala Trp Glu Ile Pro Arg 260 265 270 Glu Ser Leu Arg Leu Glu Val Lys Leu Gly Gln Gly Cys Phe Gly Glu 275 280 285 Val Trp Met Gly Thr Trp Asn Gly Thr Thr Arg Val Ala Ile Lys Thr 290 295 300 Leu Lys Pro Gly Thr Met Ser Pro Glu Ala Phe Leu Gln Glu Ala Gln 305 310 315 320 Val Met Lys Lys Leu Arg His Glu Lys Leu Val Gln Leu Tyr Ala Val 325 330 335 Val Ser Glu Glu Pro Ile Tyr Ile Val Thr Glu Tyr Met Asn Lys Gly 340 345 350 Ser Leu Leu Asp Phe Leu Lys Gly Glu Thr Gly Lys Tyr Leu Arg Leu 355 360 365 Pro Gln Leu Val Asp Met Ser Ala Gln Ile Ala Ser Gly Met Ala Tyr 370 375 380 Val Glu Arg Met Asn Tyr Val His Arg Asp Leu Arg Ala Ala Asn Ile 385 390 395 400 Leu Val Gly Glu Asn Leu Val Cys Lys Val Ala Asp Phe Gly Leu Ala 405 410 415 Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Phe 420 425 430 Pro Ile Lys Trp Thr Ala Pro Glu Ala Ala Leu Tyr Gly Arg Phe Thr 435 440 445 Ile Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Thr Glu Leu Thr 450 455 460 Thr Lys Gly Arg Val Pro Tyr Pro Gly Met Val Asn Arg Glu Val Leu 465 470 475 480 Asp Gln Val Glu Arg Gly Tyr Arg Met Pro Cys Pro Pro Glu Cys Pro 485 490 495 Glu Ser Leu His Asp Leu Met Cys Gln Cys Trp Arg Lys Glu Pro Glu 500 505 510 Glu Arg Pro Thr Phe Glu Tyr Leu Gln Ala Phe Leu Glu Asp Tyr Phe 515 520 525 Thr Ser Thr Glu Pro Gln Tyr Gln Pro Gly Glu Asn Leu 530 535 540 <210> 5 <211> 64 <212> PRT <213> Artificial Sequence <400> 5 Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu Ser Arg Thr Glu Thr Asp 1 5 10 15 Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln Ile Val Asn Asn Thr Arg 20 25 30 Lys Val Asp Val Arg Glu Gly Asp Trp Trp Leu Ala His Ser Leu Ser 35 40 45 Thr Gly Gln Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp 50 55 60 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <400> 6 Tyr Asp Tyr Glu Ser Arg Thr Glu Thr Asp Leu 1 5 10

Claims

1. Use of a c-Src SH3 RT-loop antagonist in the preparation of a composition, characterized in that, The composition is used for: preventing and / or treating thrombosis, and the prevention and / or treatment of thrombosis does not affect bleeding or improves bleeding while achieving antithrombosis; wherein, the c-Src SH3 RT-loop antagonist does not antagonize, does not affect, or basically does not affect the binding or interaction between integrin β3 and the c-Src SH3 n-loop region; wherein, the composition further comprises an additional antithrombotic drug, and the additional antithrombotic drug is selected from the group consisting of: aspirin, clopidogrel, eptifibatide, xuesaitong, ginkgo biloba tablets, or a combination thereof; and the antagonist is DCDBS84 or a pharmaceutically acceptable salt thereof:

2. The use according to claim 1, characterized in that, The antagonist is a c-Src SH3 RT-loop region-specific antagonist.

3. The use according to claim 1, characterized in that, The prevention and / or treatment of thrombosis includes one or more of the following characteristics: (a) interfering with the interaction between integrin β3 and c-Src; (b) inhibiting platelet spreading on solid-phase fibrinogen; and / or (c) inhibiting platelet aggregation and / or adhesion.

4. The use according to claim 1, characterized in that, The c-Src SH3 RT-loop antagonist includes antagonizing the amino acids at positions R95 and / or E97.

5. The use according to claim 1, wherein The dissociation constant KD value of the described antagonist interacting with the E97A mutant c-Src protein is denoted as KD E97A The dissociation constant KD value of the described antagonist interacting with the wild-type c-Src protein is denoted as KD wt and KD E97A / KD wt The ratio of ≥ 5; wherein, the amino acid sequence of the E97A mutant c-Src protein is as shown in SEQ ID No:1, and the E at the 100th position is mutated to A.

6. The use according to claim 1, characterized in that, The dissociation constant KD value of the described antagonist interacting with the E97A mutant c-Src protein is denoted as KD E97A , and the dissociation constant KD value of the described antagonist interacting with the wild-type c-Src protein is denoted as KD wt , and KD E97A / KD wt The ratio of is ≥ 10; wherein, the amino acid sequence of the E97A mutant c-Src protein is as shown in SEQ ID No:1, and the E at the 100th position is mutated to A.

7. The use according to claim 1, characterized in that, The dissociation constant KD value of the described antagonist interacting with the E97A mutant c-Src protein is denoted as KD E97A The dissociation constant KD value of the described antagonist interacting with the wild-type c-Src protein is denoted as KD wt and the ratio of KD E97A / KD wt is ≥ 20; wherein, the amino acid sequence of the E97A mutant c-Src protein is as shown in SEQ ID No:1, and the E at the 100th position is mutated to A.

8. The use according to claim 1, characterized in that, The dissociation constant KD value of the described antagonist interacting with the E97A mutant c-Src protein is denoted as KD E97A The dissociation constant KD value of the described antagonist interacting with the wild-type c-Src protein is denoted as KD wt and KD E97A / KD wt The ratio of ≥ 40; wherein, the amino acid sequence of the E97A mutant c-Src protein is as shown in SEQ ID No:1, and the E at the 100th position is mutated to A.

9. The use according to claim 1, characterized in that, The c-Src SH3 RT-loop is selected from (A) a polypeptide having an amino acid sequence as shown in SEQ ID NO.:

3.

10. The use according to claim 1, characterized in that, The thrombosis includes thromboses in cardiovascular and cerebrovascular diseases.

11. The use according to claim 1, characterized in that, The thrombosis is a thrombosis in cardiovascular and cerebrovascular diseases, selected from the group consisting of: myocardial infarction thrombosis, cerebral infarction thrombosis, ischemic stroke, atherosclerotic thrombosis, or a combination thereof.

12. An in vitro non-therapeutic method for interfering with the interaction between integrin β3 and c-Src proteins, characterized in that, including the steps of: (a) in the presence of a c-Src SH3 RT-loop antagonist, contacting integrin β3 and c-Src protein, thereby interfering with the interaction between integrin β3 and c-Src protein; and the c-Src SH3 RT-loop antagonist is DCDBS84 or a pharmaceutically acceptable salt thereof:

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