CN1237184C - Target for medicine against SARS-Cov and medicine screening method and medicine against SARS - Google Patents

Abstract

The invention provides a drug action target for screening drugs for treating and/or preventing SARS virus infection, the target is a fragment of SARS virus N protein Val235-Pro369 comprising the interaction active site of SARS virus N protein and human CypA and a human body Three-dimensional structural model of the CypA complex. The present invention also provides a method for constructing the drug action target, a method for using the drug action target to screen a drug for treating and/or preventing SARS virus infection, and confirming that cyclosporine derivatives are effective in treating and/or preventing SARS virus infection through screening. new uses on .

Description

Anti-SARS-CoV drug target, drug screening method and anti-SARS drug

technical field

The present invention relates to screening drugs with bioinformatics and molecular biology methods, in particular to the use of SARS coronavirus nucleocapsid protein (N protein) and human cyclophilin A (CypA) interaction model as a drug action target at the molecular and cellular levels Methods of screening drugs.

Background technique

Infectious atypical pneumonia is called Severe Acute Respiratory Syndrome (Severe Acute RespiratorySydrome, SARS). Studies have found that a new type of coronavirus is likely to be the cause. The virus has been isolated from patients and deceased patients in Hong Kong and Canada. Anti-hMPV antibodies have also been found in the serum of some SARS patients. On April 16, 2003, the World Health Organization (WHO) officially confirmed that the SARS virus was the cause of infectious atypical pneumonia. More than 90% of SARS patients infected with SARS virus can be cured, and the fatality rate is about 10%. Electron micrographs of respiratory tract sections and cell cultures from SARS patients showed the presence of coronavirus particles. This is a new type of coronavirus, which is different from other known members of the coronavirus genus. The virus can cause pathological effects in green monkey kidney cells (VERO-E6), and the virus replication can be inhibited by the serum of recovered people infected with SARS. Immunofluorescence assays (IFA) can be used to detect the presence of SARS virus in cultured cells using infected cells and serum from recovered patients. This method shows a specific response. Studies in the United States, Canada and Hong Kong have shown that the serum of non-SARS patients cannot react with the new coronavirus; (Feline Infectious Peritonitis virus, FIPV), 229E human coronavirus super-antiserum can inhibit the growth of cultured virus. Sequencing of the virus by several laboratories showed that the new virus is related to the genus of coronavirus, but it is different from other coronavirus groups of the same genus, and is a new variant of coronavirus. SARS coronavirus belongs to the coronavirus (Coronaviridae) line in the nest virus (Nidovirales) family of the positive strand RNA virus (ssRNA positivestrand viruses) family in terms of species classification. It is an emerging subclass of the coronavirus family. In March 2003, scientists discovered the SARS coronavirus (SARS Coronaviruses, SARS-CoV), the culprit that caused SARS, and successfully completed the complete sequencing of the SARS-CoV genome. The SARS-CoV genome consists of 29727 nucleotides and 11 open reading frames (Opening Reading Frames). Its genome structure is very similar to other coronaviruses. However, the comparison of genetic history and sequence alignment shows that the characteristics of SARS-CoV are not completely similar to those of previously discovered coronaviruses. It not only exhibits the characteristics shared by other coronaviruses, but also has the characteristics unique to SARS-CoV itself (Paul A.Rota, M.Steven Oberste, Stephan S.Monroe, W.Allan Nix, Ray Campagnoli, et al.Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science (Scienceexpress) 1 May, 2003; Marco A.Marra, Steven J.M.Jones, Caroline R.Astell, Robert A.Holt, Angela Brooks-Wilson, et al.The genome sequence of the SARS-associated coronavirus. Science (Scienceexpress)lMay, 2003). The scientists simultaneously performed genome sequencing of 29,751 bases of the Toronto, Canada (Tor2) isolate of a SARS-related coronavirus. The genome sequence revealed that this coronavirus is not closely related to known coronaviruses (including human coronavirus HcoVOC43, HCoV-229E). Phylogenetic analysis of predicted viral proteins showed that the coronavirus was not closely similar to any of the three known coronaviruses. The genome sequence will contribute to the understanding of the mechanism of SARS virus infection, the diagnostic detection of potential animal hosts (using PCR and immunoassays), as well as the development of antiviral agents (including neutralizing antibodies) and the identification of vaccine epitopes (Marco A. Marra, Steven J.M. Jones, Caroline R. Astell, Robert A. Holt, Angela Brooks-Wilson, et al. The genome sequence of the SARS-associated coronavirus. Science (Scienceexpress) 1 May, 2003).

But so far, people have not fully studied the infection mechanism of SARS-CoV virus. In order to reduce the infection rate and mortality, there is a very urgent clinical demand for specific drugs that can prevent and/or treat SARS-CoV infection.

Therefore, the first object of the present invention is to provide a drug target for rapid screening of drugs for the treatment and/or prevention of SARS virus infection.

The second object of the present invention is to provide a method for constructing the drug target.

The third object of the present invention is to provide a method for screening drugs for the treatment and/or prevention of SARS virus infection with this drug action target.

The fourth object of the present invention is to provide a drug for treating and/or preventing SARS virus infection screened out with the target of the drug.

