CN1472332A - Target for medicine against Sars-Cov and medicine screening method and medicine against Sars - Google Patents
Target for medicine against Sars-Cov and medicine screening method and medicine against Sars Download PDFInfo
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Abstract
A medicine action target for screening the medicine used to prevent and/or treat SARS virus infection contains SARS virus N protein Val235-Pro369 fragment at active site where SARS virus N protein is mutually acting on human CypA, and the 3D model of human CypA compound. A method for configuring said target and a process for screening said medicines are also disclosed. Its screening proves that the ciclosporin derivative can be used to treat and/or prevent SARS virus infection.
Description
Technical field
The present invention relates to be specifically related to be marked on as drug target the method for molecule and cell levels screening of medicaments with sars coronavirus nucleocapsid protein (N albumen) and people's cyclophilin A (CypA) interaction model with biology information technology and molecular biology method screening of medicaments.
Background technology
Severe acute respiratory syndrome be called severe acute respiratory syndrome (Severe Acute RespiratorySydrome, SARS).Discover that a kind of novel coronavirus is the cause of disease most probably, in Hong Kong and Canadian patient and dead patient's corpse, all isolated this virus, in some patients' SARS serum, also found the antibody of anti-hMPV.On April 16th, 2003, The World Health Organization's official confirmation SARS virus is the cause of disease of severe acute respiratory syndrome.The people who infects SARS virus among the severe acute respiratory syndrome patient more than 90% can self-healing, and lethality rate is about about 10%.Patient's SARS the respiratory tract section and the electron micrograph of cell culture show, have the coronavirus particle.This is a kind of novel coronavirus, different with other known members in the coronavirus genus, this virus can cause that the pathology effect takes place green monkey kidney cell (VERO-E6), virus replication can be suppressed by the serum that SARS infects the rehabilitation people, can utilize the cell of infection and rehabilitation patient's serum to carry out immunofluorescence assay (Immunofluorescence assays, IFA) detect the situation that cell infection in the culturing cell has SARS virus, this method shows specific reaction.Studies show that of the U.S., Canada and Hong Kong, non-patient's SARS serum can not react with novel coronavirus; Infectivity gastroenteritis virus (Transmissible Gastroenteritis Virus, TGEV), murine hepatitis virus (Murine HepatitisVirus, MHV), feline infectious peritonitis virus (Feline Infectious Peritonitis virus, FIPV), 229E human corona virus's the super antiserum(antisera) viral growth that can suppress to cultivate.Several laboratories show that to the order-checking of virus new virus is relevant with coronavirus genus, but are different from other coronavirus of generic group, are the new mutation of coronavirus.The coronavirus (Coronaviridae) that sars coronavirus belongs in kind classification in nido virus (Nidovirales) family of positive chain RNA virus (ssRNA positivestrand viruses) family is.It is an emerging subclass in the coronavirus family.In March, 2003, scientists has found to cause arch-criminal's sars coronavirus of SARS, and (SARS Coronaviruses SARS-CoV), and has successfully finished the genomic complete order-checking of SARS-CoV.The SARS-CoV genome is made up of 29727 Nucleotide, 11 open reading frame (Opening Reading Frames).Its genome structure and other coronavirus are closely similar.But show the feature of SARS-CoV and feature and the incomplete similarity of the former coronavirus of finding by more hereditary history and sequence alignment, it not only shows the total feature of other coronavirus, also has the distinctive feature of SARS-CoV (Paul A.Rota itself, M.Steven Oberste, Stephan S.Monroe, W.Allan Nix, Ray Campagnoli, et al.Characterization of a novel coronavirus associated with severe acute respiratorysyndrome.Science (Sciencexpress) 1 May, 2003; Marco A.Marra, Steven J.M.Jones, Caroline R.Astell, Robert A.Holt, Angela Brooks-Wilson, et al.Thegenome sequence of the SARS-associated coronavirus.Science (Sciencexpress) 1May, 2003).Scientist has carried out the gene order-checking of relevant coronavirus Toronto (Tor2) strain isolated 29751 bases of SARS simultaneously.Genome sequence disclose this coronavirus and known coronavirus (comprise human coronary virus HcoVOC43, HCoV-229E) no closely related.The kind of prediction viral protein is an analysis revealed, and this coronavirus is all non-similar closely to known three races coronavirus.Genome sequence will help human the understand mechanism of SARS virus infection, potential animal host's diagnostic detection (using PCR and immunity test), simultaneously also help to develop anti-virus formulation (comprising neutralizing antibody) and find out vaccine antigen determinant (Marco A.Marra, Steven J.M.Jones, Caroline R.Astell, Robert A.Holt, Angela Brooks-Wilson, et al.The genome sequence of the SARS-associatedcoronavirus.Science (Sciencexpress) 1 May, 2003).
But up to now, people are also very insufficient to the infection mechanism research of SARS-CoV virus, and for reducing infection rate and mortality ratio, the specific medicament that the SARS-CoV that can prevent and/or treat is infected has very urgent need clinically.
Therefore, first purpose of the present invention provides the drug effect target that a kind of rapid screening treats and/or prevents the SARS virus infection medicine.
Second purpose of the present invention provides this drug target target construction process.
The 3rd purpose of the present invention provides the method that treats and/or prevents the SARS virus infection medicine with this drug effect target screening.
The 4th purpose of the present invention provides the medicine that treats and/or prevents the SARS virus infection that filters out with this drug effect target.
