CN100478030C - SARS resisting medicine action target and its preventing and treating medicine - Google Patents
SARS resisting medicine action target and its preventing and treating medicine Download PDFInfo
- Publication number
- CN100478030C CN100478030C CNB2003101025652A CN200310102565A CN100478030C CN 100478030 C CN100478030 C CN 100478030C CN B2003101025652 A CNB2003101025652 A CN B2003101025652A CN 200310102565 A CN200310102565 A CN 200310102565A CN 100478030 C CN100478030 C CN 100478030C
- Authority
- CN
- China
- Prior art keywords
- sacp
- sars
- virus
- sequence
- coronavirus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000003814 drug Substances 0.000 title claims abstract description 44
- 230000009471 action Effects 0.000 title abstract description 5
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 claims description 81
- 208000015181 infectious disease Diseases 0.000 claims description 12
- 101100338269 Caenorhabditis elegans his-41 gene Proteins 0.000 claims description 8
- 235000018417 cysteine Nutrition 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 125000000539 amino acid group Chemical group 0.000 claims description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 3
- 239000000428 dust Substances 0.000 claims description 2
- 241000315672 SARS coronavirus Species 0.000 abstract description 57
- 102000005927 Cysteine Proteases Human genes 0.000 abstract description 56
- 108010005843 Cysteine Proteases Proteins 0.000 abstract description 56
- 239000003112 inhibitor Substances 0.000 abstract description 7
- 230000009385 viral infection Effects 0.000 abstract description 7
- 239000004593 Epoxy Substances 0.000 abstract 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical class OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 abstract 1
- 241000711573 Coronaviridae Species 0.000 description 41
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 33
- 241000700605 Viruses Species 0.000 description 32
- 241000711475 Feline infectious peritonitis virus Species 0.000 description 27
- 150000001875 compounds Chemical class 0.000 description 24
- 238000000034 method Methods 0.000 description 21
- 241000711466 Murine hepatitis virus Species 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 239000000758 substrate Substances 0.000 description 17
- -1 oligosaccharide compound Chemical class 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 14
- 102000035195 Peptidases Human genes 0.000 description 13
- 108091005804 Peptidases Proteins 0.000 description 13
- 229940079593 drug Drugs 0.000 description 13
- 235000002639 sodium chloride Nutrition 0.000 description 13
- DKMROQRQHGEIOW-UHFFFAOYSA-N Diethyl succinate Chemical compound CCOC(=O)CCC(=O)OCC DKMROQRQHGEIOW-UHFFFAOYSA-N 0.000 description 12
- 230000003197 catalytic effect Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 230000007062 hydrolysis Effects 0.000 description 12
- 238000006460 hydrolysis reaction Methods 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 11
- 241000711450 Infectious bronchitis virus Species 0.000 description 10
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 10
- 239000004365 Protease Substances 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 235000019419 proteases Nutrition 0.000 description 10
- 241000711467 Human coronavirus 229E Species 0.000 description 9
- SRVFFFJZQVENJC-IHRRRGAJSA-N aloxistatin Chemical compound CCOC(=O)[C@H]1O[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)NCCC(C)C SRVFFFJZQVENJC-IHRRRGAJSA-N 0.000 description 9
- 229940024606 amino acid Drugs 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 241000711443 Bovine coronavirus Species 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 244000309467 Human Coronavirus Species 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 208000005577 Gastroenteritis Diseases 0.000 description 6
- 108010076039 Polyproteins Proteins 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 238000002864 sequence alignment Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 241001292005 Nidovirales Species 0.000 description 5
- 125000000217 alkyl group Chemical group 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000000857 drug effect Effects 0.000 description 5
- 230000002458 infectious effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 206010003757 Atypical pneumonia Diseases 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 229910052736 halogen Inorganic materials 0.000 description 4
- 150000002367 halogens Chemical class 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000003032 molecular docking Methods 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- PNEYBMLMFCGWSK-UHFFFAOYSA-N Alumina Chemical compound [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 241000271566 Aves Species 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 125000002769 thiazolinyl group Chemical group 0.000 description 3
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 3
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 3
- GQEZCXVZFLOKMC-UHFFFAOYSA-N 1-hexadecene Chemical compound CCCCCCCCCCCCCCC=C GQEZCXVZFLOKMC-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 208000001528 Coronaviridae Infections Diseases 0.000 description 2
- LTLYEAJONXGNFG-DCAQKATOSA-N E64 Chemical compound NC(=N)NCCCCNC(=O)[C@H](CC(C)C)NC(=O)[C@H]1O[C@@H]1C(O)=O LTLYEAJONXGNFG-DCAQKATOSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 125000004450 alkenylene group Chemical group 0.000 description 2
- 239000007900 aqueous suspension Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 238000007622 bioinformatic analysis Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 239000002852 cysteine proteinase inhibitor Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000009931 harmful effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 238000000329 molecular dynamics simulation Methods 0.000 description 2
- 238000000324 molecular mechanic Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- LEACJMVNYZDSKR-UHFFFAOYSA-N 2-octyldodecan-1-ol Chemical compound CCCCCCCCCCC(CO)CCCCCCCC LEACJMVNYZDSKR-UHFFFAOYSA-N 0.000 description 1
- 101800000535 3C-like proteinase Proteins 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 241000867607 Chlorocebus sabaeus Species 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 241000342334 Human metapneumovirus Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-L L-tartrate(2-) Chemical compound [O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O FEWJPZIEWOKRBE-JCYAYHJZSA-L 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 102000007399 Nuclear hormone receptor Human genes 0.000 description 1
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010035742 Pneumonitis Diseases 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- WNLRTRBMVRJNCN-UHFFFAOYSA-L adipate(2-) Chemical compound [O-]C(=O)CCCCC([O-])=O WNLRTRBMVRJNCN-UHFFFAOYSA-L 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001447 alkali salts Chemical class 0.000 description 1
- DCAYPVUWAIABOU-UHFFFAOYSA-N alpha-n-hexadecene Natural products CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 1
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical compound [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000013528 artificial neural network Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- 229940050390 benzoate Drugs 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012928 buffer substance Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 239000007766 cera flava Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940001468 citrate Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108700010904 coronavirus proteins Proteins 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- GXGAKHNRMVGRPK-UHFFFAOYSA-N dimagnesium;dioxido-bis[[oxido(oxo)silyl]oxy]silane Chemical compound [Mg+2].[Mg+2].[O-][Si](=O)O[Si]([O-])([O-])O[Si]([O-])=O GXGAKHNRMVGRPK-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- VFNGKCDDZUSWLR-UHFFFAOYSA-L disulfate(2-) Chemical compound [O-]S(=O)(=O)OS([O-])(=O)=O VFNGKCDDZUSWLR-UHFFFAOYSA-L 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 229950005627 embonate Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 229950007655 esilate Drugs 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000000887 face Anatomy 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229940050411 fumarate Drugs 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-L fumarate(2-) Chemical compound [O-]C(=O)\C=C\C([O-])=O VZCYOOQTPOCHFL-OWOJBTEDSA-L 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229960005150 glycerol Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940102223 injectable solution Drugs 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical group CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 229940001447 lactate Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000391 magnesium silicate Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229940099273 magnesium trisilicate Drugs 0.000 description 1
- 229910000386 magnesium trisilicate Inorganic materials 0.000 description 1
- 235000019793 magnesium trisilicate Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 229940042472 mineral oil Drugs 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 108020004017 nuclear receptors Proteins 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 239000012053 oil suspension Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 150000003891 oxalate salts Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
- IUGYQRQAERSCNH-UHFFFAOYSA-M pivalate Chemical compound CC(C)(C)C([O-])=O IUGYQRQAERSCNH-UHFFFAOYSA-M 0.000 description 1
- 229950010765 pivalate Drugs 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 239000001818 polyoxyethylene sorbitan monostearate Substances 0.000 description 1
- 235000010989 polyoxyethylene sorbitan monostearate Nutrition 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229940113124 polysorbate 60 Drugs 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- SNOOUWRIMMFWNE-UHFFFAOYSA-M sodium;6-[(3,4,5-trimethoxybenzoyl)amino]hexanoate Chemical compound [Na+].COC1=CC(C(=O)NCCCCCC([O-])=O)=CC(OC)=C1OC SNOOUWRIMMFWNE-UHFFFAOYSA-M 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000001587 sorbitan monostearate Substances 0.000 description 1
- 235000011076 sorbitan monostearate Nutrition 0.000 description 1
- 229940035048 sorbitan monostearate Drugs 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000001562 sternum Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 229950004288 tosilate Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- DCEMCPAKSGRHCN-JCYAYHJZSA-N trans-2,3-epoxysuccinic acid Chemical class OC(=O)[C@@H]1O[C@H]1C(O)=O DCEMCPAKSGRHCN-JCYAYHJZSA-N 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 229940099259 vaseline Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000003871 white petrolatum Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to the use of SARS cysteine proteinase (SACP) as the action target of SARS virus infection preventing and/or treating medicine. The present invention also relates to trans-epoxy butanedioic acid derivative as SACP inhibitor for preventing and/or treating SARS virus infection.
