Computer-assisted drug screening method, system or equipment based on PARP-1 and PAD4

Abstract

The invention belongs to the technical field of computer-aided drug screening, discloses a computer-aided drug screening method, system or equipment based on PARP-1 and PAD4, and provides a screening method, system and equipment for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 and a computer readable storage medium for the field for the first time. The invention provides an efficient, accurate and rapid screening method for research and development of new drugs for resisting lung cancer thrombosis, and provides a new research direction and strategy for development of new drugs for reducing tumor thrombosis without increasing bleeding risk.

Description

Technical Field

[0001] The present invention belongs to the technical field of computer-assisted drug screening, and specifically, relates to a computer-assisted drug screening method, system or device based on PARP-1 and PAD4. Background Art

[0002] Domestic and foreign studies have shown that the incidence of thrombosis in cancer patients is 4-7 times that of healthy people, and 20%-30% of the first thrombotic events are related to cancer. Such patients have a poor prognosis and a significantly reduced survival rate. Thrombosis is the second leading cause of death in cancer patients, second only to cancer progression itself. During autopsy, thrombosis can be found in half of the deceased cancer patients. Therefore, the use of antithrombotic drugs to prevent thrombosis has become a necessary means of clinical tumor treatment. However, since antithrombotic drugs such as low molecular weight heparin, eptifibatide, and rivaroxaban can damage the body's normal coagulation function, long-term use of drugs is accompanied by the risk of bleeding.

[0003] Antithrombotic treatment and bleeding risk in cancer patients have always been an unresolved clinical problem. Tumor thrombosis is often caused by factors such as changes in coagulation factors, increased platelet adhesion, and reduced fibrinolysis caused by the tumor itself. Drugs targeting the coagulation system and platelets have become the only means of preventing and treating thrombosis. Although it is known in the art that inflammation and thrombosis are related, it is not clear whether inflammation can promote tumor thrombosis and the mechanism by which inflammation promotes tumor thrombosis, and the research and development of related drugs are limited. Therefore, the art urgently needs to develop a new and efficient screening method for screening new drugs that reduce tumor thrombosis. Summary of the invention

[0004] In order to overcome the above-mentioned technical problems existing in the art, the object of the present invention is to provide a computer-assisted drug screening method, system or device based on PARP-1 and PAD4.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides a screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4, the screening method comprising:

[0007] Obtain PARP-1 protein and PAD4 protein data;

[0008] Selecting the spatial structure of the PARP-1 protein and PAD4 protein complex, and determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex as the binding site of the targeted drug;

[0009] A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

[0010] Furthermore, the computer-assisted drug screening method comprises:

[0011] Obtaining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex;

[0012] Based on the spatial structure of the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0013] Molecular docking is performed on the screened small molecule compounds with PARP-1 protein and PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptors PARP-1 protein and PAD4 protein to obtain the score of each small molecule compound, and the candidate drugs for anti-lung cancer thrombosis are obtained by sorting according to the scores.

[0014] Furthermore, the method for determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex comprises:

[0015] Constructing n truncated expression mutants of PARP-1, where n is a natural number greater than or equal to 1;

[0016] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PARP-1 and PAD4;

[0017] The spatial structure of the key region for the interaction between PARP-1 and PAD4 is the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex.

[0018] Furthermore, the screening method further comprises:

[0019] Obtain lung cancer cell line models and / or lung cancer animal models;

[0020] The screened anti-lung cancer thrombosis candidate drug is used to treat the lung cancer cell line model and / or lung cancer animal model to verify the effectiveness of the anti-lung cancer thrombosis candidate drug.

[0021] The second aspect of the present invention provides a screening system for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4, the screening system comprising:

[0022] Protein data acquisition unit, to acquire PARP-1 protein and PAD4 protein data;

[0023] A binding site determination unit, which selects the spatial structure of the PARP-1 protein and PAD4 protein complex, and determines the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and the PAD4 protein complex as the binding site of the targeted drug;

[0024] A candidate drug screening unit, which uses a computer-assisted drug screening method to obtain a candidate drug targeting the binding site;

[0025] The computer-assisted drug screening method comprises:

[0026] Obtaining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex;

[0027] Based on the spatial structure of the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0028] Performing molecular docking on the screened small molecule compounds with PARP-1 protein and PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptors PARP-1 protein and PAD4 protein to obtain the score of each small molecule compound, and ranking them according to the score to obtain candidate drugs for anti-lung cancer thrombosis;

[0029] Optionally, the method for determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex comprises:

[0030] Constructing n truncated expression mutants of PARP-1, where n is a natural number greater than or equal to 1;

[0031] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PARP-1 and PAD4;

[0032] The spatial structure of the key region for the interaction between PARP-1 and PAD4 is the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex;

[0033] Optionally, the screening method further comprises: a drug effectiveness verification unit, obtaining a lung cancer cell line model and / or a lung cancer animal model; treating the lung cancer cell line model and / or the lung cancer animal model with the screened anti-lung cancer thrombosis candidate drug to verify the effectiveness of the anti-lung cancer thrombosis candidate drug.

[0034] The third aspect of the present invention provides a computer device for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4, the computer device comprising:

[0035] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 described in the first aspect of the present invention is implemented.

[0036] The fourth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, and when the computer program is executed by a processor, the screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 as described in the first aspect of the present invention is implemented.

[0037] A fifth aspect of the present invention provides a method for regulating PAD4 activity, the method comprising:

[0038] Obtaining an in vitro cell system, in vitro tissue system or in vitro organ system containing PAD4;

[0039] The in vitro cell system, in vitro tissue system or in vitro organ system containing PAD4 is treated with PARP-1, a PARP-1 promoter or a PARP-1 inhibitor to regulate the activity of PAD4.

[0040] A sixth aspect of the present invention provides any of the following products:

[0041] (1) An anti-lung cancer thrombosis drug screened according to the screening method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 according to the first aspect of the present invention;

[0042] (2) A pharmaceutical composition for reducing tumor thrombosis without increasing the risk of bleeding, the pharmaceutical composition comprising Olaparib; optionally, the tumor is lung cancer.

[0043] A sixth aspect of the present invention provides any of the following applications:

[0044] (1) Use of Olaparib, an anti-tumor drug targeting PARP-1, in the preparation of a drug for reducing tumor thrombosis without increasing the risk of bleeding; optionally, the tumor is lung cancer;

[0045] (2) Application of PARP-1, PARP-1 promoters or PARP-1 inhibitors in regulating PAD4 activity in vitro.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The present invention first discovered that PARP-1 has potential physiological and pathological effects in regulating PAD4 activity. PARP-1 promotes histone H3 citrullination and lung cancer-induced NETs production by regulating PAD4 activity, thereby leading to lung cancer thrombosis. Based on this, the present invention provides a screening method, system, device and computer-readable storage medium for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 in the art. The present invention provides an efficient, accurate and rapid screening method for the research and development of new anti-lung cancer thrombosis drugs, and also provides a new research direction and strategy for the development of new drugs that reduce tumor thrombosis without increasing the risk of bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : A schematic diagram of a screening method for anti-thrombotic drugs for lung cancer based on PARP-1 and PAD4;

[0049] Figure 2 : Schematic diagram of a screening system for anti-thrombotic drugs for lung cancer based on PARP-1 and PAD4;

[0050] Figure 3 : Schematic diagram of a computer device for screening anti-thrombotic drugs for lung cancer based on PARP-1 and PAD4;