Summary of the invention
The drug effect target is meant the key function biomacromolecule that can play a decisive role in disease generation and the evolution on most critical pathology link, comprise cell-membrane receptor, proteolytic enzyme, hormone and cytokine, ionic channel, DNA, RNA, nuclear receptor etc.The core of modern medicines research is to be designated as the basis with drug target, and screening or design and target complementary structure, can excitement or the biological function that mediates of antagonism target have the medicine guide thing of therapeutic action.The pathology link of most critical in disease generation and the evolution is found and optionally blocked to this novel drugs based on mechanism and structure, develops the specific drugs of high-efficiency low-toxicity side effect.
The present inventor has carried out deep bioinformatic analysis to the gene expression product of SARS-CoV, thereby has discerned the approach of SARS virus infection human body; Verify this action pathway with molecular biology and bio-physical method then; According to this action pathway SCREENED COMPOUND, and carry out the test of virus levels, the pharmaceutically-active target of conclusive evidence severe acute respiratory syndrome (SARS) prevents and/or treats the severe acute respiratory syndrome medicine with this drug effect target screening, thereby has finished the present invention.
It is provided by the invention that to be used to screen the drug effect target that treats and/or prevents the SARS virus infection medicine be to duplicate in SARS coronavirus, transcribe, the nucleocapsid protein (SARS Nucleocapsid) that plays a crucial role in translation and the assembling process, host's (human body) action protein matter people's cyclophilin A (Cyclophilin A) and interaction approach between the two, described target is SARS virus N albumen Val235-Pro369 segment and the human body CypA mixture 3 d structure model that comprises SARS virus N albumen and human body CypA interaction avtive spot, and wherein the proteic sequence signature of target SARS virus N is: 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 signature of target human body CypA is: MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKGSCFHRIIPGF MCQGGDFTRH NGTGGKSIYG EKFEDENFIL KHTGPGILSMANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNGKTSKKITIAD CGQLE
In the drug effect target provided by the invention, target SARS virus N protein function regional sequence is numbered: 235 VSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFG28,9 DQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKD PQFK349 DNVILLNKHIDAYKTFPPTEP
In the described drug effect target, target human body CypA protein function regional sequence is numbered:
Arg55、Ile57、Phe60、Met61、Gln63、Gly72、Thr73、Gly74、Ala101、Asn102、Ala103、Gln111、Phe113、Trp121、Leu122、Lys125、His126
In the described drug effect target, SARS virus N albumen and human body CypA interaction avtive spot comprise:
The loop that N albumen: Trp302 forms to the Phe315 residue, wherein main amino-acid residue is Trp302, Pro303, Gln304, Ile305, Ala306, Aln307, Phe308, Ala309, Pro310, Ser311, Ala312, Ser313, Ala314 and Phe315; Human body CypA:Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125 and His126.
Drug target target construction process provided by the invention may further comprise the steps:
1) with bioinformatics method the protein among the SARS-CoV and human body protein are interacted and carry out network analysis, disclose the interaction of SARS-CoV N albumen and people CypA;
2) make up the proteic three-dimensional structure of SARS-CoV virus N with homologous protein mould construction method, calculate the interaction of SARS-CoV virus N albumen and human body CypA with molecule simulation method, make up the structure of N albumen and human body CypA mixture, establish the zone of these two protein-interactings;
3) express N albumen and human body CypA with molecular biology method, usefulness surface plasma laser resonant biosensor technique is studied the interaction of N albumen and human body CypA, discloses to exist intensive to interact between these two albumen;
4) compound database is searched with the computer virtual screening method in the zone that mutually combines at N albumen and human body CypA, finds to stop N albumen and human body CypA bonded compound the foundation of conclusive evidence drug target target.
Provided by the inventionly treat and/or prevent the method for SARS virus infection medicine, comprise the steps: with medicine action target screening
1) binding constant of mensuration material standed for and CypA is selected the compound with strong avidity;
2) compound be will be selected and SARS-CoV virus N albumen and the test of human body CypA bonded suppressed;
3) test is selected compound to being subjected to the provide protection of SARS-CoV virus infected cell.
Description of drawings
Fig. 1 shows the sequence alignment of SARS-CoV virus N albumen Val235-Pro369 segment and HIV-1 virus CA albumen Pro401-Tyr545.Identical or the conservative residue of " * " representative among the figure, ". " represents the replacement of conserved residues, and ": " represents semiconservative replacement, and similarity is 47.8%.
Fig. 2 shows SARS-CoV virus N albumen Val235-Pro369 segment (zonal structure) and CypA (spherical surface structure) mixture 3 d structure model.
Fig. 3 shows hydrogen bond and the hydrophobic interaction between SARS-CoV virus N albumen and the CypA.Wherein the meaning of symbol is as follows:
The part key
The non-part residue that relates in the hydrophobic contact
Fig. 4 shows ARS-CoV virus N albumen and human body CypA interaction dynamics test result.Used instrument is BIACORE3000, and analysis software is Kinetic Analysis among the Application Wizard.CypA is fixed on the CM5 chip during mensuration, and the N protein concentration is followed successively by: 0.214nM, 1.07nM, 5.35nM, 10.7nM, 21.4nM, 42.8nM, 85.63nM, 0.171uM, 0.343uM, 0.685uM and 1.37uM.
