CN113967257A - c-Src SH3 RT-loop as target for resisting thrombus - Google Patents

c-Src SH3 RT-loop as target for resisting thrombus Download PDF

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CN113967257A
CN113967257A CN202010725703.6A CN202010725703A CN113967257A CN 113967257 A CN113967257 A CN 113967257A CN 202010725703 A CN202010725703 A CN 202010725703A CN 113967257 A CN113967257 A CN 113967257A
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src
loop
antagonist
leu
amino acid
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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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Shanghai Institute of Materia Medica of CAS
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Shanghai Institute of Materia Medica of CAS
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Priority to PCT/CN2021/108371 priority patent/WO2022017532A1/en
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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    • C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • 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
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    • G01—MEASURING; TESTING
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
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    • 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)
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    • 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

Abstract

The invention relates to a c-Src SH3 RT-loop as a target for resisting thrombus. Specifically, the invention provides an application of a c-Src SH3 RT-loop antagonist in preparing a composition or a preparation, wherein the composition or the preparation is used for: (a) the interaction of interferon beta 3 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 invention discovers for the first time that the pharmaceutical composition or preparation taking the RT-loop region of the c-Src SH3 structural domain as the target point can effectively treat thrombotic diseases without increasing the bleeding risk.

Description

c-Src SH3 RT-loop as target for resisting thrombus
Technical Field
The invention belongs to the field of molecular biology and biomedicine, and particularly relates to a c-Src SH3 RT-loop as a target for resisting thrombus.
Background
Cardiovascular and cerebrovascular thrombotic diseases such as myocardial infarction, cerebral infarction and the like seriously affect the life and health of human beings. When the vascular endothelium is damaged or the atherosclerotic plaque is broken, the platelets are activated and cause pathological thrombosis through a series of functions of adhesion, extension, aggregation and the like, so that ischemic necrosis of heart and brain tissues in an affected blood vessel distribution area is caused, and the life of a patient can be seriously threatened. Therefore, the antiplatelet therapy becomes the first choice for treating cardiovascular and cerebrovascular thrombotic diseases.
Classical antiplatelet drugs including Cyclooxygenase (COX) inhibitor aspirin are the most widely studied and applied antiplatelet drugs in antiplatelet therapy at present, and mainly inhibit arachidonic acid Cyclooxygenase (COX) to irreversibly acetylate Ser-529 and Ser-516, thereby blocking the synthesis of TXA2 and playing the antiplatelet role. Common adverse reactions of aspirin are gastrointestinal discomfort and gastrointestinal bleeding, with the risk of bleeding being dose-related. To avoid bleeding side effects, high amounts of antithrombotic agents cannot be used. Another class of classical antiplatelet drugs are Adenosine Diphosphate (ADP) P2Y12 receptor antagonists, including the thienopyridines, with Ticlopidine (Ticlopidine), Clopidogrel (Clopidogrel) and Prasugrel (Prasugrel) being representative drugs. Non-thiophene pyridines, representative drugs are Ticagrelor (Ticagrelor) and Cangrelor (Cangrelor). Among them, clopidogrel, the second generation P2Y12 receptor antagonist, is widely used and can irreversibly inhibit platelet ADP receptors, thereby inhibiting platelet aggregation induced by ADP release from activated platelets, and bleeding remains its main side effect. It follows that classical antiplatelet drugs, while able to exert a good antithrombotic effect, cannot exert a deep antithrombotic effect with sufficient dosage due to the limitation of bleeding side effects.
In order to be able to target more specifically the receptors of platelets involved in thrombosis, receptor antagonists targeting integrin α IIb β 3 have been developed. The integrin alpha IIb beta 3 is used as a final common path for mediating platelet activation aggregation and thrombosis and is a main target point of antithrombotic drug research. In fact, the research of using integrin α IIb β 3 as the target of antithrombotic drugs has made an important progress, and at present, the main focus is on integrin α IIb β 3 receptor antagonist drugs, and good clinical efficacy has been obtained. Currently, there are three types of integrin α IIb β 3 receptor antagonist antiplatelet drugs approved by the U.S. Food and Drug Administration (FDA) for clinical antithrombotic therapy, namely abciximab, eptifibatide, and tirofiban. Such α IIb β 3 receptor antagonists specifically exert antithrombotic effects through the interaction of interferon α IIb β 3 with its ligands. However, this strategy still has significant problems, and the α IIb β 3 receptor antagonist drugs block bidirectional signal transduction by preventing integrin α IIb β 3 from binding its ligand, i.e., exert antithrombotic effects while affecting normal hemostatic functions. Review of clinical trials has shown that severe intracranial bleeding occurs in about 2% of patients treated with integrin α IIb β 3 antagonist drugs, gastrointestinal bleeding occurs in about 15% of patients, peritoneal bleeding occurs in about 5-10% of patients, and significant bleeding occurs in about 60-80% of patients at the femoral artery puncture site. Like classic antithrombotic drugs aspirin and clopidogrel, the integrin alpha IIb beta 3 antagonist exerts an effective antithrombotic effect and also causes an increase in bleeding risk of patients, which is the most common and important side effect of the current antithrombotic drugs. Therefore, the clinical antithrombotic agent dosage should be selected with consideration of the risk of bleeding side effects, so that it is difficult to achieve better antithrombotic effect by increasing the antithrombotic agent dosage at present. In studies with death as an end-point event, it is difficult to find an appropriate dose threshold to reduce mortality due to thrombotic and hemorrhagic deaths. Therefore, by developing a new generation of antithrombotic drugs that do not affect the normal hemostatic function, it would be possible to obtain a stronger antithrombotic effect with a low risk, which represents the development direction of antithrombotic drugs.
Targeting platelet external-internal signals rather than inhibiting the bidirectional signal transduction function of the integrin alpha IIb beta 3 intact receptor distinguishes the antithrombotic effect from the normal hemostatic function, and can realize deep antithrombotic without increasing the bleeding risk. Integrin beta 3/Src interaction plays an important role in platelet-outward-inward signal transduction. This can be achieved by designing polypeptides or small molecules that target the β 3/Src interaction, specifically dissociating the β 3/Src interaction. Previous researches find that the RGT tripeptide of the cytoplasmic tail end of the synthesized integrin beta 3 can specifically inhibit platelet external-internal signals and related platelet functions; RGT tripeptide gene knockout mice at the cytoplasmic tail of integrin beta 3 can also exert inhibitory effects on platelet ecto-endo signaling and related functions by disrupting the beta 3/Src interaction. Since inhibition of platelet ecto-endo signaling can be achieved by integrin beta 3 mimetics, can new small molecule drug development be achieved by targeting sequences on c-Src that interact with beta 3? The c-Src SH3 domain has been found to interact with integrin beta 3, but the specific site of action is not known. According to the invention, through technical means such as Co-IP and Surface Plasmon Resonance (SPR), the RT loop of c-Src SH3 has the tendency of combining integrin beta 3, while the n-Src loop has the tendency of classical combination, and the classical combination participates in the kinase activity and other functions of c-Src. Therefore, the novel antithrombotic drug is designed by taking the RT loop of the c-Src SH3 as a target spot, and is expected to realize deep antithrombotic and hardly affect the normal hemostatic function and the activity and function of the c-Src. Therefore, there is a need in the art to develop a specific target for the prevention or treatment of thrombosis.
Disclosure of Invention
The invention aims to provide application of a c-Src SH3 RT-loop antagonist (antaconist) in antithrombotic aspect.
In a first aspect of the invention there is provided the use of a c-Src SH3 RT-loop antagonist (antaconist) for the preparation of a composition or formulation for:
(a) the interaction of interferon beta 3 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.
In another preferred embodiment, said c-Src is human (including human) c-Src.
In another preferred example, the c-Src SH3 RT-loop is a human (including human) c-Src SH3 RT-loop.
In another preferred embodiment, the "interaction of interferon beta 3 and c-Src" is selected from the group consisting of:
(a1) reducing the binding of integrin beta 3 to the RT-loop region of c-Src SH 3;
(a2) blocking the binding of integrin beta 3 and the RT-loop region of c-Src SH 3.