Summary of the invention

Drug targets refer to the key functional biomacromolecules that can play a decisive role in the most critical pathological links in the process of disease occurrence and development, including cell membrane receptors, proteases, hormones and cytokines, ion channels, DNA, RNA, nuclear receptors wait. The core of modern drug research is based on the drug target, screening or designing drug leads that are complementary to the target structure, can stimulate or antagonize the biological function mediated by the target, and have therapeutic effects. This new drug discovery based on mechanism and structure selectively blocks the most critical pathological links in the process of disease occurrence and development, and develops specific drugs with high efficiency and low side effects.

The present inventor has conducted in-depth bioinformatics analysis on the gene expression products of SARS-CoV, thereby identifying the pathway of SARS virus infecting the human body; then using molecular biology and biophysical methods to verify this pathway of action; according to this pathway of action The compound is screened, and the virus level test is carried out to confirm the drug target of infectious atypical pneumonia (SARS), and the drug action target is used to screen the drug for preventing and/or treating infectious atypical pneumonia, thereby completing the present invention.

The drug action target that is used for screening treatment and/or prevention SARS virus infection drug provided by the present invention is nucleocapsid protein (SARS Nucleocapsid) that plays a key role in SARS coronavirus replication, transcription, translation and assembly process, host (Human body) action protein Cyelophilin A (Cyelophilin A) and the interaction pathway between the two, the target is the SARS virus N protein Val235-Pro369 fragment comprising the interaction active site of the SARS virus N protein and human CypA和人体CypA复合物三维结构模型，其中靶标SARS病毒N蛋白的序列特征为：MSDNGPQSNQ RSAPRITFGG PTDSTDNNQN GGRNGARPKQ RRPQGLPNNT ASWFTALTQH GKEELRFPRGQGVPINTNSG PDDQIGYYRR ATRRVRGGDG KMKELSPRWY FYYLGTGPEA SLPYGANKEG IVWVATEGALNTPKDHIGTR NPNNNAATVL QLPQGTTLPK GFYAEGSRGG SQASSRSSSR SRGNSRNSTP GSSRGNSPARMASGGGETAL ALLLLDRLNQ LESKVSGKGQ QQQGQTVTKK SAAEASKKPR QKRTATKQYN VTQAFGRRGPEQTQGNFGDQ DLIRQGTDYK HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYHGAIKL DDKDPQFKDNVILLNKHIDA YKTFPPTEPK KDKKKKTDEA QPLPQRQKKQ PTVTLLPAAD MDDFSRQLQNSMSGASADST QA

The sequence characteristics of the target human CypA are: MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKGSCFHRIIPGF MCQGGDFTRH NGTGGKSIYG EKFEDENFIL KHTGPGILSMANAGNTNGS QFFICTAKTE WLDGKHVQFG KVKEGMNIVE GSERFKITGLENGKTS

Among the drug action targets provided by the present invention, the sequence number of the functional region of the N protein of the target SARS virus is: 235 VSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFG289 DQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFK349 DNKVILLNTE

Among the drug action targets, the sequence number of the target human CypA protein functional region is:

Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125, His126

Among the drug targets, the interaction active site between SARS virus N protein and human CypA includes:

N protein: a loop composed of residues from Trp302 to Phe315, in which the main amino acid residues are Trp302, Pro303, Gln304, Ile305, Ala306, Aln307, Phe308, Ala309, Pro310, Ser311, Ala312, Ser313, Ala314 and Phe315;

Human CypA: Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125 and His126.

The method for constructing the drug action target provided by the present invention comprises the following steps:

1) Use bioinformatics methods to conduct network analysis on the interaction between proteins in SARS-CoV and human proteins, and reveal the interaction between SARS-CoV N protein and human CypA;

2) Use the homologous protein modeling method to construct the three-dimensional structure of the SARS-CoV virus N protein, use the molecular simulation method to calculate the interaction between the SARS-CoV virus N protein and human CypA, construct the structure of the N protein and human CypA complex, and establish The region where these two proteins interact;

3) Express N protein and human CypA by molecular biology methods, and use surface plasmon laser resonance biosensing technology to study the interaction between N protein and human CypA, revealing that there is a strong interaction between the two proteins;

4) For the region where N protein binds to human CypA, use computer virtual screening method to search the compound database, find compounds that prevent N protein from binding to human CypA, and confirm the establishment of drug targets.

The method provided by the invention for screening treatment and/or preventing SARS virus infection drug with drug action target comprises the following steps:

1) Determine the binding constant between the candidate and CypA, and select the compound with stronger affinity;

2) The selected compound is tested to inhibit the combination of SARS-CoV virus N protein and human CypA;

3) Test the protective effect of the selected compounds on cells infected with SARS-CoV virus.

Description of drawings

Figure 1 shows the sequence alignment of the Val235-Pro369 fragment of the SARS-CoV virus N protein with the HIV-1 virus CA protein Pro401-Tyr545. In the figure, "*" represents identical or conservative residues, "." represents substitution of conservative residues, and ":" represents semi-conservative substitution, with a similarity of 47.8%.

Fig. 2 shows the three-dimensional structure model of the complex of SARS-CoV virus N protein Val235-Pro369 fragment (ribbon structure) and CypA (spherical surface structure).