Description
Technical field
The present invention relates to discern infectivity resistant atypical pneumonia (Severe Acute RespiratorySyndrome, SARS) drug effect target and prevent and/or treat medicine, the drug effect target of identification is that the coronavirus (Coronaviruses) that causes severe acute respiratory syndrome is duplicated, transcribe, necessary key protein hydrolytic enzyme (SARS cysteineproteinase in translation and the assembling process, SACP), preventing and/or treating medicine is organic molecular compound or polypeptide compound or oligosaccharide compound or monoclonal antibody or the nucleoside compound that acts on this target enzyme SACP, and contains their pharmaceutical composition.
Background technology
Severe acute respiratory syndrome be called severe acute respiratory syndrome (Severe AcuteRespiratory Sydrome, SARS).Discover, a kind of novel coronavirus is the cause of disease most probably, but other co-factors such as human world matter pneumonitis virus (humanmetapneumovirus might be arranged also, hMPV) make that sb.'s illness took a turn for the worse, in Hong Kong and Canadian patient and dead patient's corpse, all isolate 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.94% people who infects SARS virus can spontaneous recovery among the severe acute respiratory syndrome patient, and fatality rate is about 6%.Patient's SARS the respiratory tract section and the electron micrograph of cell culture show, have the coronavirus granule.This is a kind of novel coronavirus, different with other known members in the coronavirus genus, this virus can cause that the pathological changes 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 cultured 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; Infectiousness gastroenteritis virus (TransmissibleGastroenteritis Virus, TGEV), murine hepatitis virus (Murine Hepatitis Virus, MHV), feline infectious peritonitis virus (Feline Infectious Peritonitis virus, FIPV), 229E human corona virus's the super antiserum viral growth that can suppress to cultivate.Several laboratorys show that to the order-checking of virus new virus is relevant with coronavirus genus, but are different from generic other coronavirus 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 (ssRNApositive-strand 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 (SARSCoronaviruses SARS-CoV), and has successfully finished the genomic complete order-checking of SARS-CoV.The SARS-CoV genome has 29727 nucleotide, 11 open reading frame (Opening Reading Frames) are formed.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 (Rota et al. itself, Characterization of a Novel Coronavirus Associated with SevereAcute Respiratory Syndrome, Paul A.Published online 1 May 2003; 10.1126/science.1085952).Scientist has carried out the gene order-checking of relevant coronavirus Toronto (Tor2) separated strain 29751 bases of SARS simultaneously.Genome sequence disclose this coronavirus and known coronavirus (comprise human coronary virus HcoV-OC43, HCoV-229E) no closely related.The kind system of prediction virus protein the analysis showed that, 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 reservoir'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 (The Genome Sequence of theSARS-Associated Coronavirus, Marco A.Marra et al., Publishedonline 1 May 2003; 10.1126/science.1085953).
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 rate, the specific medicament that the SARS-CoV that can prevent and/or treat is infected has very urgent need clinically.
Summary of the invention
The objective of the invention is the gene expression product of SARS-CoV is carried out deep bioinformatic analysis, identification infectivity resistant atypical pneumonia (Severe Acute RespiratorySyndrome, SARS) pharmaceutically-active target and prevent and/or treat medicine.
Method by the utilization bioinformatics, the present invention has carried out complete sequence analysis to SARS-CoV coronavirus protein sequence, to 11 SARS-CoV BJ01, SARS-CoV BJ02, SARS-CoV BJ03, the SARS-CoV BJ04 that has checked order, the protein sequence of the SARS-CoV coronavirus of SARS-CoVCUHK-W1, SARS-CoV GZ01, SARS-CoV HKU-39849, SARS-CoVTaiwan, SARS-CoV Tor2, SARS-CoV Urbani and SARS-CoV Vietnam carries out the multisequencing compare of analysis, found that one section sequence in the SARS-CoV virus:
1 11 21 31 41 51
| | | | | |
1 SGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDTVYCPR HVICTAEDML NPNYEDLLIR 60
61 KSNHSFLVQA GNVQLRVIGH SMQNCLLRLK VDTSNPKTPK YKFVRIQPGQ TFSVLACYNG 120
121 SPSGVYQCAM RPNHTIKGSF LNGSCGSVGF NIDYDCVSFC YMHHMELPTG VHAGTDLEGK 180
181 FYGPFVDRQT AQAAGTDTTI TLNVLAWLYA AVINGDRWFL NRFTTTLNDF NLVAMKYNYE 240
241 PLTQDHVDIL GPLSAQTGIA VLDMCAALKE LLQNGMNGRT ILGSTILEDE FTPFDVVRQC 300
301 SGVTFQ
Conservative at all SARS-CoV virus camber, this section sequence is identical in 11 SARS-CoV coronavirus that all have checked order.The result of sequence alignment shows that this albumen does not have independently signal peptide, it is to be one of 7074 amino acid whose polyproteins (putative orflab polyprotein) independently functional domain since the 250th nucleotide code length, and this functional domain is positioned at the amino acid whose position, 3241 aminoacid positions to 3547 of 7074 aminoacid polyproteins of SARS-CoV virus.The present invention with this albumen be defined as the SARS cysteine proteinase (SARS Cysteine Proteinase, SACP).The corresponding segment of this polyprotein and murine hepatitis virus (murinehepatitis virus) has homology.
The protein sequence of the coronavirus of the protein sequence of SARS-CoV coronavirus and other kinds is carried out the multisequencing compare of analysis, correlated sequence comprises: infectiousness gastroenteritis virus (Transmissible Gastroenteritis Virus, TGEV), murine hepatitis virus (MurineHepatitis Virus, MHeV), bovine coronavirus (bovine coronavirus, BcoV), feline infectious peritonitis virus (Feline Infectious Peritonitis virus, FIPV), human corona virus 229E (human coronavirus 229E, HcoV), Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) and avian infectious bronchitis virus (Avian Infectious Bronchiyis Virus, AIBV).Comparing result and homology thereof are as follows, the method for sequence contrast and homology analysis employing be the protein sequence homology comparison method that provides of American National bio information center (NCBI) (NCBI, BLASTp).
1, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to murine hepatitis virus (Murine Hepatitis Virus, MHeV), identical sequence (Identities)=154/306 (homogeny 50%), homologous sequence (Positives)=198/306 (homology 64%), sequence gap (Gaps)=3/306 (0%).
SACP：1 SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDMLNPNYEDLLIR 60
SG KM P+ KVE C+V VT G TLNGLWLDD VYCPRHVIC++ DM +P+Y +LL R
MHeV：1 SGIVKMVSPTSKVEPCIVSVTYGNMTLNGLWLDDKVYCPRHVICSSADMTDPDYPNLLCR 60
SACP：61 KSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYNG 120
++ F V +G + L V+ + MQ C L L V NP TPKY F ++PG+TF+VLA YNG
MHeV：61 VTSSDFCVMSGRMSLTVMSYQMQGCQLVLTVTLQNPNTPKYSFGVVKPGETFTVLAAYNG 120
SACP：121 SPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGK 180
P G + +R +HTIKGSFL GSCGSVG+ + D V F YMH +EL TG H GTD G
MHeV：121 RPQGAFHVTLRSSHTIKGSFLCGSCGSVGYVLTGDSVRFVYMHQLELSTGCHTGTDFSGN 180
SACP：181 FYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYE 240
FYGP+ D Q Q D T T+NV+AWLYAA+ N WF+ + +L +FN+ AM +
MHeV：181 FYGPYRDAQVVQLPVQDYTQTVNVVAWLYAAIFNRCNWFVQSDSCSLEEFNVWAMTNGFS 240
SACP：241 PLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC 300
+D V L L++ TG+ V + AA+K L+G G+ ILGS +LEDE TP DV +Q
MHeV：241 SIKADLV--LDALASMTGVTVEQVLAAIKR-LHSGFQGKQILGSCVLEDELTPSDVYQQL 297
SACP：301 SGVTFQ 306
+GV Q
MHeV：298 AGVKLQ 303
2, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to bovine coronavirus (bovine coronavirus, BcoV), identical sequence (Identities)=149/306 (homogeny 48%), homologous sequence (Positives)=196/306 (homology 64%), sequence gap (Gaps)=3/306 (0%).