In another preferred embodiment, the integrin beta 3 comprises integrin alpha IIb beta 3.
In another preferred embodiment, the antagonist is a c-Src SH3 RT-loop region-specific antagonist.
In another preferred embodiment, the "c-Src SH3 RT-loop region-specific antagonist" means that the antagonist antagonizes (or affects) the binding of the RT-loop regions of integrin beta 3 and c-Src SH3, but does not antagonize (or affect) or does not substantially antagonize the binding of the n-Src loop regions of integrin beta 3 and c-Src SH 3.
In another preferred embodiment, the c-Src SH3 RT-loop antagonist does not antagonize (or affect) or does not substantially affect the binding (or interaction) of integrin beta 3 to the c-Src SH3 n-loop region.
In another preferred embodiment, the antagonist has a dissociation constant KD (expressed as KD) for interaction with the R95A mutant c-Src proteinR95A) Dissociation constant Kd value (expressed as KD) for the interaction of said antagonist with wild-type c-Src proteinwt) Ratio (KD)R95A/KDwt) Is not less than 5, preferably not less than 10, more preferably not less than 20, and most preferably not less than 40.
In another preferred embodiment, the antagonist has a dissociation constant KD (expressed as KD) for interacting with the mutant c-Src protein E97AE97A) Dissociation constant, KD, values (denoted KD) for the interaction of said antagonist with wild-type c-Src proteinwt) Ratio (KD)E97A/KDwt) Is not less than 5, preferably not less than 10, more preferably not less than 20, and most preferably not less than 40.
In another preferred example, the amino acid sequence of the C-Src protein with the R95A mutant is shown in SEQ ID No. 1, and the R at position 98 is mutated into A.
In another preferred example, the 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 into A.
In another preferred embodiment, the c-Src SH3 RT-loop antagonist is a dual RT-loop region and n-Src loop region antagonist.
In another preferred embodiment, the c-Src SH3 RT-loop antagonist comprises antagonism of amino acids at positions R95 and/or E97.
In another preferred embodiment, the antagonist is selected from the group consisting of: small molecule antagonists, antisense nucleotides, mirnas, sirnas, or combinations thereof.
In another preferred embodiment, the antagonist comprises: DCDBS84 or a pharmaceutically acceptable salt thereof:
Figure BDA0002601580040000041
in another preferred embodiment, the antagonist is a structural derivative of DCDBS84, or other c-Src SH3 targeted small molecule candidate compounds.
In another preferred embodiment said 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.
In another preferred embodiment, the C-Src SH3 domain has an RT-loop region.
In another preferred example, the RT-loop region of the c-Src SH3 domain or the gene encoding the same is of mammalian (including human and murine) origin.
In another preferred embodiment, said c-Src protein is selected from the group consisting of:
(A) a polypeptide with an amino acid sequence shown as SEQ ID No. 1;
(B) a c-Src protein derivative formed by substituting, deleting or adding one or more (usually 1 to 60, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 5) amino acid residues to the amino acid sequence shown in SEQ ID No. 1, or an active fragment thereof;
(C) c-Src protein derivatives having a homology of not less than 90%, preferably not less than 95%, more preferably not less than 98%, most preferably not less than 99% to the amino acid sequence shown in SEQ ID No. 1, or active fragments thereof.
In another preferred embodiment, the c-Src SH3 domain is selected from the group consisting of:
(A) a polypeptide with an amino acid sequence shown as SEQ ID No. 2;
(B) a c-Src SH3 domain derivative formed by substitution, deletion or addition of one or more (usually 1-10, preferably 1-5, more preferably 1-3, most preferably 1-2) amino acid residues to the amino acid sequence shown in SEQ ID No. 2, or an active fragment thereof;
(C) a c-Src SH3 domain derivative, or an active fragment thereof, having a sequence homology of greater than or equal to 90%, preferably greater than or equal to 95%, more preferably greater than or equal to 98%, most preferably greater than or equal to 99% to the amino acid sequence shown in SEQ ID No. 2.
In another preferred embodiment, the c-Src SH3 RT-loop is selected from the following group:
(A) a polypeptide with an amino acid sequence shown as SEQ ID No. 3;
(B) an RT-loop region 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 to the amino acid sequence shown in SEQ ID NO. 3;
(C) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 3 and having a mutation selected from the group consisting of: R95A and E97A.
In another preferred embodiment, the antagonist or composition or formulation does not increase or does not substantially increase the risk of bleeding (otherwise referred to as "ameliorating bleeding").
In another preferred embodiment, the thrombus comprises cardiovascular and cerebrovascular disease thrombus; more preferably, the thrombus is cardiovascular and cerebrovascular disease thrombus selected from the following group: myocardial infarction thrombus, cerebral infarction thrombus, ischemic stroke, atherosclerotic thrombus, or a combination thereof.
In another preferred embodiment, the prevention and/or treatment of thrombosis does not affect or improve bleeding while achieving antithrombotic effect.
In another preferred embodiment, said improving bleeding comprises 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.
In another preferred embodiment, the hemostatic function comprises a platelet hemostatic function.
In another preferred embodiment, said hemostasis comprises physiological hemostasis.
In another preferred embodiment, said inhibiting aggregation of platelets comprises inhibiting biphasic aggregation of platelets.
In another preferred embodiment, said inhibiting aggregation of platelets comprises one phase aggregation without inhibiting platelets.
In another preferred embodiment, the composition comprises a pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and a safe and effective amount of the antagonist.
In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: solid dosage forms, liquid preparations, semi-solid preparations.
In another preferred embodiment, the composition is selected from the group consisting of: oral preparation and injection.
In another preferred embodiment, the composition or formulation is in a dosage form selected from the group consisting of: tablet, granule, capsule, injection, infusion solution, paste, gel, solution, microsphere or pellicle.
In another preferred embodiment, the composition or formulation further comprises other antithrombotic agents (e.g., aspirin).
In another preferred embodiment, the additional antithrombotic agent is selected from the group consisting of: aspirin, clopidogrel, eptifibatide, Xuesaitong, ginkgo biloba leaves, or a combination thereof.
In a second aspect of the invention there is provided the use of a c-Src SH3 RT-loop agonist (agonst) for the preparation of a composition or formulation for use in therapy
(a) Promotes integrin beta 3 and c-Src interactions;
(b) promoting platelet spreading on solid-phase fibrinogen;
(c) promoting aggregation and adhesion of platelets; and/or
(d) Promoting blood coagulation.
In a third aspect of the invention, there is provided a method of interfering with the interaction of integrin beta 3 and c-Src protein, comprising the steps of:
(a) integrin beta 3 and c-Src proteins are contacted in the presence of a c-Src SH3 RT-loop antagonist, thereby interfering with the interaction of integrin beta 3 and c-Src proteins.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, in step (a), cells expressing integrin beta 3 and c-Src protein are cultured in the presence of a c-Src SH3 RT-loop antagonist and the binding of integrin beta 3 and c-Src protein is determined.
In another preferred embodiment said c-Src protein comprises wild-type c-Src protein, mutant c-Src protein.
In another preferred embodiment, said mutant c-Src protein comprises: R95A mutant c-Src protein, E97A mutant c-Src protein, or a combination thereof.
In a fourth aspect of the invention, there is provided a method of antithrombotic (or inhibition of platelet aggregation and/or adhesion) comprising the steps of: administering to a subject in need thereof a c-Src SH3 RT-loop antagonist.
In another preferred embodiment, the subject is a human or non-human mammal (rodent, rabbit, monkey, livestock, dog, cat, etc.).
In a fifth aspect of the present invention, there is provided a method of screening for an antithrombotic candidate compound, the method comprising the steps of:
(a) contacting integrin beta 3 and c-Src proteins in the presence of a test agent in a test group, and observing whether integrin beta 3 in the test group binds to the RT-loop region of the c-Src SH3 domain; contacting integrin beta 3 and c-Src protein in the control group in the absence of said test agent and observing whether integrin beta 3 in said control group binds to the RT-loop region of the c-Src SH3 domain;
wherein, if the test group has a significantly lower degree or amount of binding of integrin beta 3 to the RT-loop region of the c-Src SH3 domain than the control group, the test agent is a candidate compound against thrombus,
wherein the candidate compound is a c-Src SH3 RT-loop antagonist.