Fig. 5 shows that ciclosporin A suppresses ARS-CoY virus N albumen and human body CypA binding kinetics test result.CypA is fixed on the CM5 chip, and the proteic concentration of N is that the concentration of 1.37 μ M ciclosporin As (CsA) is followed successively by 832,1664,2496 and 3328 μ M.
Fig. 6 shows the provide protection of ciclosporin A to the Vreo-E6 cell of infection SARS-CoV virus.
Detailed Description Of The Invention
The present inventor uses bioinformatics method to the protein among the SARS-CoV and human body protein phase The network of mutual effect is analyzed, the nucleocapsid protein of discovery SARS-CoV (SARS Nucleocapsid, Hereinafter to be referred as N albumen) and human body in human Cyclophilin A (Cyclophilin A is hereinafter to be referred as CypA) phase is arranged Mutual effect; The present invention has made up the three-dimensional knot of SARS-CoV virus N albumen with homologous protein mould construction method Structure, the interaction of having calculated SARS-CoV virus N albumen and human body CypA with molecule simulation method, Make up the structure of N albumen and human body CypA compound, established the zone of these two protein-interactings; Expressed N albumen and human body CypA with molecular biology method, passed with the surface plasma laser resonant is biological Sense (SPR) technical research the interaction of N albumen and human body CypA, find to exist between these two albumen Strong interaction; For the zone that N albumen and human body CypA mutually combine, sieve with computer virtual Choosing method has been searched compound database, find existing immune suppressive cyclosporin A can stop N albumen with The combination of human body CypA; Measure ciclosporin A with the SPR technology and stoped N albumen and human body CypA In conjunction with activity; Screened the activity that ciclosporin A suppresses virus infections Vero-E6 cell in virus levels, Prove conclusively approach and the target of this SARS-CoV virus infections human body, and set up corresponding molecular water The screening technique of gentle cellular level.
1, bioinformatic analysis and SARS-CoV virus N albumen and human body CypA interact and simulate: 1994, Luban etc. found that CypA can integrate with HIV-1 virus nuclear, with HIV-1 virus dimerization The effect of capsid territory (Luban, J. in the Gap polyprotein; Bossolt, K.L.; Franke, E.K.; Kalpana, G.V.; Goff, S.P.Human immunodefficiency 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 1994,372,359-362; Thali, M.; Bukovsky, A.; Kondo, E.; Rosenwirth, B.; Walsh, C.T.; Sodroski, J.; Go ¨ ttlinger, H.G. Functional association of Cyclophilin A with HIV-1 virions.Nature 1994,372, 363-365), and then in the virus maturation process, polyprotein resolves into stromatin, capsid protein (CA But these albumen still combine CypA and capsid protein albumen) and nucleocapsid protein (N albumen), Approximately with 1: 10 ratio combination. Therefore, CypA and capsid protein directly is combined in HIV-1 virus (Gitti, R.K. play an important role in ripe, the viral formation that copies with reovirion; Lee, B.M.; Walker, J.; Summers, M.F.; Yoo, S.; Sundquist, W.I.Structure of the Amino-terminal core domain of the HIV-1 capsid protein.Science 1996,273, 231-235; Gamble, T.R.; Vajdos, F.F.; Yoo, S.; Worthylake, D.K.; Houseweart, 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 immunodefficiency virus type 1 Gag polyprotein to Cyclophilin A is mediated by the central region of capsid and Requires Gag dimerizaion.J.Virol.1996,70,4299-4310; Ott, D.E.Cellular Proteins in HIV virions.Rev.Med.Virol.1997,7,167-180). Albumen-protein-interacting The analysis showed that N albumen and the human body CypA of SARS-CoV virus have interaction. At SARS-CoV Do not find CA albumen in the virus, imagination is both capsid protein, the N albumen of SARS-CoV virus One of function be with HIV-1 virus in the CA protide seemingly, be combined with CypA, to the maturation of virus, The formation that copies with reovirion of virus plays an important role. Therefore, the present invention has carried out further order Row compare and the structure mould is built.
Sequence analysis shows that the CA albumen homology of SARS-CoV virus N albumen and HIV-1 virus Height, but complete sequence is relatively, both similitudes are still very low, and sequence similarity is 37.6%, homology is 26.9%, the sequence gap is less than 4.8%. Yet, further analyze and find SARS-CoV virus N albumen The Val235-Pro369 segment and the calmodulin binding domain CaM of HIV-1 virus CA albumen and CypA Trp302-Phe315 has higher similitude, and sequence similarity is 42.2%, homology is 24.4%, order The row gap is less than 4.1%. This sequence analysis shows that SARS-CoV virus N albumen may pass through Regional be combined with CypA (the seeing Fig. 1) of Val235-Pro369.
Crystal structure (egg according to HIV-1 virus CA albumen Pro401-Tyr545 segment and CypA compound White matter three-dimensional structure database PDB is numbered 1AK4), mould of the present invention has been built SARS-CoV virus N egg White Val235-Pro369 segment and CypA compound 3 d structure model (seeing Fig. 2). The structural model that mould is built Show, have one section with the effect of human body CypA active pocket one section on the N albumen of SARS-CoV virus Loop.
2, SARS-CoV virus N albumen and human body CypA interaction avtive spot: by SARS-CoV Virus N albumen Val235-Pro369 segment and CypA compound 3 d structure model (accompanying drawing 2) can obtain Get some information of SARS-CoV virus N albumen and human body CypA interaction avtive spot. SARS-CoV virus N albumen and human body CypA calmodulin binding domain CaM are to form from Trp302 to the Phe315 residue Loop, the main residue that forms the CypA binding pocket is: Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125, His126. The hydrogen bond and the hydrophobic interaction branch that form between two albumen Do not list in Fig. 3 and table 1 and table 2.