SACP：1 SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDMLNPNYEDLLIR 60
SG KM P+ KVE C+V VT G TLNGLWLDD VYCPRHVIC+A DM NP+Y +LL R
BcoV：3247 SGIVKMVNPTSKVEPCIVSVTYGNMTLNGLWLDDKVYCPRHVICSASDMTNPDYTNLLCR 3306
SACP：61 KSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYNG 120
++ F V +L V+ + MQ C+L L V N +TPKY F ++PG+TF+VLA YNG
BcoV：3307 VTSSDFTVLFDRLSLTVMSYQMQGCMLVLTVTLQNSRTPKYTFGVVKPGETFTVLAAYNG 3366
SACP：121 SPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGK 180
P G +MR ++TIKGSFL GSCGSVG+ I DCV F YMH+EL TG H GTD G
BcoV：3367 KPQGAFHVTMRSSYTIKGSFLCGSCGSVGYVIMGDCVKFVYMHQLELSTGCHTGTDFNGD 3426
SACP：181 FYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYE 240
FYGP+ D Q Q D ++N +AWLYAA++N WF+ ++ DFN+ A+ +
BcoV：3427 FYGPYKDAQVVQLPVQDYIQSVNFVAWLYAAILNNCNWFVQSDKCSVEDFNVWALSNGFS 3486
SACP：241 PLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC 300
+D V+L++TG++++AA+K L+NG GR I+GS EDE TP DV+Q
BcoV：3487 QVKSDLV--IDALASMTGVSLETLLAAIKR-LKNGFQGRQIMGSCSFEDELTPSDVYQQL 3543
SACP：301 SGVTFQ 306
+G+ Q
BcoV：3544 AGIKLQ 3549
3, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to Porcine epidemic diarrhea virus (Poreine Epidemic Diarrhea Virus, PEDV), identical sequence (Identities)=137/306 (homogeny 44%), homologous sequence (Positives)=199/306 (homology 64%), sequence gap (Gaps)=6/306 (1%).
SACP：1 SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTA-EDMLNPNYEDLLI 59
+G RKMA PSG VE C+V+V G LNGLWL D V CPRHVI ++ ++ +Y ++
REDV：2998 AGLRKMAQPSGVVEKCIVRVCYGNMALNGLWLGDIVMCPRHVIASSTTSTIDYDYALSVL 3057
SACP：60 RKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYN 119
R H+F + +GNV L V+ +M+ LL++KV+ +N TPKY + ++PG++F++LACY+
PEDV：3058 RL--HNFSISSGNVFLGVVSATMRGALLQIKVNQNNVHTPKYTYRTVRPGESFNILACYD 3115
SACP：120 GSPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEG 179
G+ +GVY MR N+TI+GSF+NG+CGS G+NI+ V FCY+H+EL+G H G+DL+G
PEDV：3116 GAAAGVYGVNMRSNYTIRGSFINGACGSPGYNINNGTVEFCYLHQLELGSGCHVGSDLDG 3175
SACP：180 KFYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNY 239
YG + D+ T Q G + T NVLA+LYAA+ING W+L+ ++ FN A+
PEDV：3176 VMYGGYEDQPTLQVEGASSLFTENVLAFLYAALINGSTWWLSSSRIAVDRFNEWAVHNGM 3235
SACP：240 EPLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQ 299
T+D L+A+TG+ V + A+++ L +N G+ ILG T L DEFT +VVRQ
PEDV：3236 T--TVGNTDCFSILAAKTGVDVQRLLASIQSLHKN-FGGKQILGHTSLTDEFTTGEVVRQ 3292
SACP：300 CSGVTFQ 306
GV Q
PEDV：3293 MYGVNLQ 3299
4, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to infectiousness gastroenteritis virus (Transmissible Gastroenteritis Virus, TGEV), identical sequence (Identities)=134/306 (homogeny 43%), homologous sequence (Positives)=186/306 (homology 60%), sequence gap (Gaps)=4/306 (1%).
SACP：1 SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDMLNPNYEDLLIR 60
SG RKMA PSG VE C+V+V+ G LNGLWL D V CPRHVI + + NYE+ +
TGEV：2879 SGLRKMAQPSGLVEPCIVRVSYGNNVLNGLWLGDEVICPRHVIASDTTRV-INYENEMSS 2937
SACP：61 KSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYNG 120
H+F V NV L V+ + L LKV+ NP TP++KF I+ G++F++LACY G
TGEV：2938 VRLHNFSVSKNNVFLGVVSARYKGVNLVLKVNQVNPNTPEHKFKSIKAGESFNILACYEG 2997
SACP：121 SPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGK 180
P VY MR TIKGSF+ G+CGSVG+ ++ + F YMHH+EL G H G++ EG+
TGEV：2998 CPGSVYGVNMRSQGTIKGSFIAGTCGSVGYVLENGILYFVYMHHLELGNGSHVGSNFEGE 3057
SACP：181 FYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYE 240
YG + D+ + Q GT+ + NV+A+LYAA+ING+RWF+ + +L +N A ++
TGEV：3058 MYGGYEDQPSMQLEGTNVMSSDNVVAFLYAALINGERWFVTNTSMSLESYNTWAKTNSFT 3117
SACP：241 PLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC 300
L+ D L+A+TG +V + ++ L G GRTIL L DEFTP +V+RQ
TGEV：3118 ELSS--TDAFSMLAAKTGQSVEKLLDSIVR-LNKGFGGRTILSYGSLCDEFTPTEVIRQM 3174
SACP：301 SGVTFQ 306
GV Q
TGEV：3175 YGVNLQ 3180
5, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to feline infectious peritonitis virus (Feline Infectious Peritonitis virus, FIPV), identical sequence (Identities)=134/306 (homogeny 43%), homologous sequence (Positives)=184/306 (homology 60%), sequence gap (Gaps)=4/306 (1%).
SACP：1 SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDMLNPNYEDLLIR 60
SG RKMA PSG VE C+V+V G LNGLWL D V CPRHVI + + NYE+ L
FIPV：11 SGLRKMAQPSGVVEPCIVRVAYGNNVLNGLWLGDEVICPRHVIASDTSRV-INYENELSS 69
SACP：61 KSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYNG 120
H+F + N L V+ + L LKV+ NP TP++KF ++PG++F++LACY G
FIPV：70 VRLHNFSIAKNNAFLGVVSAKYKGVNLVLKVNQVNPNTPEHKFKSVRPGESFNILACYEG 129
SACP：121 SPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGK 180
P VY MR TIKGSF+ G+CGSVG+ ++ + F YMHH+EL G H G++LEG+
FIPV：130 CPGSVYGVNMRSQGTIKGSFIAGTCGSVGYVLENGTLYFVYMHHLELGNGSHVGSNLEGE 189
SACP：181 FYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYE 240
YG + D+ + Q GT+ + NV+A+LYAA+ING+RWF+ + TL +N A ++
FIPV：190 MYGGYEDQPSMQLEGTNVMSSDNVVAFLYAALINGERWFVTNTSMTLESYNAWAKTNSFT 249
SACP：241 PLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC 300
+ D L+A+TG +V + + L G GRTIL L DEFTP +V+RQ
FIPV：250 EIVS--TDAFNMLAAKTGYSVEKLLECIVR-LNKGFGGRTILSYGSLCDEFTPTEVIRQM 306
SACP：301 SGVTFQ 306
GV Q
FIPV：307 YGVNLQ 312
6, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to human corona virus 229E (human coronavirus 229E, HcoV), identical sequence (Identities)=124/306 (homogeny 40%), homologous sequence (Positives)=186/306 (homology 60%), sequence gap (Gaps)=6/306 (1%).
SACP：1 SGFRKMAFPSGKVEGCMWQVTCGTTTLNGLWLDDTVYCPRHVICT-AEDMLNPNYEDLLI 59
+G RKMA PSG VE C+V+V G T LNGLWL D VYCPRHVI + ++ ++E ++
HcoV：2966 AGLRKMAQPSGFVEKCVVRVCYGNTVLNGLWLGDIVYCPRHVIASNTTSAIDYDHEYSIM 3025
SACP：60 RKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYN 119
R H+F + +G L V+G+M L++KV +N TP++ F ++ G+ F++LACY+
HcoV：3026 RL--HNFSIISGTAFLGVVGATMHGVTLKIKVSQTNMHTPRHSFRTLKSGEGFNILACYD 3083
SACP：120 GSPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEG 179
G GV+ MR N TI+GSF+NG+CGS G+N+ V F YMH +EL +G H G+ +G
HcoV：3084 GCAQGVFGVNMRTNWTIRGSFINGACGSPGYNLKNGEVEFVYMHQIELGSGSHVGSSFDG 3143
SACP：180 KFYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNY 239
YG F D+ Q + +T+NV+A+LYAA++NG W+L + +N A +
HcoV：3144 VMYGGFEDQPNLQVESANQMLTVNVVAFLYAAILNGCTWWLKGEKLFVEHYNEWAQANGF 3203
SACP：240 EPLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQ 299
+ + D L+A+TG+ V + A+ ++L NG G+ILG +L DEF+ +VV+Q
HcoV：3204 TAMNGE--DAFSILAAKTGVCVERLLHAI-QVLNNGFGGKQILGYSSLNDEFSINEVVKQ 3260
SACP：300 CSGVTFQ 306
GV Q
HcoV：3261 MFGVNLQ 3267
7, SARS cysteine proteinase (SARS Cysteine Proteinase, SACP) to avian infectious bronchitis virus (Avian Infectious Bronchiyis Virus, AIBV), identical sequence (Identities)=127/316 (homogeny 40%), homologous sequence (Positives)=179/316 (homology 56%), sequence gap (Gaps)=19/316 (6%).