In another preferred embodiment, the drug to be tested is a compound, a protein drug or a gene drug.
In another preferred embodiment, in step (a), the test is performed in a cell-free system.
In another preferred embodiment, in step (a), the test is performed in a system with cells expressing integrin beta 3 and c-Src proteins.
In another preferred embodiment, the cells are platelets.
In a sixth aspect of the present invention, there is provided a method of screening for an antithrombotic candidate compound, the method comprising the steps of:
(a) contacting NITYRGT peptide and c-Src protein in the presence of a test substance in a first test group and observing the amount of a first complex formed by NITYRGT peptide and c-Src protein in the test group;
contacting NITYRGT peptide and c-Src protein in the absence of the test substance in a first control group, and observing the amount of a first complex formed by NITYRGT peptide and c-Src protein in the control group;
wherein if the amount of the first complex in the first test group is significantly less than the amount of the first complex in the first control group, the test agent is indicative of a candidate compound for combating thrombosis,
wherein the candidate compound is a c-Src SH3 RT-loop antagonist.
In another preferred example, the method further comprises:
(b) contacting an RLP1 polypeptide and a c-Src protein in the presence of said test agent in a second test group and observing the amount of a second complex formed between an RLP1 polypeptide and the c-Src protein in said test group;
contacting an RLP1 polypeptide and a c-Src protein in the absence of said test substance in a second control group, and observing the amount of a second complex formed between an RLP1 polypeptide and the c-Src protein in said control group;
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, the candidate compound is suggested to be a c-Src SH3 RT-loop-specific antagonist (i.e., to act predominantly with the RT-loop region and to have substantially no effect with the n-loop region);
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, the candidate compound is suggested to be a dual antagonist of c-Src SH3 RT-loop and n-loop regions (i.e., to have an effect on both the RT-loop and n-loop regions).
In another preferred embodiment, the phrase "substantially less than" means that the ratio of the amount or degree of binding or amount of binding (designated C1) of the complex in the test group to the amount or degree of binding or amount of binding (designated C0) of the complex in the control group (C1/C0) is less than or equal to 1/2, preferably less than or equal to 1/3, more preferably less than or equal to 1/4, and most preferably less than or equal to 1/5.
In another preferred embodiment, the term "equivalent" means that the ratio (C1/C0) of the amount of the complex or the degree of binding or the amount of binding (designated as C1) in the test group to the amount of the complex or the degree of binding or the amount of binding (designated as C0) in the control group is 0.8 to 1.2.
In a seventh aspect of the invention, there is provided a c-Src mutein, said mutein having an amino acid mutation at one or more positions selected from the group consisting of: 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.
In another preferred embodiment, the mutein has amino acid mutations at positions selected from the group consisting of: 95 th position, 97 th position, or a combination thereof.
In another preferred embodiment, said c-Src mutein has a mutated amino acid mutation selected from the group consisting of: R95A, E97A, or a combination thereof.
In another preferred embodiment, said c-Src mutein further comprises amino acid mutations at positions selected from the group consisting of: 116 bits, 118 bits, 131 bits, or a combination thereof.
In another preferred embodiment, said c-Src mutein has a mutated amino acid mutation selected from the group consisting of: G116A, W118A and Y131A.
In an eighth aspect of the invention there is provided a polynucleotide encoding a c-Src mutein according to the seventh aspect of the invention.
In a ninth aspect of the invention, there is provided a vector comprising a polynucleotide as described in the eighth aspect of the invention.
In a tenth aspect of the invention, there is provided a host cell comprising a vector according to the ninth aspect of the invention or a genome comprising a polynucleotide according to the eighth aspect of the invention in combination.
In an eleventh aspect of the present invention, there is provided a detection kit, comprising:
(i) and (3) a detection reagent for detecting the RT-loop region of the c-Src SH3 structural domain and the coding gene thereof.
In another preferred embodiment, the detection reagent comprises a reagent that detects the amount of c-Src protein or mRNA.
In another preferred embodiment, the detection reagent comprises a reagent for detecting whether the RT-loop region has an amino acid mutation or a nucleotide mutation.
In another preferred embodiment, the detection reagent detects the presence or absence of an amino acid mutation at position 95, 96, 97, 98, 99, 100 of the c-Src protein and/or the presence or absence of a nucleotide mutation corresponding to said amino acid mutation.
In another preferred embodiment, the amino acid mutations include: R95A, E97A, or combinations thereof
In a twelfth aspect of the invention there is provided the use of a test kit according to the eleventh aspect of the invention for the preparation of a diagnostic kit for assessing whether a test subject, such as a thrombotic patient, is suitably treated with a c-Src SH3 RT-loop antagonist.
In another preferred embodiment, the diagnostic kit is further used for assessing the risk of thrombus in a test subject.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 shows a structural mimetic diagram of the binding of RGT peptide, NITYRGT peptide, and a classical binding peptide containing PXXP domain (APPIPPPPR) to the c-Src SH3 domain.
FIG. 2 shows the amino acid sequence of the c-Src SH3 domain and the corresponding secondary structure pattern.
FIG. 3 shows the differences in the interaction of each mutant with β 3, as detected using Co-IP, after transfection of the c-Src SH3 mutant (R95A, E97A, G116A, W118A and Y131A) in 293T β 3 cells (integrin β 3 transfected in 293T cells).
FIG. 4 shows the binding of RLP1 polypeptide (containing PXXPdomain, binding to c-Src SH3 is considered classical binding) to c-Src SH3 mutants (R95A, E97A, G116A, W118A and Y131A).
FIG. 5 shows a structural mimic diagram of the binding of RGT peptide, the designed synthetic small molecule compound DCDBS84, and the classical binding peptide containing the PXXP domain (APPIPPPPR) to the c-Src SH3 domain.
FIG. 6 shows that the nuclear magnetic resonance experiment detects the binding site of DCDBS84 in the c-Src SH3 domain.
FIG. 7 shows a binding site map analyzed based on the chemical shift interference (CSP) of amino acid sites based on the results of nuclear magnetic resonance experiments.
FIG. 8 shows the binding constants of DCDBS84 with c-Src SH3(WT) and the respective mutants (R95A, E97A, G116A, W118A, and Y131A) as measured using Surface Plasmon Resonance (SPR).
FIG. 9 shows the binding constants of RLP1 peptide (containing PXXPdomain, binding to c-Src SH3 is considered classical binding) c-Src SH3(WT) and the respective mutants (R95A, E97A, G116A, W118A and Y131A) detected using SPR.
FIG. 10 shows the construction of gene targeting c-SrcE97AGeneral strategy profile of transgenic mice.
FIG. 11 shows c-SrcE97AAnd (5) identifying the genotype of the transgenic mice.
FIG. 12 shows the detection of c-Src using the Co-IP methodE97ATransgenic mice were able to dissociate integrin beta 3 from c-Src interactions.
FIG. 13 shows c-SrcE97AThe transgenic mice were able to reduce thrombocyte aggregation induced by thrombobin.
FIG. 14 shows c-SrcE97AStatistical map of the ability of transgenic mice to reduce thrombocyte aggregation induced by thrombobin.
FIG. 15 shows c-SrcE97ATransgenic mice were able to reduce the spreading pattern of platelets on solid phase fibrinogen.
FIG. 16 shows c-SrcE97ATransgenic mice are able to reduce the consequences of platelet adhesion to solid phase fibrinogen.
FIG. 17 shows a FeCl3In an induced carotid injury model, c-Src compared to WT control miceE97AThe transgenic mice can obviously inhibit thrombosis.
FIG. 18 shows c-Src compared to WT control mice in a mouse tailgating experimentE97ATransgenic mice did not increase mouse bleeding time.