Table 1.SARS-CoV virus N albumen and CypA interaction of hydrogen bond
| The CypA residue | The effect atom | SARS-CoV virus N albumen | The effect atom | Distance () |
| Trp121 Arg55 Arg55 Gln63 Gly72 | N 1 N 2 N 1 N 2 O | Phe308 Gln307 Gln307 Ile305 Ile305 | O O O O N | 3.31 2.73 3.01 2.76 2.78 |
Table 2.SARS-CoV virus N albumen and CypA hydrophobic interaction
| The CypA residue | The effect atom | SARS virus N protein residues | The effect atom | Distance () |
| Trp121 Trp121 Phe60 Phe60 Phe60 Phe60 Phe60 Phe60 Phe113 Phe113 Phel13 Phe113 | C 2 C 2 C 2 C 2 C C 2 C 2 C C 1 C 2 C 1 C | Pro310 Pro310 Ala309 Ala309 Ala309 Ala309 Ala309 Phe308 Gln307 Gln307 Gln307 Gln307 | C C C C C C C C C C C C | 3.59 3.85 3.58 3.56 3.70 3.42 3.84 3.70 3.46 3.82 3.17 3.30 |
| Phe113 Phe113 Phe113 Phe113 Phe113 Phe60 His126 His126 Leu122 Leu122 Phe60 His126 Arg55 Asn102 Asn102 Ala101 Ala103 Gly72 Asn71 Thr73 Thr73 Asn71 Arg69 Arg69 Thr73 Thr73 Arg69 Thr73 | C C C 2 C 1 C 1 C C 1 C 2 C 2 C 1 C C 1 C C C C C C C C 2 C C C C C 2 C C C 2 | Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Gln307 Ala306 Ile305 Ile305 Ile305 Ile305 Gln304 Trp302 Trp302 Trp302 Trp302 Trp302 Trp302 Trp302 Trp302 Trp302 Trp302 | C C C C C C C C C C C C C C 2 C 2 C 2 C 1 C C 2 C 3 C 3 C 3 C 3 C 3 C 3 C 3 C 3 C 2 | 3.75 3.65 3.77 3.71 3.89 3.74 3.49 3.90 3.79 3.85 .88 3.75 3.47 3.00 3.33 3.34 3.58 3.88 3.76 3.37 3.37 3.55 3.59 3.76 2.96 2.98 3.15 3.49 |
3, the expression of SARS-CoV virus N albumen and human body CypA and the mensuration that interacts: will make up Good pQE30/SARS_N Plasmid Transformation is in Escherichia coli M15, with IPTG (concentration 0.8mM) Inducing, is twin antibiotic in ampicillin and kanamycins, and temperature is 30 ℃, and expression time is 10 hours Under the condition, express SARS-CoV virus N albumen. With NTA-His column chromatography preliminary purification SARS-CoV virus N albumen, recycling FPLC-gel filtration is further purified SARS-CoV virus N albumen. Utilize the e. coli bl21 (DE3) of pET11/CyPA Plasmid Transformation through IPTG (0.5mM) Abduction delivering human body CypA albumen (expressing 25 ℃ of temperature) utilizes 50% saturated ammonium sulphate albumen to get To the albumen crude product, obtain sterling with Mono-Q and the molecular sieve human body CypA albumen of further purifying.
Use surface plasma laser resonant (SPR) technology to obtain SARS-CoV virus N albumen and the interactional dissociation constant (K of human body CypAD) be 7.65 * 10-8M, show SARS-CoV virus N albumen with Human body CypA interacts stronger combination. Measurement result is seen Fig. 4.
4, for N albumen and the interactional virtual screening of human body CypA: determine SARS-CoV virus Behind N albumen and human body CypA interaction mode and the calmodulin binding domain CaM, the present invention is with CypA and SARS-CoV The surface conjunction pocket of virus N albumen effect is target, and is virtual with molecular docking (Molecular Docking) Screening (Virtual Screening) method has been searched the drug data base CMC of MDL company, existing chemical combination The chemical combination of thing database ACD, medicine report database MDDR and Shanghai Pharmaceutical Inst., Chinese Academy of Sciences The matter sample storehouse has obtained a collection of compound of being selected that has strong affinity with CypA, comprises CypA's The part ring [[(E)-(2S, 3R, 4R)-3-hydroxy-4-methyl-2-methylamino-6-eight alkene acyls]-the amino butyryl of L-2--N-first Base glycyl-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] (ciclosporin A, Cyclosporin A). Molecular simulation shows that ciclosporin A can stop determines SARS-CoV virus N albumen and human body CypA In conjunction with.
5, cyclosporine stops N albumen and human body CypA binding: utilize surface plasma resonance biological Sensing technology BIACORE3000 research ciclosporin A stops SARS-CoV virus N albumen and human body CyPA combination, experimental result as shown in Figure 5, the result shows the increase along with the concentration of ciclosporin A, SARS-CoV virus N albumen is combined reduction with human body CyPA, show that ciclosporin A can suppress SARS-CoV virus N albumen is combined with human body CypA.