SACP：1 SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDMLNPNYEDLLIR 60
+GF+K+A PS VE C+V V+ LNGLWL D++YCPRHV+ ++D+L
AIBV：2799 AGFKKLAAPSSAVEKCIVSVSYRGNNLNGLWLGDSIYCPRHVL---GKFSGDQWDDVLNL 2855
SACP：61 KSNHSFLVQAGN-VQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYN 119
+NH F V GN V L V+ ++ +L L+ +N +TPKYKF++ G +F++ Y
AIBV：2856 ANNHEFEVVTGNGVTLAVVSRRLRGAVLILQTAIANAETPKYKFMKANCGDSFTIACSYG 2915
SACP：120 GSPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEG 179
G+ G+Y MR N TI+ SFL G+CGSVGFNI+ V+F YMHH+ELP +H GTDL G
AIBV：2916 GTVIGLYPVTMRSNGTIRASFLAGACGSVGFNIERGVVNFYYMHHLELPNALHTGTDLNG 2975
SACP：180 KFYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVI-------NGDRWFLNRFTTTLNDFNL 232
FYG VD+AQ D +T N++AWLYAA+I + +W L T +++D+N
AIBV：2976 AFYGGCVDEEVAQRVPPDNLVTNNIVAWLYAAIISVKESSFSTPKW-LESTTVSVDDYNK 3034
SACP：233 VAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCAALKELL--QNGMNGRTILGSTILEDE 290
A + P + + LSA TG+ D+C L+ ++ + ILG EDE
AIBV：3035 WAGDNGFTPFSTSTA--ITKLSAITGV---DVCKLLRTIMVKSSQWGNEPILGQYNFEDE 3089
SACP：291 FTPFDVVRQCSGVTFQ 306
TP V Q GV Q
AIBV：3090 LTPESVFNQVGGVRLQ3105
Find each sequence and SARS cysteine proteinase (SARS Cysteine Proteinase by these sequence contrasts and homology analysis, SACP) homology is very high, the sequence homogeny all greater than 40%, homology all greater than 55%, the sequence gap is all less than 6%, points out the albumen of these sequences representatives to carry out similar function in virus replication.
Find that by sequence contrast and homology analysis the coronavirus (Coronaviridae) that belongs in nido virus (Nidovirales) family of positive chain RNA virus (ssRNA positive-strandviruses) family with the homologous sequence of SARS cysteine proteinase SACP is on kind is classified.The sequence homology comparison does not find that the albumen of other family and the proteic homology of SACP surpass 10%.Show that these albumen are distinctive key proteins in nido virus (Nidovirales) family.
According to people (John Ziebuhr et al. such as John Ziebuhr, Virus-encodedproteinases and proteolytic processing in the Nidovirales.J Gen Virol.2000,81:853-79.) be to comprise infectiousness gastroenteritis virus (Transmissible Gastroenteritis Virus to the coronavirus in the Nidovirales family (Coronaviridae), TGEV), murine hepatitis virus (Murine Hepatitis Virus, MHeV), bovine coronavirus (bovinecoronavirus, BcoV), feline infectious peritonitis virus (Feline Infectious Peritonitisvirus, FIPV), human corona virus 229E (human coronavirus 229E, HcoV) and avian infectious bronchitis virus (Avian Infectious Bronchiyis Virus, AIBV) analysis result, belong to cysteine proteinase (CysteineProteinase is also referred to as 3C-like Proteinase) with the sequence of SACP albumen homology.
The function of cysteine proteinase is in the coronavirus family: this protease is the distinctive albumen of a class that the coronavirus genome encoding is expressed, and compares homology with the similar protease of other non-coronavirus and cellular expression and similarity is all extremely low.It and coronavirus rna replicon enzyme synthetic has extremely closely gets in touch, its prominent features be in sophisticated virion not RNA polymerase exist, but after virus is invaded host cell, just synthesize.After invading host cell, coronavirus is at first by the open reading frame expressed rna replicative enzyme precursor that accounts for its genome nearly 2/3rds, this precursor contains 7200 aminoacid of having an appointment, and then the effect of the cysteine proteinase of expressing at virus self coding is following separates into more than ten and have functional protein, and coronavirus is finished duplicating, transcribe, translate and assembling of self under these proteic regulation and control.Therefore the cysteine proteinase of coronavirus duplicating, transcribe, translating and assembling and all play crucial effects to virus.
According to sequence alignment result and SARS cysteine proteinase (SARS CysteineProteinase, SACP) prompting of other proteolytic enzyme function in the homology coronavirus family, the present invention asserts the function of SARS cysteine proteinase SACP, SARS cysteine hydrolysising protease SACP is an albumen that contains 306 amino acid residues, and wherein about 110 residues of c-terminus have significant effects to its catalyzing hydrolysis function.The cysteine proteinase difference of its catalyzing hydrolysis avtive spot and other coronavirus family is to lack the catalysis triplet His of high conservative, Asp (or Glu), acidic residues Asp among the Cys (or Glu), His provides basic group for catalytic site in SARS cysteine hydrolysising protease, and Cys provides nucleophilic group for catalytic site.Its substrate land is similar to the cysteine proteinase of other coronavirus family, but the triplet as catalytic core in the substrate combination: become Tyr-X-His by Gly-X-His, the His residue that is absolutely necessary wherein, the variation in this site will cause active forfeiture.
The substrate of the main catalyzing hydrolysis of SARS cysteine proteinase SACP is characterized as, the two peptide bond sites that formed by Gln|Ser, Gln|Ala, GIn|Asn, Gln|Gly and Gln|Cys (| representative be connected with peptide bond).
On the basis of assert SARS cysteine proteinase SACP function, the method that the present invention adopts secondary structure prediction and homology mould to build has been set up secondary structure and the three-D space structure of SARS cysteine proteinase SACP.
Neural net method (Rost B, the predicting one-dimensional protein structure by profile-based neuralnetworks.Methods Enzymol 1996 of people's development such as secondary structure prediction employing Rost; 266:525-39) predict the outcome and see accompanying drawing 1.Predict the outcome and show that SARS cysteine proteinase structure adds the β lamellar structure for the α spiral.
The method that the foundation of three-D space structure uses Clustal X sequence comparison software version 1.81 to provide is carried out the sequence search contrast, found that the proteic A chain of infectiousness gastroenteritis virus TGEV, the three-dimensional structure database code is that the sequence homology of PDB:1LVO and SARS cysteine proteinase SACP is 46.5%, the sequence of the two is compared (PDB:1LVOA chain-ordering and NCBI:NP_828863.1 sequence), identify the conservative and homologous zone of sequence between the two, the result is presented at accompanying drawing 2.
With TGEV protein A chain structure PDB:1LVO A is template, and the method mould of building with the homology mould is built the three-D space structure of SARS cysteine proteinase SACP, the results are shown in accompanying drawing 3.SACP is similar to the three dimensional structure of TGEV protein A chain, comprises three functional areas in the structure, comprising two β lamellar structure enrichment region D1, D2 and a αLuo Xuanjiegou enrichment region D3.Flexible region between D1 and D2 is catalytic activity site and the substrate land of SACP.
The present invention finds among the SARS cysteine proteinase SACP that crucial avtive spot is No. 41 histidine His41 and No. 145 cysteine Cys145, and the distance between sulphur atom between them and ε 2 nitrogen-atoms is 3.93 dusts.Distance between Asp186, Arg40 is 2.53 dusts, can generate rational salt bridge between Asp186 and Arg40.What accompanying drawing 4 showed is SACP and TGEV avtive spot situation relatively.
Substrate binding site and the TGEV of SACP are close, and the two can overlap substantially preferably.The TGEV substrate binding site constitutes by being numbered 18 aminoacid such as 41,47,51,139,160,161,162,163,164,165,166,167,171,186,187,188,189,190, the contrast of SACP and TGEV catalytic activity residue sees Table 1, in the β of SACP structure lamellar structure enrichment region D1, it is little that the residue relevant with catalytic activity constitutes the variation of the first active subunit district (seeing Table 1), only there is a site to undergo mutation, become Leu by Ile, little to the active influence of the catalyzing hydrolysis of SACP.It is more obvious that the residue relevant with catalytic activity constitutes the variation that the second active subunit district (seeing Table 1) takes place in the β of SACP structure lamellar structure enrichment region D2, there are 4 residues that variation (seeing Table 1) has taken place in 6 residues, but these variant sites residue side chains all point to the direction opposite with the land, and the geometry of substrate binding site is not produced obvious change.