Detailed Description
The inventor of the present invention has conducted extensive and intensive studies, and unexpectedly found that the inhibition of the target site of the RT-loop region of the c-Src SH3 domain can effectively treat thrombosis without increasing the bleeding risk.
Specifically, in the experiments of the present invention, it was determined whether NITYRGT, RGT peptides and the β 3 cytoplasmic tail of integrin α IIb β 3 are distinguished in the binding region of c-Src SH3 from the binding region of classical binding peptides (containing PXXP domain) in c-Src SH 3. The classical binding of PXXP domain-containing polypeptides to c-Src SH3 involves c-Src kinase activity (to which W118-dominated n-Src loops are known to contribute significantly) and the associated signal transduction to multiple functions of the cell, whereas the β 3/c-Src interaction is thought to be a relatively weak non-classical binding, the main function of which is to participate in platelet "out-in" signal transduction and associated thrombosis. By analyzing the binding of NITYRGT, RGT and PXXP domain-containing polypeptide (APPIPPPPR) to the c-Src SH3 domain by structural modeling, it was found that the binding of NITYRGT to c-Src SH3 is biased towards RT-loop, while the binding of PXXP domain polypeptide (APPIPPPPR) to c-Src SH3 is biased towards n-Src loop. Furthermore, by cloning mutants R95A, E97A, G116A, W118A and Y131A of c-Src SH3, the interaction between c-Src WT and each mutant and alpha IIb beta 3 is detected by applying a Co-IP experiment, and R95A, E97A, G116A, W118A and Y131A are found to be capable of weakening the interaction between beta 3/c-Src indeed, the most obvious weakening degree is E97A, and the region where E97 is located is RT-loop of c-Src SH 3. Since c-Src SH3 can interact with PXXP domain containing proteins, this association is called classical association, the invention synthesizes a PXXP containing polypeptide RLP1(RKLPPRPSK), and detected the interaction of RLP1 with c-Src SH3 wild-type and mutants, respectively, as a result, it was found that the binding site of RLP1 is inclined to W118, and this region is mainly the region where n-Src loop of c-Src SH3 is located.
The small molecule DCDBS84 targeting c-Src SH3 is screened out through experiments, and whether the direct action target of DCDBS84 in c-Src SH3 is distinguished from the classical combined target is detected. The Nuclear Magnetic Resonance (NMR) and chemical shift migration analysis show that the binding sites of DCDBS84 and c-Src SH3 mainly comprise R95, E97, W118, W119, Y131 and the like. Furthermore, by applying a Surface Plasmon Resonance (SPR) experiment, the binding constants of DCDBS84 and c-Src SH3 mutants of R95A, E97A, G116A, W118A, Y131A and Wild Type (WT) are detected, and R95A and E97A are found to obviously weaken the binding of DCDBS84 and c-Src SH3, which indicates that R95 and E97 are mainly involved in the binding of DCDBS84 and c-Src SH 3. Meanwhile, the invention also uses SPR to detect the binding constants of the classical binding peptide RLP1 and mutants R95A, E97A, G116A, W118A, Y131A of c-Src SH3 and Wild Type (WT), and as a result, the G116A, W118A and Y131A mutations are found to obviously weaken the binding of RLP1 and c-Src SH3, and the suggestion is that G116, W118 and Y131 are mainly involved in the binding of RLP1 and c-Src SH 3. The experiment further proves that the binding site of the small molecule DCDBS84 on c-Src SH3 is mainly located in RT-loop, and E97 is taken as a main target. Meanwhile, the structural simulation of the binding site of DCDBS84 and c-Src SH3 also suggests that the binding site mainly consists of E97 and RT-loop amino acids around the E97.
In addition, c-Src was experimentally constructedE97ATransgenic mice, genotyping WT mice and c-SrcE97AMutating mouse, separating blood platelet from mouse, Co-IP experiment confirming c-SrcE97AThe mutant mouse can dissociate the interaction of beta 3/c-Src in platelets, and inhibit the functions of platelets mediated by signal transduction in an outward direction, such as aggregation, extension and adhesion of the platelets. Of particular importance, c-SrcE97AMutant mice can be in FeCl3Thrombosis was inhibited in the induced thrombosis model and bleeding time was not increased in tail-clipped bleeding experiments compared to wild-type mice. The results show that: the c-Src SH3 RT-loop and the amino acid site mainly comprising E97 can become a novel antithrombotic target which does not influence the normal hemostasis function, and target information is provided for the research and development of novel antithrombotic drugs.
Term(s) for
As used herein, the terms "comprises," "comprising," "includes," "including," and "including" are used interchangeably and include not only closed-form definitions, but also semi-closed and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, the term "anti-thrombotic" includes the prevention and/or treatment of thrombosis.
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, "preventing" also includes delaying the onset of a disease and/or its attendant symptoms and reducing the risk of acquiring a disease in a subject.
"treatment" as used herein includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination, or reversal. In some embodiments, the compositions or pharmaceutical compositions of the invention reduce, inhibit and/or reverse associated diseases (e.g., tumors) and complications thereof, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80% by inhibiting the mitochondrial oxidative phosphorylation pathway as compared to levels observed in the absence of the compositions, kits, food or nutraceutical kits, active ingredient combinations described herein.
Src and c-Src
Src was initially found in Rous sarcoma retrovirus (retrovirus Rous sarcoma virus) as an oncogene protein, and it was subsequently found that v-Src, which is highly conserved in cells and homologous thereto, is ubiquitous.
The Src kinase family is proteins having tyrosine kinase (PTK) activity, wherein c-Src is an important component of the Src kinase family.
Herein, unless otherwise specified, amino acid sequences are numbered in order from N-terminus to C-terminus.
The amino acid sequence of the human c-Src is shown in SEQ ID No. 1:
SEQ ID NO.:1:
Figure BDA0002601580040000131
Figure BDA0002601580040000132
(SEQ ID No:1, SH3 domain underlined, in italics, RT-loop region)
SH3 Domain and RT-Loop region
As used herein, "SH 3", "SH 3 domain", "SH 3 domain protein", "SH 3 protein" are used interchangeably.
The amino acid sequence of a representative wild-type human c-Src SH3 domain is shown in SEQ ID No. 2:
Figure BDA0002601580040000134
Figure BDA0002601580040000135
(SEQ ID No:2, corresponding to positions 87-144 of SEQ ID No: 1).
As used herein, in human c-Src, R95, E97, T98, L100, D117, W118, W119, a138 are based on numbering the amino acids (SEQ ID No.:2) of the following SH3 domain, as shown below at positions 84 to 141:
Figure BDA0002601580040000133
as used herein, in the R95A, E97A mutant c-Src proteins in human c-Src, R95A, E97A are based on numbering the amino acids (SEQ ID No.:2) of the following SH3 domain, positions 84 to 141 as shown below:
Figure BDA0002601580040000141
as described herein, R95A refers to the mutation from the R amino acid to the A amino acid at amino acid position 95 in the amino acid numbering, E97A refers to the mutation from the E amino acid to the A amino acid at amino acid position 97 in the amino acid numbering, and the amino acid mutations at other positions are as described above.
As used herein, the term "c-Src SH3 RT-loop" is used interchangeably with "RT-loop region of c-Src SH3 domain". The human RT-loop region is located in the human c-Src SH3 structural domain, and the amino acid sequence is shown as YDYESRTETDL (SEQ ID No:3)
Integrin and integrin beta 3/Src interactions
Integrin alpha IIb beta 3 is a transmembrane heterodimer composed of alpha IIb and beta 3 subunits through non-covalent bonds, is mainly expressed on the surfaces of platelets and megakaryocytes, is a main membrane receptor on the surfaces of the platelets, can mediate bidirectional signal transduction of the platelets, and therefore plays a key role in platelet activation, maintenance of normal functions of the platelets and thrombosis. Platelet activators such as thrombin, ADP and the like cause configuration change of integrin alpha IIb beta 3 after acting with corresponding receptors, and lead to the increase of affinity with ligands thereof, namely soluble fibrinogen and the like, the process is internal and external signal transduction, and the marker events are unstable adhesion, free fibrinogen binding, reversible aggregation and the like of platelets; the integrin alpha IIb beta 3 is activated and combined with a ligand to activate the outside-in signal transduction, and the marked events comprise stable adhesion, extension, irreversible aggregation, fibrin clot retraction and the like of platelets, and finally promote the platelets to mutually aggregate and form stable thrombus to finish the physiological or pathological processes of hemostasis and thrombus formation. It is now well recognized that the achievement of hemostasis and thrombosis requires the co-participation of both inside-out and outside-in signal transduction, and that the pathological process of thrombosis requires the augmentation of platelet emboli under conditions of high impact force against blood flow, and therefore, thrombosis is relatively more dependent on outside-in signal transduction.