6, the anti-SARS-CoV virus activity test of ciclosporin A: for proving conclusively above-mentioned SARS-CoV virus The correlation of N albumen and human body CypA action pathway and SARS-CoV virus infections human body, the present invention's choosing Selected the ciclosporin A that can stop SARS-CoV virus N albumen to be combined with human body CypA at molecular level Carry out the test of virus levels.
Test philosophy: with the Vero-E6 cell as virus host cell (permissive cell), specimen pair The protective effect of virus infected cell, detecting index is cell degeneration reaction (CPE) and observation infection cell Protective rate.
Method of testing: the Vero-E6 cell is inoculated in 96 well culture plates, puts 37 ℃, the 5%CO2 incubator Cultivate, add the SARS virus of different diluted concentrations and the ciclosporin A of 2.5mg/m and 1250ug/ml, Observe CPE, and measure OD value, calculation sample anti-SARS virus active function with neutral red staining.
Test result: in the anti-SARS virus activity experiment of virus-cellular level model, have with difference The SARS virus of effect concentration infects the Vero-E6 cell, when adding 2.5mg/m and 1250ug/ml concentration The prolection that has obvious inhibition SARS virus to infect the Vero-E6 cell during ciclosporin A (the results are shown in Fig. 6). Show SARS-CoV virus N albumen and human body CypA action pathway and SARS-CoV virus Infect human body and have clear and definite correlation.
As mentioned above, one aspect of the present invention is found SARS-CoV virus by analysis of biological information N albumen and human body CypA have interaction. Sequence analysis shows, SARS-CoV virus N albumen with The CA albumen homology of HIV-1 virus is higher, but complete sequence relatively, and both similitudes are still very low, order The row similitude is 37.6%, homology is 26.9%, the sequence gap is less than 4.8%. Yet, further analyze Find, the Val235-Pro369 segment of SARS-CoV virus N albumen and HIV-1 virus CA albumen and The calmodulin binding domain CaM Trp302-Phe315 of CypA has higher similitude, and sequence similarity is 42.2%, same Source property is 24.4%, the sequence gap is less than 4.1%. Prompting SARS-CoV virus N albumen and HIV-1 disease Poison CA albumen has similar function, namely behind SARS-CoV virus infections human body, and the SARS-CoV disease The maturation that directly is combined in SARS-CoV virus of poison N albumen and human body CypA, copy and new virus Play an important role in the formation of particle.
Crystal according to human body CypA and HIV-1 virus CA albumen Trp302-Phe315 segment compound Structure, mould have been built human body CypA and SARS-CoV virus N albumen Val235-Pro369 segment compound The three-dimensional result model. The result shows, SARS-CoV virus N albumen can with the land of human body CypA Be the loop that forms from Trp302 to the Phe315 residue, its main interaction of hydrogen bond and hydrophobic interaction See that respectively accompanying drawing 3 is subordinate list 1 and subordinate list 2, the binding pocket of CypA and SARS-CoV virus N albumen By Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125 and His126 residue form.
On the basis of determining SARS-CoV virus N albumen and the interactional mode of human body CypA and structural model, the present invention adopts plasma surface laser resonant bio-sensing (SPR) to study SARS-CoV virus N albumen and the interactional dynamic behavior of human body CypA, show that both have stronger combination (with accompanying drawing 4), binding constant (KD) be 7.65 * 10-8M。
Can preliminary judgement by The above results, SARS-CoV virus N albumen interacts relevant with SARS-CoV infection human body with human body CypA, SARS-CoV virus N albumen, human body CypA albumen and the interaction between them can be used as the method for the anti-SARS-CoV virus drugs of screening.By the principle of general drug targets conclusive evidence, must go out active compound according to the target sieving of hypothesis.So, the present invention is directed to the binding pocket of human body CypA and SARS-CoV virus N protein-interacting, with virtual screening method screened the drug data base CMC of MDL company, existing compound database ACD and I oneself compound sample storehouse, obtain a collection of compound of being selected that has strong avidity with CypA, comprised the part ciclosporin A of CypA.The present invention adopts plasma surface laser resonant bio-sensing (SPR) to study ciclosporin A inhibition SARS-CoV virus N albumen and human body CypA bonded dynamic behavior (the results are shown in Figure 5) then, the result shows, ciclosporin A can suppress SARS-CoV virus N albumen effectively and combine with human body CypA, increase along with ciclosporin A concentration, arrestin bonded intensity also increases, and has presented good dose relationship.
In order further to prove conclusively the validity that the present invention proposes anti-SARS-CoV virus drugs action target, the present invention has carried out ciclosporin A and has protected the Vero-E6 cell to be subjected to the test of SARS-CoV virus infection.As the virus host cell, the test ciclosporin A is to the provide protection of virus infected cell with the Vero-E6 cell, and detecting index is cytopathy reaction (CPE) and observation cells infected protection ratio.The result shows that the ciclosporin A of 2.5mg/m and 1250ug/ml concentration has the protection activity (the results are shown in accompanying drawing 6) that infects the Vero-E6 cell than the obvious suppression SARS virus.Show that SARS-CoV virus N albumen and human body CypA action pathway and SARS-CoV virus infection human body have clear and definite dependency.
According to the screening of above bioinformatic analysis, molecular simulation, SPR albumen-protein-interacting research, virtual screening, compound molecule level and virocyte level, the present invention determines that SARS-CoV virus N albumen, human body CypA and interaction approach thereof are to treat and/or prevent SARS-CoV medicine for treating viral infections action target.