The contrast of table 1SACP and TGEV catalytic activity residue
The bioinformatic analysis that one aspect of the present invention is undertaken by the protein sequence to SARS-CoV, having found one, to have catalyzing hydrolysis be the SARS cysteine proteinase SACP of feature with Gln|Ser, Gln|Ala, Gln|Asn, Gln|Gly, Gln|Cys two peptide bonds, and its sequence signature is:
1 11 21 31 41 51
| | | | | |
1 SGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDTVYCPR HVICTAEDML NPNYEDLLIR 60
61 KSNHSFLVQA GNVQLRVIGH SMQNCLLRLK VDTSNPKTPK YKFVRIQPGQ TFSVLACYNG 120
121 SPSGVYQCAM RPNHTIKGSF LNGSCGSVGF NIDYDCVSFC YMHHMELPTG VHAGTDLEGK 180
181 FYGPFVDRQT AQAAGTDTTI TLNVLAWLYA AVINGDRWFL NRFTTTLNDF NLVAMKYNYE 240
241 PLTQDHVDIL GPLSAQTGIA VLDMCAALKE LLQNGMNGRT ILGSTILEDE FTPFDVVRQC 300
301 SGVTFQ
Another aspect of the present invention relates to the reproduction process of cysteine proteinase SACP control virus, the proteic function of blocking-up SACP catalyzing hydrolysis, can block duplicating, transcribe, translate and assembling of SARS virus, the drug research purposes that can be used for the treatment of and/or prevent SARS virus to infect discloses SARS cysteine proteinase SACP and treats and/or prevents the action target that SARS virus infects specific medicament.Promptly the present invention relates to the purposes of SACP in screening prevention and/or SARS virus medicine.
The sequence relative analysis of system of the present invention confirms not have the homology of other family albumen and SACP to surpass 10%.This shows that SACP albumen is the distinctive crucial catalyzing hydrolysis protease of SARS virus, the function of blocking it can not produce harmful effect to the normal physiological process of the human or animal who infects SARS virus, announcement can be developed the specific medicament of high-efficiency low-toxicity side effect based on this new target drone of SARS cysteine proteinase SACP.
According to the result of above bioinformatics test, the present invention determines that SARS cysteine proteinase SACP treats and/or prevents the drug effect target that SARS virus infects.
Target is meant the key function albumen or the functional gene that can play a decisive role in disease generation and the evolution on most critical pathology link, comprise cell-membrane receptor, protease, hormone and cytokine, ion 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 therapeutical effect.This novel drugs based on mechanism and structure finds optionally to block the pathology link of most critical in disease generation and the evolution, develops the specific drug of high-efficiency low-toxicity side effect.The SARS cysteine proteinase SACP that the present invention relates to is as the purposes that treats and/or prevents SARS virus infection medicine action target.
The present invention relates on the other hand according to the drug effect target SARS cysteine proteinase SACP that determines, adopts the branch submodule to build the three-D space structure that SACP is set up in test, and determines the proteic avtive spot of its catalyzing hydrolysis.Be that SACP functional area of the present invention refers to that the SACP sequence numbering is the zone that 41,47,51,139,160,161,162,163,164,165,166,167,171,186,187,188,189, No. 190 amino acid residue constituted.Say that further SACP functional area of the present invention is that No. 41 histidine and sequence numbering is that No. 145 cysteine constitute by sequence numbering preferably, the distance that constitutes between ε 1 nitrogen-atoms of the sulphur atom of functional area Cys145 and His41 is the 3.0-6.0 dust.
The invention still further relates to the inhibitor of SACP, it comprise can with the material of SACP functional area complementary structure.Described material is optional from organic compound, polypeptide compound, and oligosaccharide compound, monoclonal antibody or nucleoside compound preferably have organic compounds or polypeptide.
The inventor has carried out treating and/or preventing the screening study of SARS virus infection medicine according to above-mentioned discovery, discovery has the trans Epoxysuccinic acid derivatives of general formula I can be specifically and target cysteine proteinase SACP complementary structure (complementary structure is seen accompanying drawing 5), the duplicating, transcribe, translate and assemble of SARS virus of target cysteine proteinase SACP mediation can be blocked, the purposes of SARS virus infection can be used for the treatment of and/or prevent.
U.S. Pat 3911111, US4333879 and US4393228 have disclosed the chemical compound that contains general formula I can be used for inhibitor (Neuronal injury inhibitor) as nerve injury and thiol protease inhibitor (Thiol protease inhibitor) and preparation method thereof.Japan Patent JP55115878 and JP60174777 have disclosed the new preparation method of chemical compound that contains general formula I.The inventor finds that in experiment the chemical compound that contains general formula I comprises its raceme or optical isomer or its officinal salt or hydrate, targeting SARS cysteine proteinase SACP specifically, thus can be used for inhibitor as SACP
Therefore, further aspect of the present invention relates to formula I chemical compound and is used for preventing and/or treating the medicine purposes that SARS virus infects in preparation.
Wherein:
R
1And R
2Independently be selected from hydrogen, C
1～C
4The straight or branched alkyl, C
2～C
4The straight or branched thiazolinyl, C
3～C
8Cycloalkyl, C
5～C
8Cycloalkenyl group can not have replacement on alkyl wherein or the alkenylene chain, can be selected from one or more following group yet and replace: halogen, C
3～C
8Cycloalkyl, C
5～C
7Cycloalkenyl group, aromatic carbocyclic ring wherein can be monocycle or dicyclo, and each ring is made up of 5～6 carbon, can not have replacement on the ring, also can be selected from following substituent group by 1-3 and replace: halogen, and nitro, methylol, trifluoromethyl, trifluoromethoxy,
R
3And R
4Independently be selected from hydrogen, C
1～C
8The straight or branched alkyl, C
2～C
8The straight or branched thiazolinyl, C
3～C
8Cycloalkyl, C
5～C
8Cycloalkenyl group, hydroxyl, C
1～C
4Alkoxyl, C
1～C
4Alkylamino can not have replacement on alkyl wherein or the alkenylene chain, can be selected from one or more following group yet and replace: halogen, phenyl, hydroxyl, C
3～C
8Cycloalkyl, C
5～C
7Cycloalkenyl group, nitro, methylol, trifluoromethyl, trifluoromethoxy, R
3And R
4Form 5～6 yuan heterocycle together, or form 5～6 yuan heterocycle, can not have replacement on the ring, also can be selected from following substituent group and replace: halogen by 1-3 with nitrogen-atoms, oxygen atom, nitro, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, '-diphosphono, C
1～C
6The straight or branched alkyl, C
2～C
6The straight or branched thiazolinyl, C
1～C
4Alkoxyl, C
2～C
4Alkene oxygen base, phenoxy group, benzyloxy, sulfoamido, sulfonyloxy methyl amido, dimethyl methyl amide groups.
The present invention also relates on the other hand and is used to prevent and/or treat the pharmaceutical composition that SARS infects, and it comprises at least a compound of Formula I or its pharmaceutical salts or its hydrate and pharmaceutical carrier or excipient.
The invention still further relates to the method that SARS infects that prevents and/or treats, it comprises needs the above-mentioned patient who prevents and/or treats with preventing and/or treating at least a formula I chemical compound of effective dose or its pharmaceutical salts or its hydrate.
The invention still further relates to its acceptable salt pharmaceutically of formula I chemical compound, it comprises inorganic salt or organic salt, and it includes but not limited to: alkali metal salt, alkali salt, hydrochlorate, hydrobromate, hydriodate, nitrate, sulfate, disulfate, phosphate, hydrophosphate, acetate, propionate, butyrate, oxalates, pivalate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, mesylate, esilate, benzene sulfonate, tosilate and embonate.
According to the present invention, the chemical compound below formula I compound or pharmaceutically acceptable salt thereof of the present invention or hydrate are preferred:
Wherein most preferred is Loxistatin, (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate
Another aspect of the present invention relates to pharmaceutical composition, and it contains raceme or the optical isomer and at least a pharmaceutically acceptable carrier of The compounds of this invention, and it can be used for interior therapeutic and has biocompatibility.Described pharmaceutical composition can be prepared into various forms according to different way of administration.The mentioned chemical compound of the present invention also can be prepared to the acceptable salt of various pharmacy, the said pharmaceutical composition of the present invention can be used to prevent and/or treat because the infectious atypical pneumonia that causes of coronavirus infection (Severe Acute RespiratorySyndrome, SARS).
Can being used to of the present invention relates to prevents and/or treats since the infectious atypical pneumonia that coronavirus infection causes (Severe Acute Respiratory Syndrome, pharmaceutical composition SARS) are meant formula I compound or pharmaceutically acceptable salt thereof of the present invention or hydrate and one or more the suitable pharmaceutically suitable carrier that comprises effective dose.The pharmaceutical carrier here includes but not limited to: ion-exchanger, aluminium oxide, aluminium stearate, lecithin, serum albumin such as human albumin, buffer substance such as phosphate, glycerol, sorbic acid, potassium sorbate, the partial glycerol ester admixture of saturated vegetable fatty acid, water, salt or electrolyte, as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, cabosil, magnesium trisilicate, polyvinylpyrrolidone, cellulosic material, Polyethylene Glycol, sodium carboxymethyl cellulose, polyacrylate, Cera Flava, lanoline.