[0051] Figure 4 :Lung cancer promotes the formation of NETs and VTE in vivo, including: A: plasma MPO-DNA levels in lung cancer patients and healthy people; B: blood cfDNA levels in lung cancer patients and healthy people; C: plasma D-Dimer levels in lung cancer patients and healthy people; D: Elisa detection of peripheral blood MPO-DNA levels in tumor-bearing mice, n=4; E: Sytox green detection of peripheral blood cfDNA levels in tumor-bearing mice, n=4; F: thrombus plane diagram of each group; G: thrombus weight statistics of each group; H: NETs immunofluorescence diagram of thrombus in each group; *P<0.05, **P<0.01, ***P<0.001;

[0052] Figure 5:Lung cancer cells promote NETs production in vitro, among which, Figure A: A549 tumor cell culture supernatant (A549-CM) stimulated human neutrophils (dHL-60), and Elisa detected the MPO-DNA level in neutrophil supernatant; Figure B: Sytox green detected the cf-DNA level in culture supernatant; Figure C: Western blot detected the CitH3 expression level in neutrophils; Figure D: Immunofluorescence detected the cfDNA morphology; Figure E: LLC tumor cell culture supernatant (LLC-CM) stimulated mouse neutrophils (BMN), and Elisa detected the MPO-DNA level in neutrophil supernatant; Figure F: Sytox green detected the cfDNA level in culture supernatant; Figure G: Western blot detected the CitH3 expression level in neutrophils; Figure H: Immunofluorescence detected the cfDNA morphology, *P<0.05, **P<0.01, ***P<0.001;

[0053] Figure 6 : PARP-1 interacts with PAD4 and regulates its activity through PARylation, wherein, Figure A: Human neutrophil dHL-60 cells were stimulated with a549-conditioned medium (A549-CM) or control medium at 37°C for 4 hours, and the expression of PAD4 was detected by Western blot; Figure B: Mouse bone marrow neutrophils were stimulated with LLC-conditioned medium (LLC-CM) or control medium at 37°C for 4 hours, and Western blot was used to detect the expression of PAD4. The expression of PAD4 was detected by blot; CD: Neutrophils were stimulated with lung cancer cell conditioned medium or control medium at 37°C for 4 hours to determine the activity of PAD4; E: PARylation of PAD4 was detected by immunoprecipitation of neutrophils stimulated with lung cancer cell conditioned medium; F: PARylation of PAD4 was detected by immunoprecipitation after PARP-1 was knocked down or inhibited by 10μM olaparib with or without stimulation of lung cancer cell conditioned medium; GH: PAD4 activity was detected after PARP-1 was knocked down or inhibited by olaparib; IJ: Neutrophils were stimulated with lung cancer cell conditioned medium or control medium at 37°C for 4 hours, and the binding of PARP-1 and PAD4 was determined by immunocoprecipitation with anti-PARP-1 antibody or anti-PAD4 antibody. The data are expressed as mean ± SEM; ***P < 0.001;

[0054] Figure 7: Knockdown or inhibition of PARP-1 in vitro blocks lung cancer-induced NETs formation, wherein, Figure AD: Human PARP-1 knockdown (PARP-1KD) neutrophils (dHL-60) were stimulated with A549 conditioned medium (A549-CM) or control medium at 37°C for 4 hours; Figure A: ELISA was used to detect the level of MPO-DNA in the supernatant; Figure B: SYTOX Green was used to detect the level of cfDNA in the supernatant; Figure C: Western blot was used to detect CitH3 expression; Figure D: cfDNA staining confocal imaging was used to detect DNA network structure; Figure EH: At 37°C, LLC conditioned medium (LLC-CM) or control medium was used to stimulate mouse bone marrow neutrophils for 4 hours with the addition of 10μM Olaparib; Figure E: ELISA was used to detect the level of MPO-DNA in the supernatant; Figure F: SYTOX Green was used to detect the level of cfDNA in the supernatant; Figure G: Western blot Blot analysis of CitH3 expression; Figure H: Confocal imaging of cfDNA staining to detect DNA network structure, data are expressed as mean ± SEM; **P < 0.01, ***P < 0.001;

[0055] Figure 8 : Clinical use of Olaparib can reduce the risk of thrombosis in cancer patients, among which, Figure AD: Analysis of D-dimer, fibrinogen degradation product (FDP), fibrinogen (FIB) and thrombin time (TT) in tumor patients taking and not taking Olaparib, data are expressed as mean ± SEM; **P < 0.01;

[0056] Fig. 9 :Olaparib blocks NETs-promoted thrombosis in lung cancer mice in vivo, where A: 5×10 6 LLC, tumor volume up to 500mm 3 , Olaparib (25 mg / kg) was intravenously injected for 5 consecutive days, and the plasma MPO-DNA level was detected by ELISA; Figure B: SYTOX Green was used to detect the cfDNA level in mouse plasma; Figure C: Thrombus formation in mice after inferior vena cava stenosis surgery; Figure D: Thrombus weight analysis; Figure E: Representative pictures of thrombus CitH3 immunofluorescence staining, data are expressed as mean ± SEM; **P < 0.01, ***P < 0.001;

[0057] Fig.10:Olaparib reduces thrombosis in lung cancer mice without bleeding risk, among which, AB: wild-type mice were intravenously injected with Olaparib (20 mg / kg), rivaroxaban (0.3 mg / kg) or eptifibatide (5 μmol / kg) for 5 consecutive days, and the tail bleeding time was measured and the bleeding volume (HGB, hemoglobin) was evaluated; C: 5×10 5 LLC. Tumor volume up to 500mm 3 , Olaparib, rivaroxaban or eptifibatide were intravenously injected for 5 consecutive days, and the plasma MPO-DNA level was detected by ELISA; D: SYTOX Green was used to detect the cfDNA level in mouse plasma; E: Photos of mouse thrombi after inferior vena cava stenosis surgery; F: Thrombus weight analysis; G: Determination of tail bleeding time; H: Assessment of blood loss; I: Representative pictures of CitH3 immunofluorescence staining of thrombi, data are expressed as mean ± SEM; **P < 0.01, ***P < 0.001, ns: no statistical difference. DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0059] In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations appearing in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or may be executed in parallel. The serial numbers of the operations, such as S101, S102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0060] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0061] Figure 1 The present invention provides a method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4. Specifically, the method comprises the following steps:

[0062] S101: Obtain PARP-1 protein and PAD4 protein data;

[0063] In one embodiment, the PARP-1 refers to poly adenosine diphosphate (ADP) ribose polymerase-1

[0064] (poly ADP-ribose polymerase-1, PARP-1), PARP-1 is an important member of the PARP family. It is the most abundant multifunctional protein post-translational modification enzyme in the PARP family. It plays an important role in DNA damage repair and is widely distributed in eukaryotic cells in various tissues of the human body.

[0065] In one embodiment, the PAD4 refers to Peptidylarginine Deiminase 4 (PAD4), which is a post-transcriptional modification enzyme. PAD4 catalyzes the removal of imino groups from arginine residues in peptide chains to generate citrulline residues. In addition, PAD4 can remove imino groups from transcriptional regulators (such as p300, histone H2A, histone H3, histone H4), playing an important role in gene regulation.

[0066] S102: selecting the spatial structure of the PARP-1 protein and PAD4 protein complex, and determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex as the binding site of the targeted drug;

[0067] In one embodiment, the method for determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex comprises:

[0068] Constructing n truncated expression mutants of PARP-1, where n is a natural number greater than or equal to 1;

[0069] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PARP-1 and PAD4;

[0070] The spatial structure of the key region for the interaction between PARP-1 and PAD4 is the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex.

[0071] In one embodiment, the binding site can be obtained by conventional methods well known to those skilled in the art, and the present invention has no particular limitation on the specific method for determining the binding site.

[0072] In one embodiment, the present invention demonstrates that PARP-1 interacts with PAD4 and regulates its activity through PARylation. The specific experimental methods and experimental results are as follows:

[0073] The specific experimental methods are as follows: (1) Immunoprecipitation: 1) Cell culture and tumor cell conditioned medium were used to stimulate neutrophils as before; 2) Neutrophil suspension was taken out and placed in EP tubes, and three tubes were set up: activation group, non-activation group, and Normal IgG group. The tubes were centrifuged at 2000 rpm and 4°C for 5 min, and the supernatant was discarded. The remaining cell pellet was added with 600 μL RIPA (containing 6 μL PI, 100:1) cell lysis buffer, pipetted and mixed, and lysed on ice for 15 min; 3) Centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was taken out. 50 μL was taken out from each tube and reserved as the input group. The remaining part was added with 20 μL Protein A / G agarose breads, inverted and mixed, and incubated at 4°C for 1-2 h. Centrifuged at 4°C and 1000 g for 5 min; 4) The supernatant was taken out, and equal amounts of PAD4 or PARP-1 antibodies (1 mg / mL) were added to the non-activated group and the activated group, and Normal IgG of the same species was added to the Normal IgG group. IgG, the operation of the three tubes must be consistent afterwards; 5) Slowly shake at 4℃ for 8h; 6) Add 30μL Protein A / G agarose beads, mix by inversion, and slowly shake at 4℃ overnight; 7) Centrifuge at 1000g, 4℃ for 5min, discard the supernatant, add 1mL 1×RIPA lysis buffer, mix by inversion, let stand on ice for 3min, centrifuge at 1000g, 4℃ for 5min; 8) Repeat step 7) 4-5 times; 9) Discard the supernatant, add 50μL 2×SDS-PAGE protein loading solution, heat at 100℃ for 10min, quickly insert the EP tube into the ice box for 5min, and centrifuge at 12000rpm, 4℃ for 5min; 10) Take the supernatant and perform Western Blot experiment to detect PAR, PAD4 or PARP-1.

[0074] (2) PAD4 enzyme activity detection: 1) Cell culture and tumor cell conditioned medium were used to stimulate neutrophils as before; 2) Cells were repeatedly frozen and thawed in liquid nitrogen; 3) ELISA kit was used for determination.

[0075] The specific experimental results are as follows: The results showed that lung cancer cells directly promoted the formation of NETs and significantly increased histone H3 citrullination ( Figure 5 ). Therefore, this example first verifies whether lung cancer stimulation affects the expression of PAD4 during NETs formation. The results show that after lung cancer cell stimulation, the expression of PAD4 did not change ( Figure 6 AB). Therefore, this example further verifies whether lung cancer cells affect the enzyme activity of PAD4. The results show that under the stimulation of lung cancer cell conditioned medium, the enzyme activity of PAD4 increased significantly ( Figure 6CD). Since PAD4 activity increased while protein expression did not change, the inventors speculated that lung cancer regulates PAD4 activity through post-translational modification of the protein. In previous studies by the inventors, it was confirmed that lung cancer cells promote the expression of neutrophil PARP-1. In addition to the function of repairing DNA damage, PARP-1 also plays a key role in post-translational modification (called PARylation) by transferring ADP ribose units to protein substrates, thereby changing protein function (such as enzyme activity). In order to illustrate whether the PARylation of PAD4 also changes after stimulation with lung cancer cell conditioned medium, this example conducted an immunoprecipitation experiment. In human and mouse neutrophils, the PARylation of PAD4 increased with the stimulation of lung cancer cells ( Figure 6 E). Furthermore, when PARP-1 was knocked down or blocked by PARP-1, the lung cancer cell-induced increase in PAD4 PARylation was significantly suppressed in human and mouse neutrophils ( Figure 6 F). Consistent with this, PAD4 activity was elevated in neutrophils stimulated by lung cancer cells, and PARP-1 knockdown or olaparib blocked the increased PAD4 activity in lung cancer cells ( Figure 6 GH).

[0076] Then, this example aims to explore whether PARP1 interacts with PAD4. Co-IP results showed that the interaction level of PARP-1 and PAD4 was low in control neutrophils. However, under the stimulation of lung cancer cell conditioned medium, the interaction between the two proteins increased significantly ( Figure 6 I and J). These results suggest that PARP-1 has potential physiopathological implications in regulating PAD4 activity, thereby promoting histone H3 citrullination to promote lung cancer-induced NETs production.

[0077] It can be seen that the research results of the embodiments of the present invention prove the close correlation between PARP1 and PAD4. PARP-1 promotes histone H3 citrullination by regulating PAD4 activity to promote the production of NETs induced by lung cancer, thereby promoting lung cancer thrombosis. That is, PARP-1 can increase the activity of PAD4, promote histone H3 citrullination, promote the production of NETs induced by lung cancer, and then promote lung cancer thrombosis. Knockdown or blocking PARP-1 can significantly inhibit the activity of PAD4, inhibit histone H3 citrullination, inhibit the production of NETs induced by lung cancer, and then inhibit lung cancer thrombosis.

[0078] Based on the above research results discovered for the first time by the present invention, the present invention provides a new screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 in the art. The candidate drugs screened by the above screening method provided by the present invention are anti-lung cancer thrombosis drugs.

[0079] S103: Using a computer-assisted drug screening method to obtain candidate drugs targeting the binding site.

[0080] In one embodiment, the computer-assisted drug screening method comprises:

[0081] Obtaining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex;

[0082] Based on the spatial structure of the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0083] Molecular docking is performed on the screened small molecule compounds with PARP-1 protein and PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptors PARP-1 protein and PAD4 protein to obtain the score of each small molecule compound, and the candidate drugs for anti-lung cancer thrombosis are obtained by sorting according to the scores.

[0084] In one embodiment, the screening method further comprises:

[0085] Obtain lung cancer cell line models and / or lung cancer animal models;

[0086] The screened anti-lung cancer thrombosis candidate drug is used to treat the lung cancer cell line model and / or lung cancer animal model to verify the effectiveness of the anti-lung cancer thrombosis candidate drug.

[0087] In one embodiment, the small molecule compound includes an antibody, a polypeptide, a protein analog, siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, a peptide analog or an inorganic small molecule compound. It should be noted that the present invention has no particular limitation on the specific type of the small molecule compound, and any small molecule compound that may be used in the computer-assisted drug screening method provided by the present invention and may produce a corresponding effect (inhibiting PARP-1 expression or inhibiting PAD4 activity) falls within the protection scope of the present invention.

[0088] In one embodiment, in molecular docking, affinity refers to how tightly a molecule binds to a receptor. A high affinity means that the binding is more stable, while a low affinity means that the binding is less stable. Affinity is usually calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0089] In order to evaluate affinity, some scoring systems or scoring methods are usually used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models, which can reflect the binding energy, interaction type and affinity between molecules and receptors.

[0090] In one embodiment, molecular docking is performed between the screened small molecule compounds and PARP-1 protein or PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptor PARP-1 protein or PAD4 protein to obtain a score for each small molecule compound, and the compounds are sorted according to the scores to obtain candidate drugs for anti-lung cancer thrombosis. Small molecule compounds with higher affinity or binding energy targeting the receptor PARP-1 protein or PAD4 protein are preferably selected as candidate drugs for anti-lung cancer thrombosis.

[0091] In one embodiment, the modes of drug screening using computer-assisted drug screening technology include one or more of the following: protein-small molecule docking, protein-protein docking, protein-nucleic acid docking, which are all well-known to those skilled in the art.

[0092] In one embodiment, the molecular libraries used in drug virtual screening include: ZINC, PubChem, DrugBank, ChEMBL, ChemDB, HMDB, BindingDB, SMPDB, ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M and Interbioscreen, etc. These databases are also commonly used for drug virtual screening. The present invention has no particular restrictions on the specific type of molecular library used in the drug virtual screening, and molecular libraries known to those skilled in the art can be used in the screening method of the present invention.

[0093] Figure 2 Schematic diagram of a screening system for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 provided by an embodiment of the present invention. Specifically, the screening system includes:

[0094] Protein data acquisition unit, to acquire PARP-1 protein and PAD4 protein data;

[0095] A binding site determination unit, which selects the spatial structure of the PARP-1 protein and PAD4 protein complex, and determines the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and the PAD4 protein complex as the binding site of the targeted drug;

[0096] A candidate drug screening unit, which uses a computer-assisted drug screening method to obtain a candidate drug targeting the binding site;

[0097] The computer-assisted drug screening method comprises:

[0098] Obtaining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex;

[0099] Based on the spatial structure of the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex, screening small molecule compounds with similar structures in a molecular database;

[0100] Performing molecular docking on the screened small molecule compounds with PARP-1 protein and PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptors PARP-1 protein and PAD4 protein to obtain the score of each small molecule compound, and ranking them according to the score to obtain candidate drugs for anti-lung cancer thrombosis;

[0101] The method for determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex comprises:

[0102] Constructing n truncated expression mutants of PARP-1, where n is a natural number greater than or equal to 1;

[0103] Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PARP-1 and PAD4;

[0104] The spatial structure of the key region for the interaction between PARP-1 and PAD4 is the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex;

[0105] The screening method also includes: a drug effectiveness verification unit, which obtains a lung cancer cell line model and / or a lung cancer animal model; and uses the screened anti-lung cancer thrombosis candidate drug to treat the lung cancer cell line model and / or the lung cancer animal model to verify the effectiveness of the anti-lung cancer thrombosis candidate drug.

[0106] Figure 3Schematic diagram of a computer device for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 provided by an embodiment of the present invention. Specifically, the computer device includes:

[0107] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 as described above is implemented in the present invention.

[0108] An embodiment of the present invention further provides a computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 as described above is implemented.

[0109] The present invention also provides a method for regulating PAD4 activity, the method comprising:

[0110] Obtaining an in vitro cell system, in vitro tissue system or in vitro organ system containing PAD4;

[0111] The in vitro cell system, in vitro tissue system or in vitro organ system containing PAD4 is treated with PARP-1, a PARP-1 promoter or a PARP-1 inhibitor to regulate the activity of PAD4.

[0112] In one embodiment, the present invention demonstrates through experiments that PARP-1 can increase the activity of PAD4, thereby promoting lung cancer thrombosis, while knocking down or blocking PARP-1 can significantly inhibit the activity of PAD4, thereby inhibiting lung cancer thrombosis.

[0113] The present invention also provides any of the following products:

[0114] (1) An anti-lung cancer thrombosis drug screened according to the screening method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 as described above in the present invention;

[0115] (2) A pharmaceutical composition for reducing tumor thrombosis without increasing the risk of bleeding, the pharmaceutical composition comprising Olaparib; optionally, the tumor is lung cancer.

[0116] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. The main active ingredient is Olaparib.

[0117] In one embodiment, the Olaparib refers to Olaparib, which is an oral poly(ADP-ribose) polymerase inhibitor, mainly used to treat certain types of ovarian cancer, breast cancer and prostate cancer. The drug is an approved drug and its safety has been clinically verified.

[0118] In one embodiment, the tumor includes, but is not limited to, any one or more of lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, squamous cell carcinoma, lymphoma, osteosarcoma, esophageal cancer, renal cell carcinoma, glioma, prostate cancer, pancreatic cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, endometrial cancer, ovarian cancer, and cervical cancer.

[0119] The embodiments of the present invention also provide any of the following applications:

[0120] (1) Application of Olaparib, an anti-tumor drug targeting PARP-1, in the preparation of a drug for reducing tumor thrombosis without increasing the risk of bleeding;

[0121] (2) Use of PARP-1, PARP-1 promoter or PARP-1 inhibitor in regulating the activity of PAD4 in vitro; optionally, the tumor is lung cancer.

[0122] In one embodiment, the present invention discovers and demonstrates for the first time the novel use of the PARP-1-targeting anti-tumor drug Olaparib as an anti-cancer thrombotic drug, and compared with traditional anti-thrombotic drugs, it has no bleeding risk, that is, Olaparib can not only prevent the occurrence and development of cancerous thrombosis, but also is not accompanied by bleeding risk.

[0123] The verification results of this verification example show that assigning inherent weights to indications can moderately improve the performance of the method relative to the default settings. Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments, and will not be repeated here.

[0124] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0125] The units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. In addition, the functional units in the various embodiments of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated units may be implemented in the form of hardware or in the form of software functional units.

[0126] A person skilled in the art can understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing the relevant hardware through a program, and the program can be stored in a computer-readable storage medium, and the storage medium can include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc. A person skilled in the art can understand that all or part of the steps in the methods of the above embodiments can be completed by instructing the relevant hardware through a program, and the program can be stored in a computer-readable storage medium, and the storage medium mentioned above can be a read-only memory, a disk or an optical disk, etc. A computer device provided by the present invention is introduced in detail above. For a person skilled in the art, according to the idea of ​​the embodiments of the present invention, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

[0127] The present invention is further described below in conjunction with specific examples. The specific examples are only used to explain the present invention and cannot be construed as limiting the present invention. It can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art. If not otherwise specified, they can be obtained from commercial sources. The experimental methods for which specific conditions are not indicated in the present invention are usually implemented and detected according to conventional conditions or according to the conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0128] Example 1 Cancer promotes neutrophil extracellular traps (NETs) and venous thrombosis

[0129] 1. Experimental Materials

[0130] Human and mouse MPO-DNA detection kits, D-Dimer detection kit: Jiangsu Enzyme Immunoassay; LLC cells (CL-0140): China Procell Life Science Co., Ltd.; anti-mouse CitH3 antibody: Abcam; Sytox green: Thermo Fisher Scientific; C57 mice: Xuzhou Medical University Animal Center; plasma from lung cancer patients and healthy people: Department of Laboratory, Affiliated Hospital of Xuzhou Medical University.

[0131] 2. Experimental methods

[0132] (1) Detection of plasma MPO-DNA, cfDNA and D-Dimer levels: 1) Collect plasma from lung cancer patients and healthy subjects and freeze at -80°C; 2) Perform the test according to the test steps provided in the test kit.

[0133] (2) Establishment of LLC lung cancer subcutaneous transplanted tumors in mice: 1) Preparation of tumor cells: When LLC cells have grown to 80-90% of the bottom of the dish, trypsinize the cells and resuspend them in PBS at 800 rpm for 5 min. Discard the supernatant to obtain the cell pellet. Repeat the resuspension and centrifugation steps twice and dilute the cells to 3×10 with PBS. 6 / mL, prepare 120μL per tube and keep on ice. 2) Mouse preparation: Prepare healthy 6-week-old C57 / 6 male mice in advance, remove the hair from the right leg to the waist and abdomen of the mice, and fully expose the inoculation site. 3) Inoculation: Wipe the inoculation site of the mice with 75% alcohol cotton balls, shake the cell suspension thoroughly, take 1mL insulin injection needle to extract 100μL cell suspension, and keep the number of LLC cells inoculated at 3×10 5 / mouse, the inoculation site was subcutaneous on the right side of the waist and abdomen, and obvious skin papules were visible after the inoculation. The control group mice were injected with an equal volume of PBS at the same site, and the day of inoculation was designated as day 0.

[0134] (3) Establishment of the inferior vena cava thrombosis model: 1) The mouse was injected with the anesthetic chloral hydrate intraperitoneally in advance. When the mouse muscles were relaxed, the limbs were inactive, the whiskers did not show any touch reaction, and the pedal reflex disappeared, it was considered to be completely anesthetized. When the mouse was completely anesthetized, the chest and abdomen hair of the mouse was removed to fully expose the chest and abdomen skin of the mouse. 2) The mouse was kept in a supine position, and the mouse abdomen was fully exposed during surgery. 37℃ gauze was prepared in advance and placed on the waist and abdomen of the mouse. The mouse intestine was slowly pulled out and placed on the preheated gauze to fully expose the abdominal field of vision. 3) The inferior vena cava was carefully separated from the surrounding tissues, and a 4mm surgical suture was slowly placed between the inferior vena cava and the tissues. A 30μm syringe needle was placed parallel to the inferior vena cava, and the suture was slowly tied with a surgical knot. 4) After the surgical knot was tied, the 30μm syringe needle was slowly withdrawn and the excess suture was cut off. 5) The mouse intestine was slowly placed in the abdominal cavity and the abdominal skin was sutured. 6) After the operation, place the mouse on a heating pad. After the mouse recovers spontaneous breathing and can move freely, put it back into the cage and observe the mouse's condition regularly. 7) The same steps are performed on the mice in the control group. 8) Collect thrombi: 48 hours later, euthanize the mice, quickly expose the inferior vena cava of the mice, collect thrombi, take pictures of the mouse thrombi, weigh and record them, and store them in tissue fixative. They can be used for subsequent experiments. Some of them are sent to institutions to prepare frozen sections.

[0135] (4) Immunofluorescence staining of CitH3 in thrombi: 1) After embedding in OTC, the frozen sections of the above thrombi were dried at room temperature. 2) Washed with PBS 3 times, 3 min / time. 3) Soaked in 0.1% Triton X-100 for 10 min to destroy the cell membrane. 4) Washed with PBS 3 times, 3 min / time. 5) Blocked with 20% goat serum for 10 min. 6) Washed with PBS 3 times, 3 min / time. 7) Prepared primary antibody at a volume ratio of 1:100 and incubated at 4℃ for 8 hours or overnight. 8) Washed with PBS 3 times, 3 min / time. 9) Prepared corresponding secondary antibody at a volume ratio of 1:200 and incubated at 37℃ for 1 hour. 10) Washed with PBS 3 times, 3 min / time in the dark. 11) Added Fluoroshield mounting medium containing DAPI dye and covered with a coverslip. 12) Detected with a Confocal microscope at the excitation wavelength corresponding to the secondary antibody.

[0136] (5) Statistical analysis: All experimental data were statistically analyzed using GraphPad Prism 6 software and expressed as mean ± standard deviation (Mean ± SD). When comparing two groups of data, the Student's t test was used; when comparing multiple groups of data, one-way analysis of variance was used, and the Tukey test was further used. P value < 0.05 was used as the standard for significant difference.

[0137] 3. Experimental results

[0138] First, the present invention collects plasma from clinical lung cancer patients and healthy people undergoing physical examinations, and detects the levels of MPO-DNA, cfDNA, and D-Dimer, a classic neutrophil extracellular trap (NETs) marker, in the plasma. The results show that the levels of MPO-DNA, cfDNA, and D-Dimer in the plasma of lung cancer patients are significantly increased ( Figure 4 AC), indicating that the plasma NETs level of lung cancer patients increased significantly and the blood was in a hypercoagulable state, which was prone to thrombosis.

[0139] Furthermore, in order to detect whether lung cancer promotes venous thrombosis (VTE) formation, the present invention constructed a subcutaneous transplant tumor model of wild-type C57 mice LLC cell lung cancer cells and incubated the mice with tumors up to 500 mm in size. 3 The mice were operated on for inferior vena cava stenosis at 14:00 pm, and venous thrombi were collected 48 hours later. The results showed that the plasma NETs markers of lung cancer mice were significantly increased compared with those of control mice ( Figure 4 DE), and the thrombus weight increased significantly ( Figure 4 FG), thrombus immunofluorescence results showed that the level of NETs in the thrombus site of lung cancer mice was increased ( Figure 4 H), indicating that lung cancer promotes the formation of NETs and VTE in vivo.

[0140] Example 2 Lung cancer cells promote NETs generation in vitro

[0141] 1. Experimental Materials

[0142] LLC cells (CL-0140), A549 cells (CL-0016) and HL-60 cells (CL-0110) were purchased from Procell Life Science Co., Ltd., China. Anti-mouse β-actin antibody: Proteintech. Anti-mouse CitH3 antibody: Abcam; Sytox green: Thermo Fisher Scientific; Horseradish enzyme-labeled goat anti-rabbit IgG (H+L): Zhongshan Jinqiao Technology Co., Ltd. BCA protein quantification kit: Beyotime Biotechnology.

[0143] 2. Experimental methods

[0144] (1) Induction of human neutrophils (dHL-60): 1) HL-60 cell culture; 2) Transfer the cell suspension into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh culture medium to resuspend the cells, and count the cells under a microscope using a hemocytometer; 3) 25 cm 2 Add 2.5×10 6 cells, and the culture medium is supplemented to 10 mL; 4) 130 μL DMSO (accounting for 1.3% of the total volume) is added and mixed; 5) the cells are placed in a 37° C., 5% CO 2 incubator and cultured for 5-7 days to become neutrophil-like cells (dHL-60).

[0145] (2) Isolation of mouse bone marrow neutrophils: 1) Sterilize surgical scissors and forceps in advance, pre-cool calcium- and magnesium-free HBSS, prepare 20mL and 1mL syringes, and 60mm culture dishes. All the following operations are performed in a clean bench. 2) Euthanize the mouse, spray an appropriate amount of 70% alcohol to soak the fur, cut the skin on its back with scissors, remove its femur and tibia, remove the clean surface muscles, and place them in a small dish filled with pre-cooled HBSS for later use. 3) Cut off both ends of the bone with scissors, hold the bone with forceps in the left hand, and hold the syringe containing 10mL HBSS in the right hand to flush the bone marrow into a 15mL centrifuge tube, then use a pipette to mix the solution evenly, filter it through a 40μm filter into a new 50mL centrifuge tube to remove impurities. 4) Centrifuge at 1800rpm for 5min. At the same time, prepare 3 mL of 1× red blood cell lysis solution and 62% Percoll separation solution for each mouse. Take a sterile 15 mL tube, add 9 mL of ddH2O and 1 mL of 10× red blood cell lysis solution (stock solution), and shake evenly. Add 9 mL of Percoll stock solution and 1 mL of 10× PBS to a sterile 15 mL tube, shake evenly to obtain 100% Percoll, then take 6.2 mL of 100% Percoll and mix with 3.8 mL of 1× PBS to obtain 62% Percoll. Mark the tube clearly and store at 4°C away from light. 5) Remove the cell supernatant after centrifugation, add 3 mL of 1× red blood cell lysis solution to each tube, shake evenly, and incubate at room temperature away from light for 6-8 minutes. During this period, you can take it out and flick the tube wall to fully lyse the red blood cells. 6) Add three times the volume of HBSS of the red blood cell lysis solution to stop the red blood cell lysis, shake evenly, centrifuge at 1800 rpm for 5 minutes, and discard the supernatant. 7) Add 3mL of calcium- and magnesium-free HBSS solution to resuspend the cell pellet and transfer it to a new 15mL tube. Use a pipette to blow away the pellet until there are no obvious clumps. Slowly add 3mL of 62% Percoll to the bottom of the cell suspension. Do not add too fast a speed to make obvious stratification. 8) Precool the centrifuge to 4℃, balance the centrifuge tube, set the speed to 0, and centrifuge at 2500rpm for 35min. 9) After centrifugation, carefully discard the supernatant in the tube, add HBSS to wash, centrifuge at 1800rpm for 5min, discard the supernatant, and the pellet is mouse neutrophils. Add 1mL of HBSS to resuspend the cell pellet and count it. 10) Take out 1×10 5 Cells were centrifuged at 1800 rpm for 5 min, the supernatant was removed, anti-mouse Ly6G and anti-mouse CD11b were diluted to 1 μg / mL with PBS, 50 μL of antibody diluent was added to resuspend the cell pellet, shaken evenly, incubated at 4°C for 30 min, washed and filtered into flow tubes, and the purity of extracted neutrophils was detected by flow cytometry. If the proportion of double-positive cells is greater than 90%, it can be used for subsequent experiments.

[0146] (3) Preparation of tumor cell conditioned medium: 1) A549 and LLC cells were cultured normally; 2) the cell density reached 90% of the bottom of the dish, and the cells were digested with trypsin to form a single cell suspension, and passaged at a ratio of 1:4; 3) the culture supernatant was collected after 48 hours, and centrifuged at 2000 rpm for 10 minutes to obtain the supernatant, which was the tumor cell conditioned medium (A549-CM, LLC-CM).

[0147] (4) Stimulation of neutrophils with tumor cell conditioned medium: 1) Wash human or mouse neutrophils once with PBS; 2) Count the cells, taking 1×10 neutrophils per tube. 6 ; 3) Add 1 mL of cancer cell conditioned medium and incubate at 37°C for 4 hours; 4) Centrifuge at 2000 rpm for 5 minutes, take the supernatant to detect MPO-DNA and cfDNA, and use the cell pellet to detect CitH3 expression by Western blot.

[0148] (5) Cell protein extraction: 1) Take an appropriate amount of lysis buffer and add PI (RIPA:PI=100:1) within a few minutes before use; 2) Take out the activated neutrophils frozen in the -80℃ refrigerator mentioned above; 3) Add 100μL RIPA lysis buffer to each EP tube, insert the EP tube into ice, and shake on a horizontal shaker for 30min; 4) After the cells are fully lysed, centrifuge at 12000rpm, 4℃, for 10min; 5) Transfer the supernatant total protein to a new 1.5mL centrifuge tube and store in a -80℃ refrigerator for later use.

[0149] (6) BCA method for protein concentration detection: 1) Take 1.2 mL of protein standard solution and add it to a tube of protein standard. After fully dissolving, prepare a 25 mg / mL protein standard solution; 2) Take an appropriate amount of protein standard solution (25 mg / mL) and dilute it with PBS to a final concentration of 0.5 mg / mL protein standard working solution; 3) Prepare an appropriate amount of BCA working solution at a ratio of 50 volumes of BCA reagent A to 1 volume of BCA reagent B and mix thoroughly; 4) Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the protein standard working solution to the standard wells of a 96-well plate, and add ddH2O to each well to make up the volume to 20 μL; 5) Add 1 μL of the protein solution to be tested to each well of the sample well, add 19 μL of ddH2O, and set up 3 replicate wells for each sample; 6) Add 200 μL of The BCA working solution was placed at 37°C for 30 min; 7) the OD value was detected at a wavelength of 562 nm using an ELISA reader; 8) a standard curve was drawn to calculate the protein concentration.

[0150] (7) Western Blot experiment: 1) Cell culture and induction activation were performed as before, and the total protein of each group was extracted by the above method; 2) The protein stock solution of each group was diluted to the same concentration with ddH2O, and the protein loading buffer was added and mixed, and then heated and boiled for 10 min to prepare the protein loading solution; 3) The protein molecular weight marker was taken out from the -20℃ freezer and slowly melted on ice; 4) The gel plate was assembled; 5) The gel was prepared; 6) The gel was poured; 7) The electrophoresis tank was installed; 8) The sample was loaded and electrophoresed; 9) The membrane was transferred; 10) The blocking was performed; 11) The primary antibody was incubated; 12) The secondary antibody was incubated; 13) The color was developed.

[0151] (8) NETs confocal detection: 1) Take neutrophils (dHL-60) on the 5th day of induction and dilute them to 1×10 5 / 100μL, 1800rpm, 5min, discard the supernatant to obtain the cell pellet; 2) Resuspend the tumor cell conditioned medium, place a poly-lysine-coated glass slide at the bottom of the 24-well plate, and add the cell suspension; incubate in a 37°C incubator; 3) After 4h, discard the supernatant of each group, incubate the glass slide with 100μL Sytoxgreen working solution, and after 20min at room temperature away from light, discard the supernatant, take the glass slide and put it on the slide in the opposite direction, seal it with nail polish, and prepare for confocal observation of NETs structure later; 4) Isolate mouse neutrophils, and use LLC cell conditioned medium as the experimental condition. The specific experimental methods and detection methods are as above.

[0152] 3. Experimental results

[0153] To further verify the effect of lung cancer cells on NETs formation at the cellular level, the present invention collected the culture supernatants of human A549 and mouse LLC lung cancer cell lines and stimulated neutrophils (human neutrophils HL-60, mouse neutrophils BMN). The results showed that compared with the control group, the levels of NETs markers MPO-DNA, cf-DNA, CitH3 and reticular free DNA in the tumor supernatant were significantly increased ( Figure 5 AH), indicating that lung cancer cells promote NETs formation in vitro.

[0154] Example 3 PARP-1 interacts with PAD4 and regulates its activity through PARylation 1. Experimental materials

[0155] Anti-PAD4 antibody: Abclonal. Anti-Poly (ADP-ribose) antibody: Enzo Life Sciences. PAD4 enzyme activity detection kit: Signal Chem. Anti-PARP-1 antibody: CST. Olaparib: MCE.

[0156] 2. Experimental methods

[0157] (1) Immunoprecipitation: 1) Cell culture and tumor cell conditioned medium were used to stimulate neutrophils as before; 2) Neutrophil suspension was taken out and placed in EP tubes, and three tubes were set up for activation group, non-activation group, and Normal IgG group. The tubes were centrifuged at 2000 rpm and 4°C for 5 min, and the supernatant was discarded. The remaining cell pellet was added with 600 μL RIPA (containing 6 μL PI, 100:1) cell lysis buffer, pipetted and mixed, and lysed on ice for 15 min; 3) Centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was taken out. 50 μL was taken out from each tube and reserved as the input group. 20 μL Protein A / G agarose breads was added to the remaining part, and the mixture was mixed by inversion at 4°C for 1-2 h, and then centrifuged at 4°C at 1000 g for 5 min; 4) The supernatant was taken out, and the same amount of PAD4 or PARP-1 antibody (1 mg / mL) was added to the non-activated group and the activated group, and Normal IgG of the same species was added to the Normal IgG group. IgG, the operation of the three tubes must be consistent afterwards; 5) Slowly shake at 4℃ for 8h; 6) Add 30μL Protein A / G agarose beads, mix by inversion, and slowly shake at 4℃ overnight; 7) Centrifuge at 1000g, 4℃ for 5min, discard the supernatant, add 1mL 1×RIPA lysis buffer, mix by inversion, let stand on ice for 3min, centrifuge at 1000g, 4℃ for 5min; 8) Repeat step 7) 4-5 times; 9) Discard the supernatant, add 50μL 2×SDS-PAGE protein loading solution, heat at 100℃ for 10min, quickly insert the EP tube into the ice box for 5min, and centrifuge at 12000rpm, 4℃ for 5min; 10) Take the supernatant and perform Western Blot experiment to detect PAR, PAD4 or PARP-1.

[0158] (2) PAD4 enzyme activity detection: 1) Cell culture and tumor cell conditioned medium were used to stimulate neutrophils as before; 2) Cells were repeatedly frozen and thawed in liquid nitrogen; 3) ELISA kit was used for determination.

[0159] 3. Experimental results

[0160] Above Figure 5 It shows that lung cancer cells directly promote NETs formation and histone H3 citrullination significantly increased. Therefore, this example first verifies whether lung cancer stimulation affects the expression of PAD4 during NETs formation. The results show that after lung cancer cell stimulation, the expression of PAD4 did not change ( Figure 6 AB). Therefore, this example further verifies whether lung cancer cells affect the enzyme activity of PAD4. The results show that under the stimulation of lung cancer cell conditioned medium, the enzyme activity of PAD4 increased significantly ( Figure 6CD). Since PAD4 activity increased while protein expression did not change, the inventors speculated that lung cancer regulates PAD4 activity through post-translational modification of the protein. In previous studies by the inventors, it was confirmed that lung cancer cells promote the expression of neutrophil PARP-1. In addition to the function of repairing DNA damage, PARP-1 also plays a key role in post-translational modification (called PARylation) by transferring ADP ribose units to protein substrates, thereby changing protein function (such as enzyme activity). In order to illustrate whether the PARylation of PAD4 also changes after stimulation with lung cancer cell conditioned medium, this example conducted an immunoprecipitation experiment. In human and mouse neutrophils, the PARylation of PAD4 increased with the stimulation of lung cancer cells ( Figure 6 E). Furthermore, when PARP-1 was knocked down or blocked by PARP-1, the lung cancer cell-induced increase in PAD4 PARylation was significantly suppressed in human and mouse neutrophils ( Figure 6 F). Consistent with this, PAD4 activity was elevated in neutrophils stimulated by lung cancer cells, and PARP-1 knockdown or olaparib blocked the increased PAD4 activity in lung cancer cells ( Figure 6 GH).

[0161] Then, this example aims to explore whether PARP1 interacts with PAD4. Co-IP results showed that the interaction level of PARP-1 and PAD4 was low in control neutrophils. However, under the stimulation of lung cancer cell conditioned medium, the interaction between the two proteins increased significantly ( Figure 6 I and J). These results suggest that PARP-1 has potential physiopathological implications in regulating PAD4 activity, thereby promoting histone H3 citrullination to promote lung cancer-induced NETs production.

[0162] Example 4 Targeting PARP-1 to block lung cancer-induced NETs formation in vitro

[0163] 1. Experimental Materials

[0164] Control shRNA and PARP-1 shRNA plasmids were purchased from Santa Cruz Corporation.

[0165] 2. Experimental methods

[0166] Lentivirus transfection of HL-60 cells: Control shRNA or PARP-1 shRNA plasmid and pMD2.G, psPAX2 plasmid were co-transfected into 293T cells for virus packaging. The supernatant was collected after 48 hours, and HL-60 cells were infected after centrifugation and filtration. Puromycin was added for selection 48 hours after infection. After 3 consecutive days of selection, the PARP-1 knockout effect was detected by Western blot.

[0167] 3. Experimental results

[0168] To investigate the effect of PARP-1 on the formation of NETs in lung cancer, PARP-1 was first knocked down in HL-60 cells. Then, the cell line was differentiated into neutrophils. After treatment with A549 conditioned medium, PARP-1 knockdown significantly inhibited the release of MPO-DNA and cfDNA induced by lung cancer cells, the expression of CitH3, and the increase of DNA network-like structures, indicating that knockdown of neutrophils inhibited the formation of NETs induced by lung cancer ( Figure 7 AD).

[0169] Next, LLC cell-conditioned medium was used to stimulate mouse bone marrow primary neutrophils (BMN) and the PARP-1 specific blocking drug Olaparib was added. The results showed that Olaparib blocked the increase in NETs formation induced by mouse lung cancer cells ( Figure 7 EH). These results suggest that PARP-1 plays a key role in the formation of NETs induced by lung cancer cells. Targeting PARP-1 therapy may reduce the risk of NET-promoted thrombosis.

[0170] Example 5 Clinical use of Olaparib can reduce the risk of thrombosis in cancer patients

[0171] Olaparib, a PARP-1 targeted inhibitor, has been approved for marketing and is clinically used for the treatment of breast cancer and ovarian cancer. Therefore, in order to study whether the clinical use of Olaparib will affect the risk of thrombosis in cancer patients, this example collected coagulation indicators of cancer patients who did and did not receive Olaparib treatment (the patient's clinical data came from the pathology system database of the Affiliated Hospital of Xuzhou Medical University). The results showed that the coagulation indicators D-dimer, fibrinogen degradation products (FDP), and fibrinogen (FIB) of tumor patients treated with Olaparib were significantly lower than those of untreated patients ( Figure 8 AC), while the thrombin time (TT) was significantly increased ( Figure 8 D), indicating that cancer patients treated with Olaparib are in a relatively low coagulable state, reducing the risk of thrombosis. These results suggest that in addition to its anti-tumor effect, Olaparib may also have a role in preventing thrombosis in clinical practice.

[0172] Example 6 Olaparib blocks NETs-promoted thrombosis in lung cancer mice

[0173] 1. Experimental methods

[0174] Experiment on the effect of drug Olaparib on thrombosis in tumor-bearing mice: (1) Tumor cells and tumor-bearing mice were prepared as above. (2) After 10 days, obvious tumor formation was observed at the inoculation site of the experimental group mice. The control group and experimental group mice were randomly divided into two groups. One group was intraperitoneally injected with the PARP-1 inhibitor Olaparib at 25 mg / kg, and the other group was injected with an equal volume of PBS for 5 consecutive days. (3) After the end of the medication, a mouse inferior vena cava thrombosis model was established, and the levels of plasma MPO-DNA and cfDNA in the mice were detected. Immunofluorescence detection of thrombosis sites was performed in the same manner as above.

[0175] 2. Experimental results

[0176] To confirm whether blocking PARP-1 in vivo can inhibit cancer thrombosis promoted by NETs, ​​tumor-free mice and tumor-bearing mice were injected with the PARP-1 blocking drug Olaparib (25 mg / kg) for 5 consecutive days, and the control group mice were injected with normal saline. All mice underwent surgery for inferior vena cava stenosis, and plasma and thrombi were collected 48 hours later. The results showed that compared with the tumor-free group, the plasma MPO-DNA and cfDNA levels of lung cancer mice were significantly increased ( Fig. 9 A and B). Take a photo of the thrombus ( Fig. 9 C), the weight of thrombus in the lung cancer group mice increased significantly ( Fig. 9 D). Olaparib significantly blocked the increase in MPO-DNA and cfDNA levels induced by lung cancer, as well as the increase in thrombus weight ( Fig. 9 AD). Thrombus immunofluorescence staining showed that the formation of NETs increased in thrombi of lung cancer exacerbation, and Olaparib could significantly reverse this phenomenon ( Fig. 9 E). These results suggest that PARP-1 plays an important role in thrombosis in mice with lung cancer and that olaparib can reverse thrombosis in mice with lung cancer. Together, these results suggest that inhibition or deletion of PARP-1 may limit lung cancer-induced thrombosis by inhibiting the formation and release of NETs.

[0177] Example 7 Olaparib reduces thrombosis in lung cancer mice without bleeding risk

[0178] 1. Experimental Materials

[0179] Eptifibatide, Rivaroxban: MCE Company.

[0180] 2. Experimental methods

[0181] (1) Effects of drugs (Olaparib, Eptifibatide, and Rivaroxban) on thrombosis: 1) Tumor cells and tumor-bearing mice were prepared as above; 2) When obvious tumor formation was visible at the inoculation site of the mice, the control group and experimental group mice were randomly assigned and injected with Olaparib 25 mg / kg, Eptifibatide 5 μmol / kg, and Rivaroxban 0.3 mg / kg for 5 consecutive days. 3) The inferior vena cava of the mice was ligated, and the operation steps were as above. 4) After 48 hours, the mice were euthanized, EDTA solution was prepared in advance, the mouse eyeballs were removed, and the mouse peripheral blood was collected at 12000 rpm for 10 minutes. The mouse plasma was collected for later experiments. The mouse thrombus was collected, and the operation steps were as above.

[0182] (2) Effects of drugs (Olaparib, Eptifibatide and Rivaroxban) on coagulation function: 1) Healthy 6-week-old adult C57 / 6 male mice were prepared in advance, the weight of the mice was recorded, and the mice were randomly divided into four groups. The experimental groups were Olaparib group, Eptifibatide group and Rivaroxban group. The mice in the Olaparib group were injected with Olaparib 25 mg / kg, the mice in the Eptifibatide group were injected with Eptifibatide 5 μmol / kg, and the mice in the Rivaroxban group were injected with Rivaroxban 0.3 mg / kg for 5 consecutive days. The control group was injected with an equal volume of PBS for 5 consecutive days. 2) After 5 days, PBS was preheated in advance, and the mice were intraperitoneally anesthetized to prepare for the subsequent coagulation function test experiment. 3) Place the mouse on a horizontal plate with the abdomen facing downward, cut off 1 cm of the mouse tail, perform a tail-cutting experiment, and place the tail on the left side of the prepared 37°C PBS tube. Record the time when the mouse tail starts bleeding, stops bleeding, and bleeds again. If the difference between the time of re-bleeding and the time of stopping bleeding exceeds 60s, the bleeding time of the mouse is considered to have ended. Otherwise, continue to record the bleeding time until the difference between the bleeding time exceeds 60s. Record the difference between the time of stopping bleeding and the time of starting bleeding, which is considered to be the bleeding time of the mouse. Collect the amount of bleeding, centrifuge at 12000rpm for 10min, resuspend the cell pellet in 100μL PBS, add them to 96-well plates, and detect the wavelength value of each well at a wavelength of 490nm, which is considered to be the relative hemoglobin amount. The two indicators point to coagulation function together. 4) To monitor the effect of drugs on the coagulation function of tumor-bearing mice, a drug-treated group of tumor-bearing mice needs to be added, and the other operation steps are the same as above.

[0183] 3. Experimental results

[0184] The mouse tail bleeding time method was used to evaluate the effect of Olaparib on hemostasis. First, tumor-free mice were injected with Olaparib (25 mg / kg), the anticoagulant drug rivaroxaban (0.3 mg / kg), and the antiplatelet drug eptifibatide (5 μmol / kg) for 5 consecutive days. The results showed that the classic antithrombotic drugs rivaroxaban and eptifibatide significantly prolonged the tail bleeding time and increased blood loss, while Olaparib had no such adverse reactions ( Fig.10 AB), indicating that Olaparib does not affect normal hemostasis. In addition, this example uses the same tail bleeding time test to explore whether the antithrombotic effect of these drugs is accompanied by bleeding risk in lung cancer mice. The results showed that Olaparib blocked the increase in plasma NETs levels in lung cancer mice ( Fig.10 CD). Although Olaparib, eptifibatide, and rivaroban can effectively reduce the aggravation of thrombosis caused by lung cancer ( Fig.10 E and F), but olaparib did not increase bleeding time or amount compared with the other two drugs ( Fig.10 G and H). Immunofluorescence staining of thrombi in each group showed that the CitH3 level in the olaparib group was reduced ( Fig.10 I). The results showed that the PARP-1 blocking drug Olaparib can effectively inhibit lung cancer thrombosis without causing bleeding adverse reactions.

Claims

1. A method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4, characterized in that: The screening method comprises: Obtain PARP-1 protein and PAD4 protein data; Selecting the spatial structure of the PARP-1 protein and PAD4 protein complex, and determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex as the binding site of the targeted drug; A computer-assisted drug screening method is used to obtain candidate drugs targeting the binding site.

2. The method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 according to claim 1, characterized in that: The computer-assisted drug screening method comprises: Obtaining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex; Based on the spatial structure of the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex, screening small molecule compounds with similar structures in a molecular database; Molecular docking is performed on the screened small molecule compounds with PARP-1 protein and PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptors PARP-1 protein and PAD4 protein to obtain the score of each small molecule compound, and the candidate drugs for anti-lung cancer thrombosis are obtained by sorting according to the scores.

3. The method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 according to claim 1, characterized in that: The method for determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex comprises: Constructing n truncated expression mutants of PARP-1, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PARP-1 and PAD4; The spatial structure of the key region for the interaction between PARP-1 and PAD4 is the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex.

4. The method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 according to claim 1, characterized in that: The screening method further comprises: Obtain lung cancer cell line models and / or lung cancer animal models; The screened anti-lung cancer thrombosis candidate drug is used to treat the lung cancer cell line model and / or lung cancer animal model to verify the effectiveness of the anti-lung cancer thrombosis candidate drug.

5. A screening system for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4, characterized in that: The screening system comprises: Protein data acquisition unit, to acquire PARP-1 protein and PAD4 protein data; A binding site determination unit, which selects the spatial structure of the PARP-1 protein and PAD4 protein complex, and determines the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and the PAD4 protein complex as the binding site of the targeted drug; A candidate drug screening unit, which uses a computer-assisted drug screening method to obtain a candidate drug targeting the binding site; The computer-assisted drug screening method comprises: Obtaining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex; Based on the spatial structure of the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex, screening small molecule compounds with similar structures in a molecular database; Performing molecular docking on the screened small molecule compounds with PARP-1 protein and PAD4 protein to calculate the affinity or binding energy of the small molecule compounds targeting the receptors PARP-1 protein and PAD4 protein to obtain the score of each small molecule compound, and ranking them according to the score to obtain candidate drugs for anti-lung cancer thrombosis; Optionally, the method for determining the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex comprises: Constructing n truncated expression mutants of PARP-1, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, an immunoprecipitation experiment was performed to obtain the key region for the interaction between PARP-1 and PAD4; The spatial structure of the key region for the interaction between PARP-1 and PAD4 is the binding site of the PARP-1 protein and the PAD4 protein in the PARP-1 protein and PAD4 protein complex; Optionally, the screening method further comprises: a drug effectiveness verification unit, obtaining a lung cancer cell line model and / or a lung cancer animal model; treating the lung cancer cell line model and / or the lung cancer animal model with the screened anti-lung cancer thrombosis candidate drug to verify the effectiveness of the anti-lung cancer thrombosis candidate drug.

6. A computer device for screening anti-thrombotic drugs for lung cancer based on PARP-1 and PAD4, characterized in that: The computer device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 described in any one of claims 1 to 4 is implemented.

7. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the screening method for anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 according to any one of claims 1 to 4 is implemented.

8. A method for regulating PAD4 activity, characterized in that: The method comprises: Obtaining an in vitro cell system, in vitro tissue system or in vitro organ system containing PAD4; The in vitro cell system, in vitro tissue system or in vitro organ system containing PAD4 is treated with PARP-1, a PARP-1 promoter or a PARP-1 inhibitor to regulate the activity of PAD4.

9. Any of the following products: (1) An anti-lung cancer thrombosis drug screened by the screening method for screening anti-lung cancer thrombosis drugs based on PARP-1 and PAD4 according to any one of claims 1 to 4; (2) A pharmaceutical composition for reducing tumor thrombosis without increasing the risk of bleeding, the pharmaceutical composition comprising Olaparib; optionally, the tumor is lung cancer.

10. Any of the following applications: (1) Use of Olaparib, an anti-tumor drug targeting PARP-1, in the preparation of a drug for reducing tumor thrombosis without increasing the risk of bleeding; optionally, the tumor is lung cancer; (2) Application of PARP-1, PARP-1 promoters or PARP-1 inhibitors in regulating the activity of PAD4 in vitro.

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