Figure 3 shows the hydrogen bonding and hydrophobic interactions between the SARS-CoV viral N protein and CypA. The meanings of the symbols are as follows:

配体键                疏水接触中涉及的非配体残基

Ligand bond Non-ligand residues involved in hydrophobic contacts

非配体键              疏水接触中涉及的相应原子

non-ligand bond Corresponding atoms involved in hydrophobic contacts

氢键及其长度

Hydrogen bonds and their lengths

Figure 4 shows the kinetic test results of the interaction between SARS-CoV virus N protein and human CypA. The instrument used is BIACORE3000, and the analysis software is Kinetic Analysis in Application Wizard. CypA was immobilized on the CM5 chip during the measurement, and the concentration of N protein was as follows: 0.214nM, 1.07nM, 5.35nM, 10.7nM, 21.4nM, 42.8nM, 85.63nM, 0.171uM, 0.343uM, 0.685uM and 1.37uM.

Figure 5 shows cyclosporin A inhibits the binding kinetics test results of SARS-CoV virus N protein and human CypA. CypA was immobilized on the CM5 chip, and the concentration of N protein was 1.37 μM, and the concentration of cyclosporine A (CsA) was 832, 1664, 2496 and 3328 μM in sequence.

Figure 6 shows the protective effect of cyclosporin A on Vreo-E6 cells infected with SARS-CoV virus.

Detailed description of the invention

The inventors used bioinformatics methods to analyze the interaction network between the proteins in SARS-CoV and human proteins, and found that the nucleocapsid protein (SARS Nucleocapsid, hereinafter referred to as N protein) of SARS-CoV and the human parent in the human body Cyclophilin A (CyclophilinA, hereinafter referred to as CypA) has interaction; the present invention constructs the three-dimensional structure of SARS-CoV virus N protein with homologous protein modeling method, calculates SARS-CoV virus N protein and human CypA with molecular simulation method The interaction between the N protein and human CypA was constructed, and the interaction region of the two proteins was established; the N protein and human CypA were expressed by molecular biology methods, and the surface plasmon laser resonance biosensing (SPR ) technology to study the interaction between N protein and human CypA, and found that there is a strong interaction between the two proteins; for the region where N protein and human CypA interact, searched the compound database with computer virtual screening method, and found that Inhibitor cyclosporine A can prevent the combination of N protein and human CypA; the activity of cyclosporine A to prevent the combination of N protein and human CypA was determined by SPR technology; cyclosporine A was screened at the virus level to inhibit virus infection Vero- The activity of E6 cells has confirmed the pathway and target of the SARS-CoV virus infecting the human body, and established corresponding screening methods at the molecular and cellular levels.

1. Bioinformatics analysis and simulation of the interaction between SARS-CoV virus N protein and human CypA: In 1994, Luban et al. found that CypA can integrate with HIV-1 virus nucleus, and in the Gap polyprotein dimerized with HIV-1 virus Capsid domain role (Luban, J.; Bossolt, K.L.; Franke, E.K.; Kalpana, G.V.; Goff, S.P. Human immunodeficiency virus type 1 Gag protein binds to Cyclophilins A and B. Cell 1993, 73, 1067-1078; Luban, J.Absconding with the chaperone: essential Cyclophilin-Gag interaction in HIV-1 virions. Cell 1996, 87, 1157-1159; Franke, E.K.; Yuan, H.E.H.; Luban, J.Specific incorporation of Cyclophilin A into HIV-1 virions. Nature 199 , 372, 359-362; Thali, M.; Bukovsky, A.; Kondo, E.; Rosenwirth, B.; Walsh, C.T.; Sodroski, J.; Gottlinger, H.G. Functional association of Cyclophilin A with HIV-1 virions. Nature 1994, 372, 363-365), and then in the process of virus maturation, the polyprotein is decomposed into matrix protein, capsid protein (CA protein) and nucleocapsid protein (N protein), but these proteins are still combined together, CypA binds to capsid protein at a ratio of approximately 1:10. Thus, direct binding of CypA to the capsid protein plays an important role in HIV-1 virus maturation, viral replication, and formation of new virions (Gitti, R.K.; Lee, B.M.; Walker, J.; Summers, M.F.; Yoo , S.; Sundquist, W.I.Structure of theamino-terminal core domain of the HIV-1 capsid protein. Science 1996, 273, 231-235; Gamble, T.R.; Vajdos, F.E.; Yoo, S.; Worthylake, D.K.; M.; Sundquist, W.I.; Hill, C.P. Crystal structure of human Cyclophilin A bound to the N-terminal domain of HIV-1 capsid. Cell 1996, 87, 1285-1294; Colgan, J.; Yuan, H.E.H.; Franke, E.K.; Luban, J.Binding of the human immunodeficiency virus type1 Gag polyprotein to Cyclophilin A is mediated by the central region of capsid and requires Gag dimerization.J.Virol.1996,70,4299-4310; Ott,D.E.CellularsHIVRevinins.rin Med. Virol. 1997, 7, 167-180). Protein-protein interaction analysis showed that the N protein of SARS-CoV virus interacted with human CypA. The CA protein was not found in the SARS-CoV virus. It is assumed that the same is the capsid protein. One of the functions of the N protein of the SARS-CoV virus is similar to the CA protein in the HIV-1 virus. It binds to CypA and regulates the maturation, Replication of the virus and formation of new virus particles play an important role. Therefore, the present invention carried out further sequence comparison and structure modeling.