The present invention relates to block the catalyzing hydrolysis protein function of SACP, block duplicating, transcribe, translate and assembling of SARS virus, the function of blocking-up SACP protease can not produce harmful effect to the normal physiological process of the human or animal who infects SARS virus, the medical usage that can be used for the treatment of and/or prevent SARS virus to infect.
The pharmaceutical composition of The compounds of this invention can be used with following any-mode: oral, spraying sucks, rectal application, nasal cavity applied medicine, buccal medication, local application, non-enterally administer, as subcutaneous, vein, intramuscular, intraperitoneal is in the sheath, in the ventricle, breastbone interior and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Preferred oral when wherein preventing SARS virus to infect, preferred intraperitoneal or intravenous administration mode when the treatment SARS virus infects.
When medicine for oral use, The compounds of this invention can be made into oral acceptable dosage form arbitrarily, includes but not limited to tablet, capsule, aqueous solution or water slurry.Wherein, the carrier that tablet uses generally comprises lactose and corn starch, also can add lubricant such as magnesium stearate in addition.The diluent that capsule preparations uses generally comprises lactose and dried corn starch.Aqueous suspension preparation then normally mixes use with active component with examples of suitable emulsifiers and suspending agent.If desired, also can add some sweeting agents, aromatic or coloring agent in the above oral formulations form.
When local medication, particularly treat local external application easy to reach and suffer from face or organ, during as eyes, skin or lower intestinal tract nervous system disease, can The compounds of this invention be made different local application's dosage forms, specify as follows according to different trouble faces or organ:
When the eye local application, The compounds of this invention can be mixed with the dosage form of a kind of micronization suspension or solution, and the carrier that uses is the Sterile Saline of isoosmotic certain pH, wherein can add also not adding preservative agent such as zephiran chloride alkoxide.For eye usefulness, also chemical compound can be made unguentum form such as vaseline paste.
When topical application, The compounds of this invention can be made into suitable ointment, lotion or cream dosage form, wherein active component is suspended or is dissolved in one or more carriers.The spendable carrier of ointment formulation includes but not limited to: mineral oil, Albolene, white vaseline, propylene glycol, polyethylene glycol oxide, polypropylene oxide, emulsifing wax and water; The spendable carrier of lotion or cream includes but not limited to: mineral oil, and sorbitan monostearate, polysorbate60, the hexadecane ester type waxes, hexadecene is fragrant and mellow, 2-octyldodecanol, benzyl alcohol and water.
The all right aseptic injection preparation form medication of The compounds of this invention comprises aseptic injection water or oil suspension or aseptic injectable solution.Wherein, spendable carrier and solvent comprise water, Ringer's mixture and isotonic sodium chlorrde solution.In addition, the fixed oil of sterilization also can be used as solvent or suspension media, as monoglyceride or two glyceride.
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 order of severity of disease and diagnosis and treatment doctor's the subjective judgment of patient's age, body weight, sex, natural health situation, nutriture, chemical compound.Preferred using dosage is between 0.01～100mg/kg body weight/day, and wherein optimal dosage is in the 1mg/kg-50mg/kg body weight/day.
Description of drawings
The secondary structure prediction of Fig. 1 .SARS cysteine proteinase SACP
The sequence alignment of Fig. 2 .SARS cysteine proteinase SACP and TGEV 1LVO, " * " number represents identical residue site among the figure, the high residue site of ": " expression conservative; ". " number represents the lower slightly residue site of conservative, and curve is represented the accuracy rate of sequence alignment.
Fig. 3. the three-D space structure of the SARS cysteine proteinase SACP that mould is built
The contrast of Fig. 4 .SARS cysteine proteinase SACP and TGEV space structure avtive spot, among the SACP, No. 41 histidine of the avtive spot of the key of SACP and TGEV (His41) and No. 145 cysteine Cys145 can overlap preferably as we can see from the figure
Fig. 5. medicine (+)-(2S; 3S)-2; 3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] molecular docking of ethyl succinate and SARS cysteine proteinase SACP; figure Chinese medicine (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate represents with mallet figure; the Key residues His41 of avtive spot; Cys145, His163 represent that with bar graph cysteine proteinase SACP represents with ribbon figure.
Fig. 6. the SARS 3CL that rp-hplc method bacterial detection is expressed
ProActive HPLC collection of illustrative plates.RT=10.7's is the peak of substrate, RT=7.6 and 8.5 be that substrate is by SARS 3CL
ProCracked primary product peak.
Fig. 7. rp-hplc method detection of drugs (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate is to the SARS3CL of bacterial expression
ProSuppress active HPLC collection of illustrative plates.RT=10.7's is the peak of substrate, RT=7.6 and 8.5 be that substrate is by SARS 3CL
ProCracked primary product peak.
Embodiment
The following examples are illustrative preferred embodiments of the present invention, and the present invention is not constituted any limitation.
Embodiment 1: the multiple comparing result of people SARS-CoV coronavirus SACP protein sequence
Correlated sequence source is from American National bio information center NCBI, they comprise that respectively from the SARS case specimen of different regions the NCBI accession number is gi|30275668 (BJ01), gi|30027618 (Vietnam), gi|29836495 (Tor2), gi|30023962 (CUHK-W1), 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 contrast, the result shows that the SACP sequence is identical in all SARS-CoV coronavirus of having checked order.The result of sequence alignment shows that SACP albumen does not have independently signal peptide, it is to be one of 7074 amino acid whose polyproteins independently functional domain since the 250th nucleotide code length, and this functional domain is positioned at 3241 aminoacid to 3547 amino acid positions of 7074 aminoacid polyproteins of SARS-CoV virus.
SACP ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
BJ01 ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
Vietnam ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
CUHK-W1 ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
Tor2 ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
BJ02 ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
HKU39849：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
Canada ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
BJ03 ：SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDTVYCPRHVICTAEDML
SACP ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
BJ01 ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
Vietnam ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
CUHK-W1 ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
Tor2 ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
BJ02 ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
HKU39849：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
Canada ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
BJ03 ：NPNYEDLLIRKSNHSFLVQAGNVQLRVIGHSMQNCLLRLKVDTSNPKTPK
SACP ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
BJ01 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
Vietnam ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
CUHK-W1 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
Tor2 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
BJ02 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
HKU39849：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
Canada ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
BJ03 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
SACP ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
BJ01 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
Vietnam ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
CUHK-W1 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
Tor2 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
BJ02 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
HKU39849：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
Canada ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
BJ03 ：YKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNHTIKGSFLNGSCGSVGF
SACP ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
BJ01 ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
Vietnam ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
CUHK-W1 ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
Tor2 ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
BJ02 ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
HKU39849：NIDYDCVSFCYMHHMELPTGVH GTDLEGKFYGPFVDRQTAQAAGTDTTI
Canada ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
BJ03 ：NIDYDCVSFCYMHHMELPTGVHAGTDLEGKFYGPFVDRQTAQAAGTDTTI
SACP ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
BJ01 ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
Vietnam ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
CUHK-W1 ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
Tor2 ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
BJ02 ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
HKU39849：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
Canada ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
BJ03 ：TLNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDIL
SACP ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
BJ01 ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
Vietnam ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
CUHK-W1 ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
Tor2 ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
BJ02 ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
HKU39849：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
Canada ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
BJ03 ：GPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTPFDVVRQC
SACP ：SGVTFQ
BJ01 ：SGVTFQ
Vietnam ：SGVTFQ
CUHK-W1 ：SGVTFQ
Tor2 ：SGVTFQ
BJ02 ：SGVTFQ
HKU39849：SGVTFQ
Canada ：SGVTFQ
BJ03 ：SGVTFQ
The protein sequence of the coronavirus of SACP sequence and other kinds carries out multisequencing and compares multiple comparing result in the embodiment 2:SARS-CoV coronavirus
CLUSTAL X is used in the sequence contrast, version 1.81 is finished, the protein sequence of the coronavirus of correlated other kinds comprises: infectiousness gastroenteritis virus (TransmissibleGastroenteritis Virus, TGEV), murine hepatitis virus (Murine Hepatitis Virus, MHeV), bovine coronavirus (bovine coronavirus, BcoV), feline infectious peritonitis virus (Feline Infectious Peritonitis virus, FIPV), human corona virus 229E (human coronavirus 229E, HcoV) and avian infectious bronchitis virus (AvianInfectious Bronchiyis Virus, AIBV).Comparing result and homology thereof " * " number are represented identical residue site, the high residue site of ": " expression conservative; ". " number represents the lower slightly residue site of conservative.Important as can be seen from the comparison result amino acid residue has comprised the Cys145 of catalytic action and His41 identical (the aminoacid sequence number is according to sars coronavirus SACP sequence numbering).
TGEV SGLRKMAQPSGLVEPCIVRVSYGNNVLNGLWLGDEVICPRHVIAS-DTTRVINYENEMSS 59
FIPV SGLRKMAQPSGVVEPCIVRVAYGNNVLNGLWLGDEVICPRHVIAS-DTSRVINYENELSS 59
HCoV AGLRKMAQPSGFVEKCVVRVCYGNTVLNGLWLGDIVYCPRHVIAS-NTTSAIDYDHEYSI 59
IBV SGFKKLVSPSSAVEKCIVSVSYRGNNLNGLWLGDTIYCPRHVLGK---FSGDQWNDVLNL 57
:*: *:. *:. ** *:* * ******.* : *****: . ::.