The present invention relates on the other hand according to drug effect target SARS-CoV virus N albumen, human body CypA and the interaction approach thereof determined, adopt molecule simulation method to set up the 3 d structure model of SARS-CoV virus N protein function segment Val235-Pro369 and human body CypA mixture, determine key position and amino-acid residue that SARS-CoV virus N albumen and human body CypA mutually combine.On this basis, SARS-CoV virus N albumen and human body CypA bonded material standed for have been obtained to block with virtual screening method, active testing and the test of inhibition Vero-E6 cell infection virus activity through the SPR molecular level filter out the target organisms depressant of functions.
The target organisms depressant of functions is for mutually combining with target SARS virus N protein function zone or target human body CypA protein function zone and blocking described SARS virus N albumen and human body CypA functional area bonded organic molecular compound or or polypeptide compound or oligosaccharide compound or monoclonal antibody or nucleoside compound.
Another aspect of the present invention has found that the ciclosporin derivative with general formula I can combine with CypA specifically; suppress combining of SARS-CoV virus N albumen and human body CypA effectively; have the protection activity that infects the Vero-E6 cell than the obvious suppression SARS virus, have the purposes that is used for the treatment of and/or prevents the SARS-CoV virus infection.
Clinically, ciclosporin A is mainly used in the rejection after inhibition organ and the tissue transplantation, also is used for the treatment of autoimmune disorder in addition, as type i diabetes, psoriasis, rheumatoid arthritis, systemic lupus erythematous etc. are all had sure curative effect.U.S. Pat 6270957 and US5840305 have disclosed the compound that contains general formula I and can act on the T lymphocyte, suppress hiv protease and suppress cellular uptake virus, disturb HIV-1 virus Gag albumen to interact, suppress CypA with CypA to be integrated into the HIV-1 virion, stop the virion maturation and duplicate; U.S. Pat 6521595, US4885276 and US19891205 have disclosed the new preparation method of compound who contains general formula I.
Each substituent definition is as follows in the ciclosporin derivative of general formula I or its pharmacy acceptable salt or the hydrate:
In the formula
R
1Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl, hydroxyl;
R
2Be H, ethanoyl, C
1-C
4Substituted alkyl acyl group, C
1-C
4Substituted alkyl;
R
3Be H, hydroxyl, hydroxy alkoxy base, hydroxyl alkylthio, C
1-C
4Alkyl sulfide ether, alkoxycarbonyl alkyl thioether group, arylalkyl thioether group, artyl sulfo, alkyl sulphonyl, hydroxyalkyl alkylsulfonyl, many alkoxyl groups replace the dimethyl sulfide base;
R
4Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl;
R
5For H, dimethyl sulfide base, many alkyl replace thiomethyl;
R
6Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl;
R
7Be H, hydroxyl, hydroxyalkyl, hydroxy alkoxy base, C
1-C
4The alkyl sulfide ether;
R
8Be H, hydroxyl, hydroxyalkyl, hydroxy alkoxy base, C
1-C
4The alkyl sulfide ether;
R
9Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl.
" pharmacy acceptable salt " described in this specification sheets can be enumerated and propionic acid particularly, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, Deng organic acid and aspartic acid, acidic amino acids such as L-glutamic acid form behind the ester salt that forms with mineral alkali again, as sodium, potassium, calcium, aluminium salt and ammonium salt, or the salt that forms with organic bases, as methylamine salt, ethylamine salt, ethanolamine salt etc., or and Methionin, arginine, basic aminoacidss such as ornithine form the hydrochloric acid behind the ester, Hydrogen bromide, hydrofluoric acid, sulfuric acid, nitric acid, the salt of mineral acids such as phosphoric acid, or and formic acid, acetate, picric acid, methylsulfonic acid, organic acid salt such as ethyl sulfonic acid.
Compound or its pharmacy acceptable salt or the hydrate of general formula I found in experiment of the present invention; can suppress combining of SARS-CoV virus N albumen and human body CypA effectively; has protection activity than obvious suppression SARS-CoV virus infection Vero-E6 cell; therefore, has the purposes that treats and/or prevents the SARS-CoV virus infection.
Most preferred is in the compound of Formula I that the inventive method filters out or its pharmacy acceptable salt or the hydrate: ciclosporin A (Cyclosporin A), ring [[(E)-(2S, 3R, 4R)-3-hydroxy-4-methyl-2-methylamino--6-eight alkene acyls]-the amino butyryl of L-2--N-methyl glycyl-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 the pharmaceutical composition that can be used for preventing and/or treating the infectious atypical pneumonia that coronavirus infection causes, it comprises at least a compound of Formula I or its pharmacy acceptable salt or its hydrate and at least a pharmaceutical carrier or the vehicle that prevents and/or treats significant quantity.
Pharmaceutical carrier described here includes but not limited to: ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein such as human serum albumin, buffer substance such as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, the partial glycerol ester mixture of saturated vegetable fatty acid, water, salt or ionogen, as protamine sulfate, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloided silica, Magnesium Trisilicate, polyvinylpyrrolidone, cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax, lanolin.
The invention still further relates to this pharmaceutical composition and prevent and/or treat infectious atypical pneumonia (Severe Acute Respiratory Syndrome, SARS) the new purposes on the medicine in preparation.