Sequence analysis shows that the N protein of SARS-CoV virus has a high homology with the CA protein of HIV-1 virus, but the overall sequence comparison shows that the similarity between the two is still very low, with a sequence similarity of 37.6% and a homology of 26.9% , The sequence gap is less than 4.8%. However, further analysis found that the Val235-Pro369 fragment of the SARS-CoV virus N protein has a high similarity with the binding region Trp302-Phe315 of the HIV-1 virus CA protein and CypA, with a sequence similarity of 42.2% and a homology of 24.4%, sequence gap less than 4.1%. This sequence analysis indicated that the SARS-CoV viral N protein may bind to CypA through the Val235-Pro369 region (see Figure 1).

According to the crystal structure of the complex of HIV-1 virus CA protein Pro401-Tyr545 fragment and CypA (the protein three-dimensional structure database PDB number is 1AK4), the present invention models the three-dimensional structure of the SARS-CoV virus N protein Val235-Pro369 fragment and CypA complex model (see Figure 2). The modeled structural model shows that there is a loop on the N protein of the SARS-CoV virus that interacts with the active pocket of CypA in the human body.

2. Active site of interaction between SARS-CoV virus N protein and human CypA: From the three-dimensional structural model of the complex of SARS-CoV virus N protein Val235-Pro369 fragment and CypA (attached figure 2), the interaction between SARS-CoV virus N protein and human CypA can be obtained. Some information on the interaction active site of human CypA. The binding region between SARS-CoV virus N protein and human CypA is a loop composed of residues from Trp302 to Phe315, and the main residues that make up the CypA binding pocket are: Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125, His126. The hydrogen bonds and hydrophobic interactions formed between the two proteins are listed in Figure 3 and Tables 1 and 2, respectively.

Table 1. Hydrogen bond interaction between SARS-CoV virus N protein and CypA CypA residues Action atom SARS-CoV virus N protein Action atom Distance () Trp121Arg55Arg55Gln63Gly72 N ¹ N ² N ¹ N ² O Phe308Gln307Gln307Ile305Ile305 OOOON 3.312.733.012.762.78

Table 2. Hydrophobic interaction between SARS-CoV virus N protein and CypA CypA residues Action atom SARS virus N protein residues Action atom Distance () Trp121Trp121Phe60Phe60Phe60Phe60Phe60Phe60Phe113Phe113Phe113Phe113 C ² C ² C ² C ² CC ² C ² CC ¹ C ² C ¹ C Pro310Pro310Ala309Ala309Ala309Ala309Ala309Phe308Gln307Gln307Gln307Gln307 CCCCCCCCCCCC 3.593.853.583.563.703.423.843.703.463.823.173.30 Phe113Phe113Phe113Phe113Phe113Phe60His126His126Leu122Leu122Phe60His126Arg55Asn102Asn102Ala101Ala103Gly72Asn71Thr73Thr73Asn71Arg69Arg69Thr73Thr73Arg69Thr73 CCC ² C ¹ C ¹ CC ¹ C ² C ² C ¹ CC ¹ CCCCCCCC ² CCCCC ² CCC ² Gln307Gln307Gln307Gln307Gln307Gln307Gln307Gln307Gln307Gln307Gln307Gln307Ala306Ile305Ile305Ile305Ile305Gln304Trp302Trp302Trp302Trp302Trp302Trp302Trp302Trp302Trp302Trp302 CCCCCCCCCCCCCC ² C ² C ² C ¹ CC ² C ³ C ³ C ³ C ³ C ³ C ³ C ³ C ³ C ² 3.753.653.773.713.893.743.493.903.793.85.883.753.473.003.333.343.583.883.763.373.373.553.593.762.962.983.153.49

3. Expression and interaction determination of SARS-CoV virus N protein and human CypA: the constructed pQE30/SARS_N plasmid was transformed into Escherichia coli M15, induced with IPTG (concentration 0.8mM), treated with ampicillin and kanamycin It is a double antibiotic, and the temperature is 30°C, and the expression time is 10 hours, and the SARS-CoV virus N protein is expressed. The N protein of SARS-CoV virus was initially purified by NTA-His column chromatography, and the N protein of SARS-CoV virus was further purified by FPLC-gel filtration. Escherichia coli BL21 (DE3) transformed with pETll/CyPA plasmid was induced to express human CypA protein (expression temperature 25° C.) by IPTG (0.5 mM), and protein crude product was obtained by 50% saturated ammonium sulfate precipitation protein, which was further purified by Mono-Q and molecular sieve. Purify human CypA protein to obtain pure product.

Using surface plasmon resonance (SPR) technology, the dissociation constant (KD) of the interaction between SARS-CoV virus N protein and human CypA is 7.65×10 ^-8 M, indicating that the SARS-CoV virus N protein interacts strongly with human CypA combination. The measurement results are shown in Figure 4.

4. Virtual screening for the interaction between N protein and human CypA: After determining the interaction mode and binding area between SARS-CoV virus N protein and human CypA, the present invention takes the surface binding pocket of CypA and SARS-CoV virus N protein as the target , using the molecular docking (Molecular Docking) virtual screening (Virtual Screening) method, searched the drug database CMC of MDL company, the existing compound database ACD, the drug report database MDDR and the compound sample library of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and obtained a batch of Selected compounds with strong affinity to CypA, including the ligand ring of CypA [[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-methylamino-6-octaenoyl ]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanine Acyl-D-alanyl-N-methyl-L-leucyl-N-methylL-leucyl-N-methyl-L-valyl] (Cyclosporin A, Cyclosporin A). Molecular modeling showed that cyclosporin A prevented the binding of the N protein of the SARS-CoV virus to human CypA.

5. Cyclosporin A prevents N protein from binding to human CypA: use surface plasmon resonance biosensing technology BIACORE3000 to study cyclosporine A to prevent SARS-CoV virus N protein from binding to human CypA, the experimental results are shown in Figure 5, the results It shows that as the concentration of cyclosporine A increases, the combination of SARS-CoV virus N protein and human CyPA decreases, indicating that cyclosporine A can inhibit the combination of SARS-CoV virus N protein and human CypA.

6. Cyclosporine A anti-SARS-CoV virus activity test: In order to confirm the correlation between the above-mentioned SARS-CoV virus N protein and human CypA action pathway and SARS-CoV virus infection of human body, the present invention has selected a drug that can prevent SARS-CoV at the molecular level. Cyclosporin A bound to human CypA by the CoV viral N protein was tested for viral levels.

Test principle: Vero-E6 cells are used as virus host cells (susceptible cells), and the protective effect of samples on virus-infected cells is tested. The detection index is cell degeneration (CPE) and the protection rate of infected cells is observed.

Test method: Inoculate Vero-E6 cells in a 96-well culture plate, culture in a 5% CO2 incubator at 37°C, add different dilutions of SARS virus and 2.5mg/ml and 1250ug/ml of cyclosporine A, and observe CPE, and measure the OD value with neutral red staining, and calculate the anti-SARS virus activity effect of the sample.

Test results: In the anti-SARS virus activity experiment of the virus-cell level model, the SARS virus with different effective concentrations was used to infect Vero-E6 cells, when 2.5mg/ml and 1250ug/ml concentrations of cyclosporine A were added, the Inhibit the protective activity of SARS virus infecting Vero-E6 cells (the results are shown in Figure 6). It shows that the interaction pathway between SARS-CoV virus N protein and human CypA has a clear correlation with SARS-CoV virus infection of human body.

As mentioned above, one aspect of the present invention finds that SARS-CoV virus N protein interacts with human CypA through biological information analysis. Sequence analysis shows that the N protein of SARS-CoV virus has a high homology with the CA protein of HIV-1 virus, but the overall sequence comparison shows that the similarity between the two is still very low, with a sequence similarity of 37.6% and a homology of 26.9% , The sequence gap is less than 4.8%. However, further analysis found that the Val235-Pro369 fragment of the SARS-CoV virus N protein has a high similarity with the binding region Trp302-Phe315 of the HIV-1 virus CA protein and CypA, with a sequence similarity of 42.2% and a homology of 24.4%, sequence gap less than 4.1%. It is suggested that the SARS-CoV virus N protein has similar functions to the HIV-1 virus CA protein, that is, after the SARS-CoV virus infects the human body, the direct combination of the SARS-CoV virus N protein and the human CypA plays an important role in the maturation and replication of the SARS-CoV virus. and play an important role in the formation of new virus particles.

According to the crystal structure of the complex of human CypA and HIV-1 virus CA protein Trp302-Phe315 fragment, a three-dimensional structure model of the complex of human CypA and SARS-CoV virus N protein Val235-Pro369 fragment was modeled. The results show that the binding region of the SARS-CoV viral N protein to human CypA is a loop consisting of residues from Trp302 to Phe315, and its main hydrogen bond interactions and hydrophobic interactions are shown in Figure 3, namely Attached Table 1 and Attached Table 2 , The binding pocket of CypA to SARS-CoV virus N protein is composed of Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125 and His126 residues.

On the basis of determining the interaction mode and structural model of the SARS-CoV virus N protein and human CypA, the present invention uses plasmonic surface laser resonance biosensing (SPR) to study the dynamics of the interaction between the SARS-CoV virus N protein and human CypA The chemical behavior shows that the two have a strong combination (see Figure 4), and the binding constant (KD) is 7.65×10 ^-8 M.

From the above results, it can be preliminarily determined that the interaction between SARS-CoV virus N protein and human CypA is related to SARS-CoV infection of human body, and the SARS-CoV virus N protein, human CypA protein and the interaction between them can be used as screening anti-SARS-CoV approach to viral medicine. According to the principles of general drug target validation, active compounds must be screened out based on hypothetical targets. Therefore, the present invention aims at the binding pocket of the interaction between human CypA and SARS-CoV virus N protein, screens the drug database CMC of MDL company, the existing compound database ACD and our own compound sample library by virtual screening method, and obtains a A batch of selected compounds with strong affinity to CypA, including cyclosporine A, a ligand of CypA. The present invention then adopts plasmonic surface laser resonance biosensing (SPR) to study cyclosporin A to inhibit the kinetic behavior of SARS-CoV virus N protein and human body CypA (results are shown in Figure 5), the results show that cyclosporine A can Effectively inhibit the combination of SARS-CoV virus N protein and human CypA, with the increase of cyclosporine A concentration, the intensity of the inhibitory protein binding also increases, showing a good dose relationship.

In order to further confirm the effectiveness of the anti-SARS-CoV virus drug target proposed by the present invention, the present invention has carried out the test that cyclosporin A protects Vero-E6 cells from SARS-CoV virus infection. Using Vero-E6 cells as virus host cells, the protective effect of cyclosporine A on virus-infected cells was tested, and the detection index was cell degeneration (CPE) and the protection rate of infected cells was observed. The result shows that the cyclosporin A of 2.5mg/m and the concentration of 1250ug/ml have obvious protective activity of inhibiting SARS virus infection Vero-E6 cell (results are shown in accompanying drawing 6). It shows that the interaction pathway between SARS-CoV virus N protein and human CypA has a clear correlation with SARS-CoV virus infection of human body.

According to the above bioinformatics analysis, molecular simulation, SPR protein-protein interaction research, virtual screening, compound molecular level and virus cell level screening, the present invention determines that SARS-CoV virus N protein, human CypA and its interaction pathway are therapeutic And/or prevent the drug action target of SARS-CoV virus infection.

Another aspect of the present invention relates to the establishment of the complex of SARS-CoV virus N protein functional fragment Val235-Pro369 and human CypA according to the determined drug target SARS-CoV virus N protein, human CypA and its interaction pathway. A three-dimensional structural model to determine the key parts and amino acid residues of the interaction between SARS-CoV virus N protein and human CypA. On this basis, a candidate that can block the combination of SARS-CoV virus N protein and human CypA was obtained by virtual screening method, and the target organism was screened out through the activity test at the molecular level of SPR and the activity test of inhibiting Vero-E6 cell infection. Functional inhibitors.

The target biological function inhibitor is an organic molecular compound or a polypeptide compound or an oligosaccharide that binds to the target SARS virus N protein functional region or the target human CypA protein functional region, and blocks the combination of the SARS virus N protein and the human CypA functional region compounds or monoclonal antibodies or nucleoside compounds.

Another aspect of the present invention finds that the cyclosporine A derivative with general formula I can specifically combine with CypA, thereby effectively inhibiting the combination of SARS-CoV virus N protein and human CypA, and has a more obvious inhibition of SARS virus The protective activity of infecting Vero-E6 cells has purposes for treating and/or preventing SARS-CoV virus infection.

Clinically, cyclosporine A is mainly used to inhibit the rejection of organ and tissue transplantation, and also for the treatment of autoimmune diseases, such as type I diabetes, psoriasis, rheumatoid arthritis, systemic erythema Lupus, etc. have a certain curative effect. U.S. Patents US6270957 and US5840305 disclose that compounds containing general formula I can act on T lymphocytes, inhibit HIV protease, inhibit cell uptake of virus, interfere with the interaction between HIV-1 virus Gag protein and CypA, and inhibit the integration of CypA into HIV-1 virus particles, Prevent virus particle maturation and replication; US patents US6521595, US4885276 and US19891205 disclose new preparation methods containing compounds of general formula I.

The definition of each substituent in the cyclosporine A derivative of general formula I or its pharmaceutically acceptable salt or hydrate is as follows:

In the formula

R ₁ is H, C ₁ -C ₄ alkyl, C ₁ -C ₄ substituted alkyl, hydroxyl;

R ₂ is H, acetyl, C ₁ -C ₄ substituted alkyl acyl, C ₁ -C ₄ substituted alkyl;

R ₃ is H, hydroxyl, hydroxyalkoxy, hydroxyalkylthio, C ₁ -C ₄ alkylsulfide, alkoxycarbonylalkylsulfide, arylalkylsulfide, arylthio, Alkylsulfonyl, hydroxyalkylsulfonyl, polyalkoxy substituted methylsulfide groups;

R ₄ is H, C ₁ -C ₄ alkyl, C ₁ -C ₄ substituted alkyl;

R ₅ is H, methyl sulfide group, polyalkyl substituted thiomethyl;

R ₆ is H, C ₁ -C ₄ alkyl, C ₁ -C ₄ substituted alkyl;

R ₇ is H, hydroxyl, hydroxyalkyl, hydroxyalkoxy, C ₁ -C ₄ alkylsulfide group;

R ₈ is H, hydroxyl, hydroxyalkyl, hydroxyalkoxy, C ₁ -C ₄ alkylsulfide group;

R ₉ is H, C ₁ -C ₄ alkyl, C ₁ -C ₄ substituted alkyl.

The "pharmaceutically acceptable salt" described in this specification specifically includes propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, etc. Salts formed with organic acids and acidic amino acids such as aspartic acid and glutamic acid and then formed with inorganic bases, such as sodium, potassium, calcium, aluminum salts and ammonium salts, or salts formed with organic bases, such as methylamine salts , ethylamine salt, ethanolamine salt, etc., or salts of inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc. after forming esters with basic amino acids such as lysine, arginine, ornithine , or salts with formic acid, acetic acid, picric acid, methanesulfonic acid, ethanesulfonic acid and other organic acids.

Experiments of the present invention have found that the compound of general formula I or its pharmaceutically acceptable salt or hydrate can effectively inhibit the combination of SARS-CoV virus N protein and human CypA, and has a more obvious inhibition of SARS-CoV virus infection Vero- The protective activity of E6 cells, therefore, has the purposes of treating and/or preventing SARS-CoV virus infection.

The most preferred compound in the compound of general formula I screened by the method of the present invention or its pharmaceutically acceptable salt or hydrate is: cyclosporin A (Cyclosporin A), ring [[(E)-(2S, 3R, 4R )-3-Hydroxy-4-methyl-2-methylamino-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl- L-Valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl -N-methyl-L-valyl].

Another aspect of the present invention also relates to a pharmaceutical composition that can be used to prevent and/or treat infectious atypical pneumonia caused by coronavirus infection, which includes at least one compound of general formula I or its compound in a preventive and/or therapeutic effective amount. A pharmaceutically acceptable salt or a hydrate thereof and at least one pharmaceutically acceptable carrier or excipient.

The pharmaceutical carriers described here include but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphate, glycerol, sorbic acid, potassium sorbate, Partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon oxide, magnesium trisilicate, polyvinylpyrrolidone , cellulosic substances, macrogol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin.

The present invention also relates to a new application of the pharmaceutical composition in preparing medicines for preventing and/or treating infectious atypical pneumonia (Severe Acute Respiratory Syndrome, SARS).

The pharmaceutical composition can be prepared into various dosage forms according to different administration routes. These dosage forms are administered in one of the following ways: oral, inhalation spray, rectal, nasal, buccal, topical, parenteral, e.g. subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, sternal Intracranial and intracranial injections or transfusions, or with the aid of an explanted reservoir. Oral or intramuscular injection is preferred when preventing SARS virus infection, and intraperitoneal or intravenous administration is preferred when treating SARS virus infection.

In addition, it should be pointed out that the dose and method of use of the compounds of the present invention depend on many factors, including the patient's age, body weight, sex, natural health status, nutritional status, activity intensity of the compound, time of administration, metabolic rate, severity of the disease, and The subjective judgment of the treating physician. If the recommended dose is 5mg-10mg/kg per day at the beginning, the maintenance dose can be reduced to 3mg/kg per day. Capsules: 0.25g/capsule. Injection 0.25g/5ml. Oral solution: 5g/50ml.

specific implementation plan

The following examples are specific preferred embodiments in order to further illustrate the present invention, but these examples are by no means any limitation to the present invention.

Example 1: Multiple alignment results of SARS-CoV virus N protein sequence

The compared sequences are from NCBI, the National Center for Bioinformatics of the United States. They come from SARS case specimens in different regions, including NCBI accession numbers gi|30275668 (BJ01), gi|30027618 (Vietnam), gi|29836495 (Tor2), gi| 30023962(CUHKW1), gi|29837498(GZ01), gi|30275667(BJ02), gi|30023953(HKU-39849), gi|30124074(Canada), gi|30173234(BJ03). The ClustalX (version 1.8) method was used for multiple sequence alignment, and the results showed that all sequenced SARS-CoV virus N protein sequences were identical.

Embodiment 2: SARS-CoV viral N protein and human body CypA complex construction

Sequence analysis shows that the homology between the full-length SARS-CoV virus N protein and the CA protein sequence of HIV-1 is not high, and the similarity is less than 30%; but a sequence (Val235-Pro369) on the SARS-CoV virus N protein The homology with the binding region (Pro401-Tyr545) of HIV-1 virus CA protein and CypA is very high, the similarity is as high as 47.8% (Figure 1). This sequence analysis indicated that the SARS-CoV viral N protein might bind to CypA through the Val235-Pro369 region.

According to the crystal structure of the complex of HIV-1 virus CA protein Pro401-Tyr545 fragment and CypA (Protein three-dimensional structure database PDB number is 1AK4), the three-dimensional structure model of SARS-CoV virus N protein Val235-Pro369 fragment and CypA complex was modeled ( See Figure 2). The modeling result model shows that there is a loop on the N protein of the SARS-CoV virus that interacts with the human CypA active pocket. The three-dimensional structural modeling of the Val235-Pro369 fragment of the SARS-CoV viral N protein was modeled using the MODELLER software of Insight II, and the quality of the structural model was evaluated with 3D-Profile and Prostat software. Loop search found that the Trp302-Phe315 region of the SARS-CoV virus N protein is similar to the HIV-1 virus CA protein and CypA binding region, so the SARS-CoV virus N protein loop region Trp302-Phe315 was docked into the CypA binding pocket to obtain a complex The initial structure is then optimized by molecular mechanics, using the force field parameters to Amber force field and Kollman-all-atom charge. The composite structure is shown in Figure 2.

Embodiment 3: SPR experimental determination of the interaction between SARS-CoV virus N protein and human CypA

CypA was immobilized on a CM5 chip, and protein N was used as the mobile phase. Use BIACORE3000 to measure the kinetic behavior of N protein and CypA, and the concentration of N protein is as follows: 0.214nM, 1.07nM, 5.35nM, 10.7nM, 21.4nM, 42.8nM, 85.63nM, 0.171uM, 0.343 uM, 0.685uM and 1.37uM. Data were analyzed with Application Wizard/Kinetic Analysis software (Figure 4). The obtained binding constant (K _D ) of the above two proteins is 7.65×10 ^-8 M.

Example 4: Virtual screening of compounds that prevent the interaction between SARS-CoV virus N protein and human CypA

The molecular docking method DOCK program was used to carry out virtual screening of MDL's drug database CMC, existing compound database ACD, drug report database MDDR, and the sample library of the National Center for New Drug Screening. The target models used in the screening were human CypA and SARS-CoV viruses The binding pocket of the N protein. The flexibility of small molecule compounds was considered during molecular docking, and 1000 candidates were first selected from the above database according to the scoring level, and the candidates were further evaluated with the scoring function of AutoDock and the Cscore scoring function, and finally 300 compounds were selected for evaluation. Screening at the molecular level. The molecular level screening method is to first determine the binding constant of the candidate and CypA to obtain the active compound.

Embodiment 5: Cyclosporin A prevents SARS-CoV virus N protein from binding test with human CypA

Through virtual screening and molecular level testing, active compounds were obtained to test for inhibiting the combination of SARS-CoV virus N protein and human CypA. In this embodiment, cyclosporine A is used to illustrate the test process, and the kinetic test method is the same as in Example 4. When testing the binding kinetics of SARS-CoV virus N protein and human CypA, different concentrations of cyclosporin A were added. The concentrations of cyclosporin A used in the present invention are 832, 1664, 2496 and 3328 μM, and the test results are shown in FIG. 5 .

Embodiment 6: Cyclosporine A inhibits SARS-CoV virus test

Using Vero-E6 cells as virus host cells (susceptible cells), the protective effect of samples on virus-infected cells was tested, and the detection indicators were cell degeneration (CPE) and the protection rate of infected cells.

The Vero-E6 cells were inoculated in a 96-well culture plate, cultured in a 37°C, 5% _CO2 incubator, and SARS virus and cyclosporin A at different dilution concentrations were added respectively, and virus control, cell control and sample control were set up. Observe the results under the microscope every day, record the CPE, and measure the OD value with neutral red staining, and calculate and evaluate the anti-SARS virus activity of the sample with reference to the control. The test results are shown in Figure 6.

Claims (3)

1. A drug target for screening treatment and/or prevention of SARS virus infection drugs, said target being the SARS virus N protein Val235-Pro369 fragment comprising SARS virus N protein and human CypA interaction active site and human CypA The three-dimensional structure model of the complex, wherein the sequence characteristics of the target SARS virus N protein are: MSDNGPQSNQ RSAPRITFGG PTDSTDNNQN GGRNGARPKQ RRPQGLPNNT ASWFTALTQH GKEELRFPRG QGVPINTNSG PDDQIGYYRR ATRRVRGGDG KMKELSPRWY FYYLGTGPEA SLPYGANKEG IVWVATEGAL NTPKDHIGTR NPNNNAATVL QLPQGTTLPK GFYAEGSRGG SQASSRSSSR SRGNSRNSTP GSSRGNSPAR MASGGGETAL ALLLLDRLNQ LESKVSGKGQ QQQGQTVTKK SAAEASKKPR QKRTATKQYN VTQAFGRRGP EQTQGNFGDQ DLIRQGTDYK HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYHGAIKL DDKDPQFKDN VILLNKHIDA YKTFPPTEPK KDKKKKTDEA QPLPQRQKKQ PTVTLLPAAD MDDFSRQLQN SMSGASADST QA The sequence characteristics of the target human CypA are: MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKG SCFHRIIPGF MCQGGDFTRH NGTGGKSIYG EKFEDENFIL KHTGPGILSM ANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNG KTSKKITIAD CGQLE The sequence number of the target SARS virus N protein functional region is: 235 VSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFG 289 DQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKDPQFK 349 DNVILLNKHIDAYKTFPPTEP The sequence number of the functional region of the target human CypA protein is: Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125, His126 The interaction active site between the SARS virus N protein and human CypA includes: N protein: a loop composed of residues from Trp302 to Phe315, in which the main amino acid residues are Trp302, Pro303, Gln304, Ile305, Ala306, Aln307, Phe308, Ala309, Pro310, Ser311, Ala312, Ser313, Ala314 and Phe315; Human CypA: Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125 and His126. 2. The method for constructing the drug action target according to claim 1, comprising the following steps: 1) Use bioinformatics methods to conduct network analysis on the interaction between proteins in SARS-CoV and human proteins, and reveal the interaction between SARS-CoV N protein and human CypA; 2) Use the homologous protein modeling method to construct the three-dimensional structure of the SARS-CoV virus N protein, use the molecular simulation method to calculate the interaction between the SARS-CoV virus N protein and human CypA, construct the structure of the N protein and human CypA complex, and establish The region where these two proteins interact; 3) Expression of N protein and human CypA by means of molecular biology, and study of the interaction between N protein and human CypA by surface plasmon laser resonance biosensing technology. Experiments have proved that there is indeed an interaction between the two proteins; 4) For the region where N protein binds to human CypA, use computer virtual screening method to search the compound database to find compounds that prevent N protein from binding to human CypA, and confirm the establishment of drug targets. 3. The method for screening and treating and/or preventing SARS virus infection medicine with the drug action target described in claim 1, comprising the steps of: 1) Determining the binding constant between the candidate and CypA, and screening the compound according to the affinity; 2) The selected compound is tested to inhibit the combination of SARS-CoV virus N protein and human CypA; 3) Test the protective effect of the selected compounds on cells infected with SARS-CoV virus.

Images