SACP KSNHSFLVQAGN-VQLRVIGHSMQNCLLRLKVDTSNPKTPKYKFVRIQPGQTFSVLACYN 119
TGEV VRLHNFSVSKNN-VFLGVVSARYKGVNLVLKVNQVNPNTPEHKFKSIKAGESFNILACYE 118
FIPV VRLHNFSIAKNN-AFLGVVSAKYKGVNLVLKVNQVNPNTPEHKFKSVRPGESFNILACYE 118
HCoV MRLHNFSIISGT-AFLGVVGATMHGVTLKIKVSQTNMHTPRHSFRTLKSGEGFNILACYD 118
BCoV VTSSDFTVLFDR-LSLTVMSYQMQGCMLVLTVTLQNSRTPKYTFGVVKPGETFTVLAAYN 119
MHV VTSSDFCVMSDR-MSLTVMSYQMQGSLLVLTVTLQNPNTPKYSFGVVKPGETFTVLAAYN 119
IBV ANNHEFEVTTQHGVTLNVVSRRLKGAVLILQTAVANAETPKYKFIKANCGDSFTIACAYG 117
.* : * *:. :. * : . *. **.:.* . *: *.: ..*
SACP GSPSGVYQCAMRPNHTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEG 179
TGEV GCPGSVYGVNMRSQGTIKGSFIAGTCGSVGYVLENGILYFVYMHHLELGNGSHVGSNFEG 178
FIPV GCPGSVYGVNMRSQGTIKGSFIAGTCGSVGYVLENGTLYFVYMHHLELGNGSHVGSNLEG 178
HCoV GCAQGVFGVNMRTNWTIRGSFINGACGSPGYNLKNGEVEFVYMHQIELGSGSHVGSSFDG 178
BCoV GKPQGAFHVTMRSSYTIKGSFLCGSCGSVGYVLMGDCVKFVYMHQLELSTGCHTGTDFNG 179
MHV GRPQGAFHVVMRSSHTIKGSFLCGSCGSVGYVLTGDSVRFVYMHQLELSTGCHTGTDFSG 179
IBV GTVVGLYPVTMRSNGTIRASFLAGACGSVGFNIEKGVVNFFYMHHLELPNALHTGTDLMG 177
* . : **.. **:.**: *:*** *: : . : * ***::** .. *.*:.: *
SACP KFYGPFVDRQTAQAAGTDTTITLNVLAWLYAAVINGDR------WFLNRFTTTLNDFNLV 233
TGEV EMYGGYEDQPSMQLEGTNVMSSDNVVAFLYAALINGER------WFVTNTSMSLESYNTW 232
FIPV EMYGGYEDQPSMQLEGTNVMSSDNVVAFLYAALINGER------WFVTNTSMTLESYNAW 232
HCoV VMYGGFEDQPNLQVESANQMLTVNVVAFLYAAILNGCT------WWLKGEKLFVEHYNEW 232
BCoV DFYGPYKDAQVVQLPVQDYIQSVNFVAWLYAAILNNCN------WFVQSDKCSVEDFNVW 233
MHV NFYGPYRDAQVVQLPVQDYTQTVNVVAWLYAAILNRCN------WFVQSDSCSLEEFNVW 233
IBV EFYGGYVDEEVAQRVPPDNLVTNNIVAWLYAAIISVKESSFSLPKWLESTTVSVDDYNKW 237
:** : * * : : *.:*:****::. :: . :: :*
SACP AMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCAALKELLQNGMNGRTILGSTILEDEFTP 293
TGEV AKTNSFTELSSTDA--FSMLAAKTGQSVEKLLDSIVR-LNKGFGGRTILSYGSLCDEFTP 289
FIPV AKTNSFTEIVSTDA--FNMLAAKTGYSVEKLLECIVR-LNKGFGGRTILSYGSLCDEFTP 289
HCoV AQANGFTAMNGEDA--FSILAAKTGVCVERLLHAIQV-LNNGFGGKQILGYSSLNDEFSI 289
BCoV ALSNGFSQVKSDLV--IDALASMTGVSLETLLAAIKR-LKNGFQGRQIMGSCSFEDELTP 290
MHV AMTNGFSSIKADLV--LDALASMTGVTVEQVLAAIKR-LHSGFQGKQILGSCVLEDELTP 290
IBV AGDNGFTPFSTSTA--ITKLSAITGVDVCKLLRTIMV-KNSQWGGDPILGQYNFEDELTP 294
* .: . . : *:: ** : : : :. * *:. : **::
BCoV SDVYQQLAGIKLQ 303
MHV SDVYQQLAGVKLQ 303
IBV ESVFNQIGGVRLQ 307
.* .* *::*
Embodiment 3:Loxistatin, (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate targeting SARS cysteine proteinase SACP
At first require (MSI company LtD with InsightII according to chirality; San Diego) makes up molecule L oxistatin; (+)-(2S; 3S)-2; 3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] three dimensional structure of ethyl succinate, obtain low energy conformations through molecular mechanics and molecular dynamics optimization.Then adopt the method for molecular docking Loxistatin to be docked to the active site of SACP, when docking except that the geometry complementations of considering Loxistatin and active site, also by rotatable key among the Loxistatin is rotated, the multiple not isomorphic map of choosing Loxistatin is to investigate the interaction of Loxistatin and SACP.At last composite structure is carried out molecular mechanics and molecular dynamics optimization, choose the minimum conformation of energy as the resulting composite structure, the interaction energy of Loxistatin and SACP is-47.83Kcal/mol in this structure, wherein the Van der Waals interaction energy is-34.23Kcal/mol, and electrostatic interaction energy is-13.60Kcal/mol.In the molecular docking display result accompanying drawing 5.As can be seen from the figure, Loxistatin is the avtive spot of targeting SACP well, the functional area that target SACP is numbered 41,47,51,139,160,161,162,163,164,165,166,167,171,186,187,188,189, No. 190 amino acid residue surrounded is advanced in effect, the model of action that adopts is His41 and the Cys145 catalytic site that occupies SACP, thereby the catalytic action of blocking-up His41 and Cys145 realizes the active inhibition of target SACP albumen catalyzing hydrolysis.
Embodiment 4: medicine (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate is to the cell toxicity test of Vero E6 cell
(+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] the method preparation that provides according to U.S. Pat 3911111 of ethyl succinate.The African green monkey kidney cell that grows up to monolayer (Vero E6) with handling well is inoculated in the 10mL Tissue Culture Flask, and 37 ℃ of absorption 2h, add the cell maintenance medium that contains 2% calf serum at sucking-off specimen fluids then, put 37 ℃ of CO
2Incubator is cultivated.The medicine that adds 1 mcg/ml, 10 mcg/ml and 100 mcg/ml is respectively observed the toxicity of medicine to Vero E6 cell after five days.Toxic experimental result to Vero E6 cell shows that under the drug level of 1 mcg/ml, 10 mcg/ml, medicine does not have toxicity to Vero E6 cell, does not influence Vero E6 cell proliferation and growth fully.
Embodiment 5: medicine (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate screening test that anti-SARS virus is infected
The test of external (in vitro) Vero E6 cell screening with the SARS virus specimen of handling well, adopts virus concentration for making 100 times of (100TCID of half Vero E6 cell infection concentration
50), attack and to grow up to vero-cell monolayer (Vero E6), be inoculated in then in the 10mL Tissue Culture Flask, two bottles of every part of specimen inoculation, 37 ℃ of absorption 2h, sucking-off specimen fluids then, flush away virus add the cell maintenance medium that contains 2% calf serum, put 37 ℃ of CO
2Incubator is cultivated.With of the protective effect of 96 orifice plate testing drugs to virus infected cell; if the medicine of the negative control of not dosing and adding 10 mcg/ml; medicine adds at least 4 holes; observe medicine after five days and infect the protective effect of Vero E6 cell preventing SARS virus; experimental result shows; under 10 mcg/ml drug level; medicine (+)-(2S; 3S)-2; 3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate can infect Vero E6 anti-SARS virus fully; the result in four holes is parallel, does not have dosing and the cell that adds in the negative control medicine hole then infects fully.Medicine (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] during ethyl succinate protection Vero E6 cell sees the following form to the anti-SARS virus result of infection.+ represent cytopathic degree.EC50＜1 μ g/ml, TC50～50 μ g/ml, therapeutic index TC50/EC50＞50.