Described pharmaceutical composition can be prepared into various formulations according to different way of administration.These formulations are used in one of following mode: oral, spraying sucks, rectal application, nasal cavity applied medicine, the cheek medication, local application, non-enterally administer, as subcutaneous, in the vein, intramuscular, intraperitoneal, sheath, in the ventricle, in the breastbone and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Preferred oral or intramuscular injection when wherein preventing SARS virus to infect, preferred intraperitoneal or intravenous administration mode when the treatment SARS virus infects.
It may be noted that in addition, the using dosage of The compounds of this invention and using method depend on all multifactor, comprise activity intensity, Time of Administration, metabolic rate, the severity of illness and diagnosis and treatment doctor's the subjective judgement of patient's age, body weight, sex, natural health situation, nutritional status, compound.Recommended doses is as being beginning 5mg～10mg/kg every day, and maintenance dose can reduce to 3mg/kg every day.Capsule: 0.25g/ grain.Injection liquid 0.25g/5ml.Oral liquid: 5g/50ml.
Specific embodiments
The following examples are specifically to select embodiment for what the present invention was further specified, but these embodiment never are any limitation of the invention.
The multiple comparison result of embodiment 1:SARS-CoV virus N protein sequence
Correlated sequence source is from American National bioinformation center NCBI, they comprise that respectively from the SARS case sample of different areas the NCBI accession number is 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).Adopt the method for ClustalX (1.8 editions) to carry out the multiple sequence comparison, the result shows that all SARS-CoV virus N protein sequences that checked order are identical.
Embodiment 2:SARS-CoV virus N albumen and human body CypA mixture make up
Sequential analysis shows that the homology of CA protein sequence of total length SARS-CoV virus N albumen and HIV-1 is not high, and similarity is less than 30%; But the one section sequence (Val235-Pro369) on the SARS-CoV virus N albumen is very high with the homology of the calmodulin binding domain CaM (Pro401-Tyr545) of HIV-1 virus CA albumen and CypA, and similarity is up to 47.8% (Fig. 1).This sequential analysis shows that SARS-CoV virus N albumen may combine with CypA by the Val235-Pro369 zone.
According to the crystalline structure (protein three-dimensional structure database PDB is numbered 1AK4) of HIV-1 virus CA albumen Pro401-Tyr545 segment and CypA mixture, mould has been built SARS-CoV virus N albumen Val235-Pro369 segment and CypA mixture 3 d structure model (see figure 2).Results model that mould is built shows, has one section one section loop with the effect of human body CypA active pocket on the N albumen of SARS-CoV virus.SARS-CoV virus N albumen Val235-Pro369 segment three-dimensional structure mould is built the MODELLER software that adopts InsightII, and with the quality of 3D-Profile and Prostat software evaluation structural models.Loop searches and finds that the proteic Trp302-Phe315 of SARS-CoV virus N zone is similar to the CypA calmodulin binding domain CaM to HIV-1 virus CA albumen, so SARS-CoV virus N albumen loop district Trp302-Phe315 is docked in the CypA binding pocket, obtain the original texture of mixture, be optimized with molecular mechanics, the force field parameter of employing is to the Amber field of force and Kollman-all-atom electric charge again.Composite structure as shown in Figure 2.
The interactional SPR measuring of embodiment 3:SARS-CoV virus N albumen and human body CypA
CypA is fixed on the CM5 chip, and N albumen is moving phase.With the dynamic behavior of BIACORE3000 mensuration N albumen and CypA, the proteic concentration of N is followed successively by as the N protein concentration: 0.214nM, 1.07nM, 5.35nM, 10.7nM, 21.4nM, 42.8nM, 85.63nM, 0.171uM, 0.343uM, 0.685uM and 1.37uM.With Application Wizard/Kinetic Analysis software analysis data (Fig. 4).Obtain the binding constant (K that above-mentioned two albumen are seen
D) be 7.65 * 10
-8M.
Embodiment 4: the virtual screening that stops SARS-CoV virus N albumen and human body CypA interacting compounds
With molecular docking method DOCK program the drug data base CMC of MDL company, existing compound database ACD, medicine report database MDDR and The National Center for Drug Screening's sample library have been carried out virtual screening, used target model is the binding pocket of human body CypA and the effect of SARS-CoV virus N albumen during screening.Considered the flexibility of micromolecular compound during molecular docking, height according to marking is selected 1000 material standed fors earlier from above-mentioned database, scoring functions and Cscore scoring functions with AutoDock are further estimated material standed for, have selected 300 compounds at last and have carried out the screening of molecular level.The molecular level screening method is a binding constant of measuring material standed for and CypA earlier, obtains active compound.
Embodiment 5: ciclosporin A stops SARS-CoV virus N albumen to combine test with human body CypA
By virtual screening and molecular level test, obtain active compound and suppress SARS-CoV virus N albumen and the test of human body CypA bonded.Present embodiment illustrates test process with ciclosporin A, and the kinetic test method is with embodiment 4.When testing SA RS-CoV virus N albumen and the behavior of human body CypA binding kinetics, add the ciclosporin A of different concns.The concentration of the used ciclosporin A of the present invention is 832,1664,2496 and 3328 μ M, and test result is seen Fig. 5.
Embodiment 6: ciclosporin A suppresses the test of SARS-CoV virus
As virus host cell (permissive cell), specimen is to the provide protection of virus infected cell with the Vero-E6 cell, and detecting index is cytopathy reaction (CPE) and cells infected protection ratio.
The Vero-E6 cell inoculation in 96 well culture plates, is put 37 ℃, 5%CO
2Incubator is cultivated, and adds the ciclosporin A of SARS virus and different weaker concns respectively, establishes virus control, cell contrast and sample contrast.Observations under the every day mirror, record CPE, and measure OD value with toluylene red dyeing, carries out the calculating and the evaluation of sample anti-SARS virus active function with reference to contrasting.Test result is seen Fig. 6.
Claims (12)
1, a kind of drug effect target that treats and/or prevents the SARS virus infection medicine for screening; SARSNCypASARSNVal235-Pro369CypA,SARSN：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 signature of target human body CypA is: MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKGSCFHRIIPGF MCQGGDFTRH NGTGGKSIYG EKFEDENFIL KHTGPGILSMANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNGKTSKKITIAD CGQLE
2, drug effect target as claimed in claim 1, wherein target SARS virus N protein function regional sequence is numbered: 235 VSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKQYNVTQAFGRRGPEQTQGNFG28,9 DQDLIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYHGAIKLDDKD PQFK349 DNVILLNKHIDAYKTFPPTEP
3, drug effect target as claimed in claim 1, wherein target human body CypA protein function regional sequence is numbered:
Arg55、Ile57、Phe60、Met61、Gln63、Gly72、Thr73、Gly74、Ala101、Asn102、Ala103、Gln111、Phe113、Trp121、Leu122、Lys125、His126
4, drug effect target as claimed in claim 1, wherein said SARS virus N albumen and human body CypA interaction avtive spot comprise:
The loop that N albumen: Trp302 forms to the Phe315 residue, wherein main amino-acid residue is Trp302, Pro303, Gln304, Ile305, Ala306, Aln307, Phe308, Ala309, Pro310, Ser311, Ala312, Ser313, Ala314 and Phe315;
Human body CypA:Arg55, Ile57, Phe60, Met61, Gln63, Gly72, Thr73, Gly74, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122, Lys125 and His126.
5, the described drug target target of claim 1 construction process may further comprise the steps:
1) with bioinformatics method the protein among the SARS-CoV and human body protein are interacted and carry out network analysis, disclose the interaction of SARS-CoV N albumen and people CypA;
2) make up the proteic three-dimensional structure of SARS-CoV virus N with homologous protein mould construction method, calculate the interaction of SARS-CoV virus N albumen and human body CypA with molecule simulation method, make up the structure of N albumen and human body CypA mixture, establish the zone of these two protein-interactings;
3) express N albumen and human body CypA with molecular biology method, usefulness surface plasma laser resonant biosensor technique is studied the interaction of N albumen and human body CypA, discloses to exist intensive to interact between these two albumen;
4) compound database is searched with the computer virtual screening method in the zone that N albumen and human body CypA are mutually combined, and finds to stop N albumen and human body CypA bonded compound the foundation of conclusive evidence drug target target.
6, treat and/or prevent the method for SARS virus infection medicine with the described drug effect target screening of claim 1, comprise the steps:
1) binding constant of mensuration material standed for and CypA is selected the compound with strong avidity;
2) compound be will be selected and SARS-CoV virus N albumen and the test of human body CypA bonded suppressed;
3) test is selected compound to being subjected to the provide protection of SARS-CoV virus infected cell.
7, a kind of target organisms depressant of functions is for target SARS virus N protein function zone or target human body CypA protein function regional complementarity and block described SARS virus N albumen and human body CypA functional area bonded organic molecular compound or or polypeptide compound or oligosaccharide compound or monoclonal antibody or nucleoside compound.
8, target organisms depressant of functions as claimed in claim 7, described biological function inhibitor is organic molecular compound or polypeptide compound.
9, claim 7 or 8 described inhibitor treat and/or prevent purposes on the medicine that SARS virus infects in preparation.
10, the derivative of ciclosporin shown in the general formula I or its pharmacy acceptable salt or hydrate treat and/or prevent purposes on the medicine that SARS virus infects in preparation
In the formula
R
1Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl, hydroxyl;
R
2Be H, ethanoyl, C
1-C
4Substituted alkyl acyl group, C
1-C
4Substituted alkyl;
R
3Be H, hydroxyl, hydroxy alkoxy base, hydroxyl alkylthio, C
1-C
4Alkyl sulfide ether, alkoxycarbonyl alkyl thioether group, arylalkyl thioether group, artyl sulfo, alkyl sulphonyl, hydroxyalkyl alkylsulfonyl, many alkoxyl groups replace the dimethyl sulfide base;
R
4Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl;
R
5For H, dimethyl sulfide base, many alkyl replace thiomethyl;
R
6Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl;
R
7Be H, hydroxyl, hydroxyalkyl, hydroxy alkoxy base, C
1-C
4The alkyl sulfide ether;
R
8Be H, hydroxyl, hydroxyalkyl, hydroxy alkoxy base, C
1-C
4The alkyl sulfide ether;
R
9Be H, C
1-C
4Alkyl, C
1-C
4Substituted alkyl.
11, purposes as claimed in claim 10, wherein said compound is: cyclosporin A, ring [[(E)-(2S, 3R, 4R)-3-hydroxy-4-methyl-2-methylamino--6-eight alkene acyls]-the amino butyryl of L-2--N-methyl glycyl-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].
12, the pharmaceutical composition that is used for the treatment of and/or prevents SARS virus to infect is comprising the claim 10 that treats and/or prevents significant quantity or 11 described compounds and at least a pharmaceutically acceptable carrier.
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