In order to be able to target more specifically the receptors of platelets involved in thrombosis, receptor antagonists targeting integrin α IIb β 3 were developed. The integrin alpha IIb beta 3 is used as a final common path for mediating platelet activation aggregation and thrombosis and is a main target point of antithrombotic drug research. In fact, the research of using integrin α IIb β 3 as the target of antithrombotic drugs has made an important progress, and at present, the main focus is on integrin α IIb β 3 receptor antagonist drugs, and good clinical efficacy has been obtained. Currently, there are three types of integrin α IIb β 3 receptor antagonist antiplatelet drugs approved by the U.S. Food and Drug Administration (FDA) for clinical antithrombotic therapy, namely abciximab, eptifibatide, and tirofiban. Such α IIb β 3 receptor antagonists specifically exert antithrombotic effects through the interaction of interferon α IIb β 3 with its ligands. However, this strategy still has significant problems, and the α IIb β 3 receptor antagonist drugs block bidirectional signal transduction by preventing integrin α IIb β 3 from binding its ligand, i.e., exert antithrombotic effects while affecting normal hemostatic functions. Clinical trials have shown that severe intracranial bleeding occurs in about 2% of patients treated with integrin α IIb β 3 antagonist drugs, gastrointestinal bleeding occurs in about 15% of patients, peritoneal bleeding occurs in about 5-10% of patients, and significant bleeding occurs in about 60-80% of patients at the femoral artery puncture site. Like classic antithrombotic drugs aspirin and clopidogrel, the integrin alpha IIb beta 3 antagonist exerts an effective antithrombotic effect and also causes an increase in bleeding risk of patients, which is the most common and important side effect of the current antithrombotic drugs. Therefore, the clinical antithrombotic agent dosage should be selected with consideration of the risk of bleeding side effects, so that it is difficult to achieve better antithrombotic effect by increasing the antithrombotic agent dosage at present. In studies with death as an end-point event, it is difficult to find an appropriate dose threshold to reduce mortality due to thrombotic and hemorrhagic deaths. Therefore, by developing a new generation of antithrombotic drugs that do not affect the normal hemostatic function, it would be possible to obtain a stronger antithrombotic effect with a low risk, which represents the development direction of antithrombotic drugs.
The integrin α IIb β 3 cytoplasmic tail, through interaction with the cytoplasmic protein c-Src, has been found to play an important role in platelet "out-in" signaling-related functions such as stable platelet adhesion and spreading on solid-phase fibrinogen, biphasic aggregation and fibrin clot retraction, while having little effect on platelet "in-out" functions such as platelet binding to soluble fibrinogen, platelet primary adhesion and one-phase aggregation. Studies have shown that the "outward-inward" signal of platelets is mainly involved in the process of platelet thrombosis, while the "inward-outward" signal plays a greater role in the function of hemostasis.
Studies have shown that the SH3 domain of Src kinase forms a constitutive bond with the three amino acids of RGT at the C-terminus of integrin β 3. In exo-endo signaling following integrin activation, Src kinase interacts with the RGT sequence at the β 3C-terminus and phosphorylation of β 3 cytoplasmic segments Y747 and Y759 occurs, an important event for exo-endo signaling. In vivo experiments prove that RGT knockout mice can avoid thrombosis caused by stimulation of carotid artery by FeCl 3; in the tail-cutting bleeding experiment, the bleeding time of some mice is prolonged, but spontaneous bleeding, postoperative hemorrhage, bloody stool, hematuria, anemia and the like do not occur. Since the β 3RGT/c-Src interaction plays such an important role in platelet "out-in" signaling, the synthetic RGT tripeptide can exert an antithrombotic effect by competing for the endogenous β 3RGT/c-Src interaction.
Use of
The invention provides a use of c-Src SH3 RT-loop antagonist (antaconist), wherein the c-Src SH3 RT-loop antagonist comprises (but is not limited to) one or more of the following uses:
(a) the interaction of interferon beta 3 and c-Src;
(b) inhibiting platelet spreading on solid-phase fibrinogen;
(c) inhibiting platelet aggregation and/or adhesion; and
(d) preventing and/or treating thrombosis.
In a preferred embodiment, integrin beta 3 includes, but is not limited to, integrin alpha IIb beta 3
In another preferred embodiment of the 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 affect the binding (or interaction) of integrin beta 3 to the c-Src SH3 n-loop region.
The specific type of the c-Src SH3 RT-loop antagonist is not particularly limited as long as the antagonist has an antagonistic effect on c-Src SH3 RT-loop. For example, the c-Src SH3 RT-loop antagonist can be a small molecule antagonist, antisense nucleotide, miRNA, siRNA and the like. Preferably, the antagonist is a compound of DCDBS 84:
Figure BDA0002601580040000161
in the present invention, c-Src protein is not particularly limited, but is preferably c-Src protein of mammals such as humans and rodents. Typically, the amino sequence of the human c-Src protein is as shown in SEQ ID No. 1. In the polypeptide shown in SEQ ID NO. 1, the 87 th to 144 th positions (SEQ ID No:2 sequence) are human c-Src SH3 structural domains. The amino sequence of the human c-Src SH3 RT-loop is as shown in SEQ ID NO. 3.
In a preferred embodiment of the invention, the c-Src SH3 RT-loop antagonist does not increase or does not substantially increase the risk of bleeding (or is referred to as "improving bleeding") during the course of antithrombotic treatment. In the present invention, said improving bleeding comprises 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.
In the invention, the thrombus comprises thrombus of 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.
Compositions or formulations, combinations and kits of active ingredients and methods of administration
The invention also provides a composition comprising a c-Src SH3 RT-loop antagonist.
The composition of the present invention is preferably a pharmaceutical composition. The compositions of the present invention may include a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid, or gel fillers that are suitable for human or animal use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant that the components of the pharmaceutical composition and the active ingredient of the drug are blended with each other and not significantly detract from the efficacy of the drug.
It is to be understood that, in the present invention, the pharmaceutically acceptable carrier is not particularly limited, and may be prepared from materials commonly used in the art, or by conventional methods, or may be commercially available. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal enhancers, colorants, flavors, stabilizers, antioxidants, preservatives, bacteriostats, pyrogen-free water, etc.
In the present invention, the dosage form of the composition is not particularly limited, and may be a solid dosage form, a liquid preparation, or a semi-solid preparation.
In the present invention, the composition and the dosage form of the preparation include, but are not limited to, oral preparations, injection preparations, and external preparations.
Typically, the dosage forms of the compositions and formulations include, but are not limited to: tablet, granule, capsule, injection, infusion solution, paste, gel, solution, microsphere or pellicle.
Typically, the injection is intravenous.
The pharmaceutical preparation should be compatible with the mode of administration, preferably oral administration, injection (e.g., intravenous injection), and is administered by administering a therapeutically effective amount of the drug to a subject (e.g., a human or non-human mammal) in need thereof. The term "therapeutically effective amount," as used herein, refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. It will be understood by those skilled in the art that the "therapeutically effective amount" may vary with the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the combination with other drugs.
In one mode of administration, a safe and effective daily dosage of the first active ingredient is generally at least about 0.1mg, and in most cases no more than about 2500 mg. Preferably, the dose is 1mg to 500 mg; a safe and effective amount of the second active ingredient is generally at least about 0.01mg, and in most cases does not exceed 2500 mg. Preferably, the dosage range is 0.1mg to 2500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention are:
the invention discovers for the first time that the inhibition of the RT-loop region target of the c-Src SH3 structural domain can effectively treat thrombus without increasing bleeding risk.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Parts and percentages are by weight unless otherwise indicated.
Examples
The amino acid sequence of the mouse c-Src is shown in SEQ ID No. 4:
SEQ ID NO.:4
MGSNKSKPKDASQRRRSLEPSENVHGAGGAFPASQTPSKPASADGHRGPSAAFVPPAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTRKVDVREGDWWLAHSLSTGQTGYIPSNYVAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKGETGKYLRLPQLVDMSAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL
the amino acid sequence of the mouse c-Src SH3 structural domain is shown as SEQ ID NO. 5:
SEQ ID NO.:5
TTFVALYDYESRTETDLSFKKGERLQIVNNTRKVDVREGDWWLAHSLSTGQTGYIPSNYVAPSD
the amino acid sequence of the mouse c-Src SH3 structural domain containing an RT-loop region is shown as SEQ ID NO. 6:
SEQ ID NO.:6
YDYESRTETDL
the structural formula of compound DCDBS84 is as follows:
Figure BDA0002601580040000191
in the examples, in human c-Src, R95, E97, T98, L100, D117, W118, W119, A138 are based on numbering the amino acids (SEQ ID NO: 2) of the following SH3 domain, as shown below at positions 84 to 141.
Figure BDA0002601580040000192
In mice, R95, E97 are based on numbering the amino acids of the SH3 domain (SEQ ID No.:5) as follows from position 84 to position 147:
84 TTFVALY DYESRTETDL SFKKGERLQI 110
111 VNNTRKVDVR EGDWWLAHSL STGQTGYIPS NYVAPSD 147
example 1 structural modeling of binding peptides to the c-Src SH3 Domain
A structural mimic of the binding of the classical binding peptide (APPPPPR) of the RGT, NITYRGT, and PXXP domains to the c-Src SH3 domain was obtained by computer structural analysis based on the RGT peptide (crystal structure), NITYRGT peptide (nuclear magnetic resonance structure), and the classical binding peptide (APPPPPR) containing the PXXP domain (nuclear magnetic resonance structure). As shown in FIG. 1, the binding of YRGT tetrapeptide from the crystal structure of the solid phase to c-Src SH3 is biased toward n-Src loop, while the binding of NITYRGT heptapeptide from the liquid-phase NMR structure to c-Src SH3 is biased toward RT-loop. The binding direction of classical binding peptides (APPIPPPPR) to c-Src SH3 is essentially perpendicular to that of NITYRGT at c-Src SH3, with a preference for n-Src loop. Based on the amino acid sequence information and secondary structure information of c-Src SH3, a structural pattern of c-Src SH3 is plotted (see FIG. 2).
Example 2: co-immunoprecipitation (Co-IP) detection of binding sites
In this example, Co-immunoprecipitation (Co-IP) was used to detect the binding site of β 3 at c-Src SH3 by constructing a mutant of integrin β 3 at the possible binding site of c-Src SH 3.
5 c-Src SH3 gene point mutation overexpression vectors, pFLAg-CMV4-Src (R95A), pFLAg-CMV4-Src (E97A), pFLAg-CMV4-Src (G116A), pFLAg-CMV4-Src (W118A) and pFLAg-CMV4-Src (Y131A) are constructed. Wherein 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 beta 4 region (as shown in FIG. 2).
The Co-immunoprecipitation assay (Co-IP) was used to detect the binding of the c-Src SH3 mutant to integrin beta 3 subunit. First 0.4ml of platelets (3X 10 concentration) are taken8/ml) lysis Protein (Protein content 500. mu.g), 50. mu.l of Protein A + G agarose beads previously washed with lysis buffer were added and incubated at 4 ℃ for 2h with rotation to remove non-specific contaminating proteins and reduce background. After incubation, centrifugation at 1000 Xg for 5min at 4 ℃ and transfer of the supernatant toIn a new centrifuge tube. To the protein supernatant, 1. mu.g of anti-Flag-tagged antibody M2(Sigma) or non-specific murine IgG (sc-2025, Santa Cruz Biotechnology, 1. mu.g) was added, and the antigen-antibody mixture was incubated overnight at 4 ℃ with rotation. The following morning 20. mu.l of Protein A + G agarose beads, previously washed with lysis buffer, were added thereto and incubated at 4 ℃ for 2h with rotation. The agarose beads-antigen-antibody complexes were then centrifuged at 1000 Xg for 5min at 4 ℃ and the supernatant was collected, washed repeatedly 5 times with 800. mu.l each time with precooled RIPA lysis buffer and allowed to stand on ice for 10 min. The agarose bead-antigen-antibody complex was then resuspended in 1 XSDS-PAGE sample buffer (100mM Tris-HCl, pH 6.8, 5% β -mercaptoethanol, 4% SDS, 20% glycol, 0.1% Bromophenol Blue) and boiled at 100 ℃ for 8min to denature the protein. The co-immunoprecipitates were detected by Western blot. As shown in FIG. 3, the R95 and E97 mutations significantly reduced β 3/c-Src binding.
Example 3 ELISA method to detect the binding of RLP1 peptide to c-Src SH3 mutant
The ligand protein molecules in the cell that interact with Src kinase generally target the Src-SH3 domain through the classical (PXXP) motif, and the RLP1 polypeptide (containing the classical PXXP motif) is a PXXP motif sequence that mimics the common multiple ligand protein molecules in the cell that interact with the Src-SH3 domain. The specific protocol was as follows, 50. mu.L of coating solution containing Flag antibody M2(Sigma) (1. mu.g/mL, diluted with 0.1M NaHCO3 pH 8.3) was added to each well of a 96-well plate and incubated overnight at 4 ℃. On day 2, wash with 1 × TBST 3 times, each for 5min, block with 5% BSA for 2h, and wash with 1 × TBST 3 times for use. pFlag-CMV4-Src (wt) and 5 additional 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 earlier in 293T cells, and 293T cells were lysed for 48h using RIPA ice, lysate supernatant was collected by centrifugation (4 ℃,18000rpm), 50 μ L lysate supernatant was added per well in Flag antibody-coated 96 well plates overnight, 4 ℃ overnight, and Western blot was relatively quantified on the supernatants of each experimental group. On day 3, wash 3 times with TBST and 50. mu.L of biotin-labeled RLP1 and control (RLA) polypeptide (1. mu.g/mL) were added to each well and incubated at 37 ℃ for 1 h. Then washed 3 times with 1 × TBST for 5min, 50 μ L of HRP-labeled avidin was added per well and incubated at 37 ℃ for 1 h. Washing with 1 × TBST for 3 times, adding TMB display solution 100 μ L per well, incubating at room temperature for 15min, adding 100 μ L sulfuric acid (1M) to terminate the reaction, and detecting the absorbance of each well with microplate reader at 450nm wavelength. The relative quantification of the absorbance values and the intensity values of the Western blot bands is shown in FIG. 4, and it can be seen that the W118 and Y131 mutations significantly reduced the binding of the RLP1 peptide to c-Src.
Example 4 NMR determination of amino acid binding sites
In this example, NMR spectra of protein (c-Src SH3) amino acid sites before and after DCDBS84 were determined by NMR.
By synthesizing various information such as a crystal structure of the RGT peptide combined with c-Src SH3, a nuclear magnetic resonance structure of NITYRGT and c-Src SH3 and the like, the micromolecule DCDBS84 targeting c-Src SH3 is screened out through computer simulation screening and analysis. A structural simulation of the combination of DCDBS84 and c-Src SH3 is shown in FIG. 5. DCDBS84 can be seen to bind mainly to the RT-loop of c-Src SH 3.
The NMR experiments were performed on a four-channel Bruker Avance III 600MHz spectrometer. Preparing a culture medium by replacing 14N with 15N to purify c-Src-SH3 protein with a 15N mark so as to carry out two-dimensional nuclear magnetic resonance; when three-dimensional nuclear magnetism is needed, 15N is needed to replace 14N, and 13C is needed to replace 12C; FIG. 6 (left panel) shows a two-dimensional 15N-HSQC experiment of the interaction between Src-SH3 and DCDBS84 with a c-Src-SH3 concentration of 50 μ M and a DCDBS84 concentration of 20 times higher; as shown in FIG. 6 (right panel), the amino acid position assignment for the 15N/13C-labeled C-Src-SH3 protein was completed by adjusting the concentration to 1.3 mM.
Example 5 analysis of binding sites by chemical Shift interference (CSP)
In the nuclear magnetic resonance experiments, binding site maps were analyzed based on the chemical shift interference (CSP) of amino acid sites.
Chemical shift disturbance (CSP) was calculated according to the formula:
Figure BDA0002601580040000221
with CSPMean + standard error was taken as baseline and greater than this as binding site. From the results shown in fig. 7, it was found that R95, E97, T98, L100, D117, W118, W119, a138 may be a binding site of SH3 to a compound.
Example 6: binding Performance of the antagonist DCDBS84 to wild-type and mutant c-Src SH3
In this example, the dissociation constants of DCDBS84 and c-Src SH3(WT) and mutants (R95A, E97A, G116A, W118A, Y131A) were determined by surface plasmon resonance experiments.
The surface plasmon resonance test was performed on a BIACORE T200 instrument (GE corporation). Purified c-Src SH3 protein (concentration 2mg/ml) was diluted to 0.1mg/ml with 10mM CH3COONa (pH 4.2) and SH3 protein was coupled to CM5 chips by standard amino coupling methods. DCDBS84 was diluted with a buffer solution (20mM Tris-HCl, pH8.0, 100mM NaCl), and injected continuously at a flow rate of 20. mu.l/s for 60 seconds, followed by dissociation for 120 seconds. Changes in response values over time were recorded and analyzed by the BIA Evaluation Software (GE Healthcare) program to obtain the dissociation constant KD of DCDBS84 and c-Src SH3 protein (WT and mutant). As shown in FIG. 8, DCDBS84 has a dissociation constant of 0.83. mu.M for wild-type c-Src SH3 protein, 48. mu.M for R95A, 35. mu.M for E97A, 8.1. mu.M for G116A, 6.24. mu.M for W118A, and 1.52. mu.M for Y131. It is suggested that R95 and E97 located in RT-loop are mainly involved in the binding of DCDBS84 and c-Src SH 3.
Further, the dissociation constant KD of the classical binding peptide RLP1 and the c-Src SH3 protein (WT and mutants) was examined as described above. As shown in FIG. 9, the dissociation constants of RLP1 and wild-type c-Src SH3 protein are 9.9. mu.M, R95A and E97A are 10.9. mu.M, G116A and W118A are 49.9. mu.M, and Y131A is 85.9. mu.M. It is suggested that G116 and W118 at n-Src loop and Y131 at the far end loop are involved mainly in the binding of RLP1 with c-Src SH 3.
The above results suggest: the main binding site of the small molecule compound DCDBS84 at c-Src SH3 tends to be dominated by R95 and E97 of RT-loop, while the main binding site of the classical binding peptide containing PXXP domain (RLP1) c-Src SH3 tends to be dominated by G116 and W118 of n-Src loop and Y131 of distal loop. It was shown that non-classical binding does distinguish from classical binding on c-Src SH 3.
Example 7 Gene targeting construction of transgenic mice
This example constructs c-Src by Gene targetingE97AThe overall strategy for mutating transgenic mice and constructing transgenic mice by gene targeting is shown in FIG. 10.
Example 8: c-SrcE97AGenotyping of transgenic mice
In this example, a primer containing a mutation (MusE99A) at the corresponding site of the mouse gene corresponding to human E97A was designed based on the sequence of the mouse Src gene (NC-000068.7), and the primer sequence was: c-SrcE97A-F:5'-GAACACCTAGTCTGCAGCCC-3',c-SrcE97A-R:5'-AGCAGAGAGAAGGAGAGG
CT-3', the length of the amplified fragment is 419bp (as shown in the upper panel of FIG. 11). The PCR product is subjected to sequencing analysis, and the position of the gene corresponding to the 99 th amino acid of the mouse is glutamic acid (E) if GAG, alanine (A) if GCG, and heterozygote if two peaks of GAG and GCG appear. The results of gene sequencing are shown in the lower panel of FIG. 11.
Example 9: co-immunoprecipitation
In this example, c-Src was detected by Co-immunoprecipitation (Co-IP)E97AThe β 3/c-Src interaction in platelets from transgenic mice.
Taking Wild Type (WT) mice and c-Src E97A3 transgenic mice are anesthetized by phenobarbital according to the weight of the mice, then the mice are subjected to heart blood collection, and 0.38% sodium citrate is used for anticoagulation. Platelet-rich plasma (PRP) was obtained by centrifugation at 300 Xg for 7min, and 1/4 volumes of ACD was added to the PRP for anticoagulation, followed by centrifugation at 500 Xg for 10min and discarding the supernatant. Platelets were washed with CGS wash (13mM sodium citrate, 120mM sodium chloride, 30mM glucose, pH 6.5) followed by Tyrode's buffer (0.1% bovine serum albumin, 5mM 4-hydroxyethylpiperazine ethanesulfonic acid, 5.5mM glucose, 137mM sodium chloride, 2mM potassium chloride, 12mM sodium bicarbonate, 0.3mM phosphateSodium dihydrogen, 1mM calcium chloride, 1mM magnesium chloride, pH 7.4) resuspend platelets. Platelets were counted using a conventional small animal blood detector (PoCH-100iV Diff) and platelet density was adjusted to 3X 108Standing at room temperature for 1 h.
The influence of the small molecular compound DCDBS84 on the interaction between beta 3 and c-Src of integrin alpha IIb beta 3 is detected by applying a Co-IP method. Taking 400 μ l of 3 × 108Platelets per ml were lysed by IP buffer (50mM Tris-HCl, pH7.4,50mM NaCl, 0.2% NP-40) on ice for 30min, centrifuged (4 ℃,12000rpm,15min) to aspirate the supernatant, and the BCA protein was quantitated. Add 50. mu.l of Protein A/G agarose beads, previously washed with IP buffer, spin incubate at 4 ℃ for 2h, centrifuge (4 ℃,1000G,5min), and transfer the supernatant to a new centrifuge tube. The anti-integrin mouse beta 3 antibody SZ-21 (1. mu.g) or non-specific murine IgG (sc-2025, Santa Cruz Biotechnology, 1. mu.g), or the rabbit monoclonal antibody c-Src antibody (36D10, #2109, Cell signaling Technology, 1. mu.g) and the non-specific rabbit IgG (#2729, Cell signaling Technology, 1. mu.g) were added to the protein supernatant and the antigen-antibody mixture was incubated at 4 ℃ overnight with rotation. The following morning 20. mu.l of Protein A/G sepharose beads washed with IP buffer in advance were added to the mixture, incubated at 4 ℃ for 2h with rotation, centrifuged (4 ℃,1000G,5min), the sepharose bead-antigen antibody complexes collected and washed 3 times with pre-cooled 1 XPBS buffer. Finally, the agarose bead-antigen-antibody complex was resuspended in 1 XSDS loading buffer and boiled at 100 ℃ for 10 min. The co-immunoprecipitates were detected by Western blot. As shown in FIG. 12, c-Src showed that when compared with WT mice, c-Src had been observed in both of the β 3 antibody IP and the c-Src antibody IPE97AThe interaction of β 3/c-Src in mouse platelets is markedly attenuated.
Example 10: c-SrcE97AEffect of transgenic mice on platelet aggregation.
Wild Type (WT) mice and c-Src were tested as described in example 9E97APlatelets were isolated from 3 transgenic mice each. Centrifuging at 300 × g for 7min to obtain PRP, centrifuging at 500 × g for 10min to obtain Platelet-poor plasma (PPP), and adjusting the Platelet concentration in PRP to 2 × 10 with PPP8/And (3) ml. Then 200. mu.l of PPP is taken to calibrate the instrumentTransmission concentrator (Chrono-Log) zero point. After subsequent incubation of each set of reagents with PRP for 60min at 37 ℃ the reaction was initiated by addition of 0.1U/ml Thrombin (Thrombin) after calibration of the zero point in a 200. mu.l/tube aggregator (37 ℃,1000 rpm agitation) and the aggregation curve recorded. As shown in FIG. 13, c-Src compares to WT miceE97AThe transgenic mice obviously inhibit the platelet biphasic aggregation without influencing the platelet biphasic aggregation, which shows that the c-SrcE97AThe transgenic mice significantly inhibited thrombosis without affecting normal physiological hemostatic effects. The statistical chart of fig. 13 is shown in fig. 14.
Example 11: c-SrcE97AEffects of transgenic mice on platelet spreading and adhesion.
Wild Type (WT) mice and c-Src were tested as described in example 9E97APlatelets were isolated from 3 transgenic mice each. Adjusting the platelet concentration to 2X 108/ml。
Add 50. mu.l fibrinogen (0.1M, pH 8.3 sodium bicarbonate dilution, 20. mu.g/ml) to a 96-well plate and coat overnight at 4 ℃. The following morning was washed 3 times with 1 XPBS and blocked with bovine serum albumin (BSA 20mg/ml) at 37 ℃ for 60 min. 50. mu.l (concentration 2X 10) of the washed platelet suspension was taken8Pieces/ml) was added to a 96-well plate and adhered in an incubator at 37 ℃ for 60 min. Nonadherent platelets were removed by 3 washes with PBS, stably adherent platelets were fixed with 4% paraformaldehyde, and washed 3 times with PBS. The platelet membrane was then perforated with 0.5% Triton X-100, and platelets were stained with 0.5. mu.g/ml phalloidin-rhodamine for 60min at 37 ℃ and washed 3 times with 1 XPBS (10 min each). After the washing, the fluorescence development was observed with a fluorescence microscope (Leica). The results are shown in FIG. 15, c-SrcE97AThe platelets of the transgenic mice were significantly less extended on solid phase fibrinogen than the WT mice.
The coating of fibrinogen in the adhesion experiment is extended in the same way as the sealing method, 50 μ l of platelet is added into a 96-well plate, and the adhesion is carried out for 60min in a 37 ℃ incubator. After adhesion was complete, the platelets were washed 5 times with PBS to remove non-adherent and non-stably adherent platelets. CCK-810. mu.l/well was added to the platelet-adherent wells and incubated at 37 ℃ for 2 h. Finally, an enzyme labeling instrument reads the OD value under the wavelength of 405nm, and the hole without the added blood platelet is used as a spaceWhite control, the amount of adherent platelets was calculated. Each sample was plated with 3 replicates and the results were averaged. As shown in FIG. 16, c-Src compares to WT miceE97AThe transgenic mouse can obviously inhibit the adhesion function of blood platelets on solid-phase fibrinogen (p)<0.01) to inhibit thrombosis.
Example 12: c-SrcE97ATransgenic mouse to FeCl3The effect of induced carotid occlusion thrombosis.
Selecting 6-8 weeks old WT and c-SrcE97ATransgenic mice were used as subjects. FeCl was used according to experimental methods reported in the literature3The carotid artery of the mouse is stimulated, resulting in endothelial damage and subsequent initiation of the thrombotic process in FeCl3The stimulated distal end detects blood flow with a doppler ultrasound probe, which drops when an upstream thrombus forms and occludes a blood vessel.
As can be seen from the results (FIG. 17), c-Src compares with WT miceE97ATransgenic mice in FeCl3The thrombosis time of the carotid artery under stimulation is obviously prolonged. Indicates that c-SrcE97ATransgenic mouse to FeCl3The induced carotid artery thrombosis has obvious inhibition effect and antithrombotic effect.
Example 13: c-SrcE97ATransgenic mice were tested for the effect on bleeding time by tail-snip.
To evaluate c-SrcE97AEffect of transgenic mice on hemostatic function selection of 6-8 week old WT and c-SrcE97ATransgenic mice were studied. The tail-cutting experiment is the process that after the blood vessel is damaged, the blood platelet maintains the normal hemostasis function and blocks the blood vessel wound. The mouse tail (tail-cut) was cut off rapidly with a sharp blade at 5mm from the tip of the tail, and then the blood exuded from the mouse tail was dipped with filter paper every 15sec without touching the mouse tail to avoid causing new damage. Bleeding stopped and 15sec did not recur as a timing criterion.
As can be seen from the results (FIG. 18), WT mice stopped bleeding at approximately 7.4min, while c-Src stopped bleeding compared to the WT groupE97AThe bleeding time of the transgenic mice is not obviously prolonged, about 8.9min, and the transgenic mice have no statistical difference compared with WTAnd (3) distinguishing. Indicating c-SrcE97AThe transgenic mice can not prolong the tail-shearing bleeding time and basically do not influence the normal physiological hemostasis effect.
Discussion of the related Art
The c-Src SH3 RT-loop antagonist described herein can reduce, inhibit or interfere with the interaction of integrin beta 3 and c-Src, thereby significantly exerting an antithrombotic effect without affecting normal physiological hemostatic effects.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Renjin Hospital affiliated to Shanghai university of transportation medical school
Shanghai Medicine Inst., Chinese Academy of Sciences
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Claims (10)

1. Use of a c-Src SH3 RT-loop antagonist for the preparation of a composition or formulation for:
(a) the interaction of interferon beta 3 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.
2. The use of claim 1 wherein the antagonist is a c-Src SH3 RT-loop region specific antagonist.
3. The use of claim 1 wherein the c-Src SH3 RT-loop antagonist does not antagonize (or affect) or substantially affects the binding (or interaction) of integrin β 3 to the c-Src SH3 n-loop region.
4. The use of claim 1, wherein said antagonist is selected from the group consisting of: small molecule antagonists, antisense nucleotides, mirnas, sirnas, or combinations thereof.
5. The use of claim 1, wherein c-Src SH3 RT-loop is selected from the group consisting of:
(A) a polypeptide with an amino acid sequence shown as SEQ ID No. 3;
(B) an RT-loop region 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 to the amino acid sequence shown in SEQ ID NO. 3;
(C) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 3 and having a mutation selected from the group consisting of: R95A and E97A.
6. Use of a c-Src SH3 RT-loop agonist (agonst) for the preparation of a composition or formulation for use in therapy
(a) Promotes integrin beta 3 and c-Src interactions;
(b) promoting platelet spreading on solid-phase fibrinogen;
(c) promoting aggregation and adhesion of platelets; and/or
(d) Promoting blood coagulation.
7. A method of interfering with the interaction of integrin beta 3 and c-Src proteins, comprising the steps of:
(a) integrin beta 3 and c-Src proteins are contacted in the presence of a c-Src SH3 RT-loop antagonist, thereby interfering with the interaction of integrin beta 3 and c-Src proteins.
8. A method of antithrombotic (or inhibition of platelet aggregation and/or adhesion) comprising the steps of: administering to a subject in need thereof a c-Src SH3 RT-loop antagonist.
9. A method of screening for an anti-thrombotic candidate compound, said method comprising the steps of:
(a) contacting integrin beta 3 and c-Src proteins in the presence of a test agent in a test group, and observing whether integrin beta 3 in the test group binds to the RT-loop region of the c-Src SH3 domain; contacting integrin beta 3 and c-Src protein in the control group in the absence of said test agent and observing whether integrin beta 3 in said control group binds to the RT-loop region of the c-Src SH3 domain;
wherein, if the test group has a significantly lower degree or amount of binding of integrin beta 3 to the RT-loop region of the c-Src SH3 domain than the control group, the test agent is a candidate compound against thrombus,
wherein the candidate compound is a c-Src SH3 RT-loop antagonist.
10. A c-Src mutein characterized in that said mutein has an amino acid mutation at one or more positions selected from the group consisting of: 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.
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