Table two viral infection resisting experimental result
| 20μg/ml | 10μg/ml | 5μg/ml | 2.5μg/ml | 1.2μg/ml | The cell contrast | Virus control |
| - | - | - | - | - | - | ++++ |
| - | - | - | - | - | - | ++++ |
| - | - | + | - | - | - | ++++ |
The SARS 3CL that experimental example 6 rp-hplc method bacterial detection are expressed
ProActivity and inhibitor activity are measured:
Have the characteristics of conservative according to the specificity of coronavirus protease catalytic substrate, design 10 peptidase activities and detect substrate, the cysteine protease hydrolysis activity is detected with the rp-hplc method.This substrate sequence is: H
2N-
SAVLQSGFRK-COOH.Get substrate 2.5mmol, protease concentration 0.7 μ mol carries out endonuclease reaction in 37 ℃ in 20 μ l 20mmol Bis-Tris-HCl (pH7.0) reaction buffers, and reaction finishes the back and adds 0.1% trifluoracetic acid-70 and ℃ deposit.Product uses 0.1% trifluoracetic acid of 5-90% concentration at Delta Pak C18 post (3.9 * 150mm; Waters) carry out rp-hplc in and separate, the 215nm wavelength detects eluted product.Inhibitor in advance with the protease of variable concentrations in 37 ℃ of preincubate 60min, add peptide substrates (final concentration is 2.5mmol) cracking 30min after, measure proteinase activity according to the hydrolysis degree of substrate.The result show medicine (+)-(2S, 3S)-2,3-epoxy-N-[1-(S)-(N-isopentyl carbamoyl-3-methyl butyl)] ethyl succinate can suppress the SARS 3CL of bacterial expression effectively
ProActivity, the inhibitory action under 10mmol concentration has reached 50%.
Table three HPLC method is measured the pharmaceutically active result
Claims (3)
1.SACP prevent and/or treat purposes in the SARS infection medicine in screening, the sequence signature of SACP and being numbered:
1 SGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDTVYCPR HVICTAEDML NPNYEDLLIR
61 KSNHSFLVQA GNVQLRVIGH SMQNCLLRLK VDTSNPKTPK YKFVRIQPGQ TFSVLACYNG
121 SPSGVYQCAM RPNHTIKGSF LNGSCGSVGF NIDYDCVSFC YMHHMELPTG VHAGTDLEGK
181 FYGPFVDRQT AQAAGTDTTI TLNVLAWLYA AVINGDRWFL NRFTTTLNDF NLVAMKYNYE
241 PLTQDHVDIL GPLSAQTGIA VLDMCAALKE LLQNGMNGRT ILGSTILEDE FTPFDVVRQC
301 SGVTFQ
2. the purposes of claim 1, wherein, target SACP functional area refers to that by sequence numbering be the zone that 41,47,51,139,160,161,162,163,164,165,166,167,171,186,187,188,189, No. 190 amino acid residue constituted.
3. the purposes of claim 1, wherein target SACP functional area refers to by sequence numbering to be that No. 41 histidine and sequence numbering is that No. 145 cysteine constitute, the distance that constitutes between ε 1 nitrogen-atoms of the sulphur atom of functional area Cys145 and His41 is the 3.0-6.0 dust.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101025652A CN100478030C (en) | 2003-05-08 | 2003-10-23 | SARS resisting medicine action target and its preventing and treating medicine |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN03143083.X | 2003-05-08 | ||
| CN03143083 | 2003-05-08 | ||
| CNB2003101025652A CN100478030C (en) | 2003-05-08 | 2003-10-23 | SARS resisting medicine action target and its preventing and treating medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1548157A CN1548157A (en) | 2004-11-24 |
| CN100478030C true CN100478030C (en) | 2009-04-15 |
Family
ID=34378738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101025652A Expired - Fee Related CN100478030C (en) | 2003-05-08 | 2003-10-23 | SARS resisting medicine action target and its preventing and treating medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100478030C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113387909B (en) * | 2020-03-13 | 2023-11-14 | 中国人民解放军军事科学院军事医学研究院 | Medical application of 2, 3-epoxysuccinyl derivative |
| CN115282154A (en) * | 2022-08-15 | 2022-11-04 | 武汉科前生物股份有限公司 | Application of 11-carbonyl-beta-acetyl boswellic acid in preparation of medicine for treating feline abdominal distension |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4333879A (en) * | 1979-02-27 | 1982-06-08 | Taisho Pharmaceutical Co., Ltd. | Epoxysuccinic acid derivatives |
-
2003
- 2003-10-23 CN CNB2003101025652A patent/CN100478030C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4333879A (en) * | 1979-02-27 | 1982-06-08 | Taisho Pharmaceutical Co., Ltd. | Epoxysuccinic acid derivatives |
Non-Patent Citations (12)
| Title |
|---|
| A 3D model of SARS_CoV 3CL proteinaseand its inhibitors design by virtual screening. Xiong Bin,等.Acta Pharmacol Sin,Vol.24 No.6. 2003 |
| A 3D model of SARS_CoV 3CL proteinaseand its inhibitors design by virtual screening. Xiong Bin,等.Acta Pharmacol Sin,Vol.24 No.6. 2003 * |
| AAP13575. Tsui,S.K.W.NCBI/BLAST. 2003 |
| AAP13575. Tsui,S.K.W.NCBI/BLAST. 2003 * |
| Characterization of a Novel Coronavirus Associated withSevere Acute Respiratory Syndrome. Paul A. Rota,等.Science,Vol.300 . 2003 |
| Characterization of a Novel Coronavirus Associated withSevere Acute Respiratory Syndrome. Paul A. Rota,等.Science,Vol.300. 2003 * |
| Coronavirus Main Proteinase (3CLpro) Structure: Basisfor Design of Anti-SARS Drugs. Kanchan Anand,等.Science,Vol.300 . 2003 |
| Coronavirus Main Proteinase (3CLpro) Structure: Basisfor Design of Anti-SARS Drugs. Kanchan Anand,等.Science,Vol.300. 2003 * |
| SARS与SARS冠状病毒. 貌盼勇，洪世雯.现代科学仪器，第4期. 2003 |
| SARS与SARS冠状病毒. 貌盼勇，洪世雯.现代科学仪器，第4期. 2003 * |
| The Genome Sequence of the SARS-Associated Coronavirus. Marco A. Marra,等.Science,Vol.300 . 2003 |
| The Genome Sequence of the SARS-Associated Coronavirus. Marco A. Marra,等.Science,Vol.300. 2003 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1548157A (en) | 2004-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods | |
| Kozlovskaya et al. | Isolation and phylogenetic analysis of SARS-CoV-2 variants collected in Russia during the COVID-19 outbreak | |
| Yan et al. | Structural biology of SARS-CoV-2: open the door for novel therapies | |
| Ng et al. | Antiviral drug discovery for the treatment of COVID-19 infections | |
| Stadler et al. | SARS—beginning to understand a new virus | |
| Hamre et al. | A new virus isolated from the human respiratory tract. | |
| US7662860B2 (en) | 3D-structure model of SARS coronavirus 3CL protease and anti-SARS drugs | |
| Domingo et al. | Historical perspective on the discovery of the quasispecies concept | |
| Yan et al. | An overview of potential inhibitors targeting non-structural proteins 3 (PLpro and Mac1) and 5 (3CLpro/Mpro) of SARS-CoV-2 | |
| Calleja et al. | Inhibitors of sars-cov-2 plpro | |
| Xu et al. | Systemic in silico screening in drug discovery for coronavirus disease (COVID-19) with an online interactive web server | |
| CN111825750A (en) | Application of ACE2 receptor protective synthetic short peptide in novel coronavirus infection | |
| CN111402968B (en) | New application of kaempferol in COVID-19 virus based on molecular simulation | |
| CN116236580B (en) | Application of old medicines such as auranofin and the like and compositions thereof in resisting single positive strand RNA viruses | |
| Islam et al. | A review on structural, non-structural, and accessory proteins of SARS-CoV-2: Highlighting drug target sites | |
| Silva et al. | Druggable targets from coronaviruses for designing new antiviral drugs | |
| Dar'ya et al. | PolypharmDB, a deep learning-based resource, quickly identifies repurposed drug candidates for COVID-19 | |
| El Kantar et al. | Derivatization and combination therapy of current COVID-19 therapeutic agents: a review of mechanistic pathways, adverse effects, and binding sites | |
| Ghaleh et al. | SARS‐CoV‐2 Non-structural protein 1 (NSP1) mutation virulence and natural selection: Evolutionary trends in the six continents | |
| Uengwetwanit et al. | Identification of novel SARS-CoV-2 RNA dependent RNA polymerase (RdRp) inhibitors: From in silico screening to experimentally validated inhibitory activity | |
| Baxi et al. | Emergence and reemergence of severe acute respiratory syndrome (SARS) coronaviruses | |
| Ubi et al. | Essentials of the COVID-19 coronavirus | |
| Narayanan et al. | Structure-based inhibitor design and repurposing clinical drugs to target SARS-CoV-2 proteases | |
| CN1237184C (en) | Target for medicine against SARS-Cov and medicine screening method and medicine against SARS | |
| Liu et al. | Distinct genetic spectrums and evolution patterns of SARS-CoV-2 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090415 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |