CN1560074A - Polypeptide of inhibiting crown virus and its derivant - Google Patents
Polypeptide of inhibiting crown virus and its derivant Download PDFInfo
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- CN1560074A CN1560074A CNA2004100186793A CN200410018679A CN1560074A CN 1560074 A CN1560074 A CN 1560074A CN A2004100186793 A CNA2004100186793 A CN A2004100186793A CN 200410018679 A CN200410018679 A CN 200410018679A CN 1560074 A CN1560074 A CN 1560074A
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Abstract
The invention discloses a polypeptide of inhibiting coronary virus and its derivatives, the composition of amino acids of the polypeptide: X-valine-leucine-glutamine-serine-glycin- phenylalanine-arginine-L, and the practice verifies that the polypeptide and its derivatives have no toxicity to kidney generation cells of Afric green monkey and a certain inhibition effect on the coronary virus, half of it have low effective concentration 2.7X to the power -5 mg/ml, good selectivity and can be applied to preparing the anti-coronary virus medicines.
Description
Technical field
The present invention relates to a kind of polypeptide and derivative thereof that suppresses virus.
Background technology
Sars coronavirus is the main Causative virus of severe acute respiratory syndrome, and very strong infectivity is arranged, and causes serious social danger.The anti-SARS medicine of state food and Drug Administration approval at present only has Interferon, rabbit " recombinanthumaninterferon's sprays " and " recombinant human interferon omega nasal spray ", pharmaceutical chemicals " injection Sivelestat sodium tetrahydrate " and " injection ulinastatin " wait a few medicine, and " the clear pest capsule of lotus flower " and herbal species such as " FUFANG BIEJIA RUANGAN PIAN " are in the clinical study.These medicines have plenty of the prevention that is used for the high risk population, it is antiviral and improve the ancillary drug of immunizing power to have plenty of Guang Spectrum, at present still do not have directly to suppress the specifics of sars coronavirus, the atypical pneumonia that is caused by sars coronavirus still is in the state that no medicine can be cured.Because infectious atypical pneumonia is to the significant damage of human health and society, the principle of applying gene group, protein science and medicinal design, development directly at the specific medicament of sars coronavirus, has major and immediate significance, economic benefit and social benefit.
Summary of the invention
The purpose of this invention is to provide a kind of polypeptide that suppresses coronavirus;
Another object of the present invention provides a kind of application of polypeptide in preparation anti-coronavirus medicine that suppresses coronavirus;
The 3rd purpose of the present invention provides a kind of polypeptide derivative that suppresses coronavirus;
Last purpose of the present invention provides a kind of application of polypeptide derivative in preparation anti-coronavirus medicine that suppresses coronavirus.
Technical scheme of the present invention is summarized as follows:
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z.The single-letter expression formula of this polypeptide is X-VLQSGFR-Z, and the trigram expression formula is X-Val-Leu-Gln-Ser-Gly-Phe-Arg-Z.
Described X is an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
Described Z is an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
With this polypeptide called after Wogu (Wei-Chou-Gan-Du) polypeptide.
A kind of polypeptide that suppresses coronavirus, the application in preparation anti-coronavirus medicine.A kind of polypeptide that suppresses coronavirus of the present invention can mix with one or more carriers according to the conventional production method of pharmaceutical field, makes required preparation, carries out one or many according to factors such as patient's age, body weight and uses.
A kind of polypeptide derivative that suppresses coronavirus, it is by shown in the following chemical expression:
Described X is an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
Described Z is an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
A kind of polypeptide derivative that suppresses coronavirus, the application in preparation anti-coronavirus medicine.A kind of polypeptide derivative that suppresses coronavirus of the present invention can mix with one or more carriers according to the conventional production method of pharmaceutical field, makes required preparation, carries out one or many according to factors such as patient's age, body weight and uses.
Effect of the present invention:
1. a kind of polypeptide that suppresses coronavirus of the present invention is the Wogu polypeptide, and virus is had certain restraining effect, and its medium effective concentration is 2.7 * 10
-5Mg/ml can prepare antiviral.
2. a kind of polypeptide that suppresses coronavirus of the present invention is that the Wogu polypeptide becomes derivative after chemically modified, can become the inhibitor of sars coronavirus main protease and the medicine of anti-sars type pneumonia.
3. a kind of polypeptide that suppresses coronavirus of the present invention is that the Wogu polypeptide becomes derivative after chemically modified, can become the inhibitor of other coronavirus.
Description of drawings
Fig. 1 is that the sars coronavirus main protease docks diagram with a kind of inhibition coronavirus polypeptide;
Fig. 2 A, B are sars coronavirus main protease and a kind of skeleton view that suppresses the different angles of coronavirus polypeptide interaction of hydrogen bond;
The aminoacid sequence of 7 aa of Fig. 3 Wogu polypeptide (SEQ ID No.1)
Embodiment:
Below in conjunction with specific embodiment the present invention is further described:
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, and wherein X is amino, and Z is a carboxyl.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
Embodiment 2
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, and wherein X is a carboxyl, and Z is amino.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
Embodiment 3
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, and wherein X is a L-Ala, and Z is amino.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
Embodiment 4
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, wherein X is glycine-phenylalanine-arginine-tyrosine-glycine-L-Ala-Serine-L-glutamic acid-methionine(Met)-L-Ala, and Z is a glycine.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, wherein X is glycine-L-Ala, and Z is glycine-arginine-phenylalanine-methionine(Met)-glycine-L-Ala-Serine-L-glutamic acid-methionine(Met)-glycine.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
Embodiment 6
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, and wherein X is a glycine, and Z is glycine-arginine-phenylalanine-methionine(Met)-glycine.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
A kind of polypeptide that suppresses coronavirus, its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, X L-Ala-Serine-L-glutamic acid-methionine(Met)-L-Ala wherein, Z is glycine-arginine-phenylalanine-methionine(Met)-glycine.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
Embodiment 8
A kind of polypeptide derivative that suppresses coronavirus, wherein X is amino, Z is a carboxyl.Synthetic according to a conventional method with Peptide synthesizer, syntheticly begin to extend to aminoterminal from carboxyl terminal.
As follows by the Wogu polypeptide to the modification protocols of polypeptide derivative:
A kind of polypeptide and the application of derivative in preparation anti-coronavirus medicine thereof that suppresses coronavirus can be made injection, tablet and capsule etc. according to a conventional method.
According to sars coronavirus main protease (SARS CoV M
Pro) three-dimensional structure of molecule and " key of distortion " theory of polypeptide drug design, design and synthesized and a kind of the sars coronavirus main protease is had restraining effect can be blocked the polypeptide X-VLQSGFR-Z that sars coronavirus duplicates (X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z); This polypeptide forms derivative after chemically modified, the experiment proved that has restraining effect to sars coronavirus, and can become the inhibitor of sars coronavirus main protease and the medicine of anti-sars type pneumonia, and to the certain restraining effect of having of other coronavirus.
1.SARS determining of coronavirus proteolytic active region
The first step of the present invention is to determine the catalytic activity zone of sars coronavirus main protease.Anand etc. have at first set up the homology model (K.Anand of sars coronavirus main protease, J.Ziebuhr, P.Wadhwani, J.R.Mesters, R.Hilgenfeld.Coronavirus main proteinase[3CLpro] structure:basis for design of anti-SARS drugs.Science 2003,300,1763-1767).The main protease gene of sars coronavirus has been cloned by the rich son of Tsing-Hua University and research group from the strain that is numbered BJ01 that Chinese Military Medical Science Institute finds, and in intestinal bacteria, obtained the efficient of proteolytic enzyme by the genetic engineering means, stably express, through further separating, purifying and crystallization, collected high-resolution diffraction data, measured the three-D space structure of this enzyme under four kinds of different conditions, and elaborated the accurate conformation of this enzyme at the substrate calmodulin binding domain CaM, and then by resolving the three-D space structure of this enzyme and the mixture of inhibitor formation, disclosed this enzyme-to-substrate bonded accurate model (H.Yang, M.Yang, Y.Ding, Y.Liu, Z.Lou, Z.Zhou, L.Sun, L.Mo, S.Ye, H.Pang, G.F.Gao, K.Anand, M.Bartlam, R.Hilgenfeld, Z.Rao.The crystal structures ofsevere acute respiratory syndrome virus main protease and its complex with an inhibitor.Proc.Natl.Acad Sci.USA.2003,100,13190-13195).Because the function of sars coronavirus main protease is the polyprotein of cutting encoding viral, in the replicative cycle of virus, play a part crucial, biological nature to virus is significant, so the three-dimensional structure of sars coronavirus main protease becomes the first-selected target based on the direct medicinal design of molecular structure.
The sars coronavirus main protease comprises 304 amino acid, be folded into three structural domains, structural domain 1 is amino-acid residue 8-99, structural domain 2 is amino-acid residue 100-193, structural domain 3 is amino-acid residue 184-199, proteolytic enzyme and the polypeptide bonded active region pocket-like position between structural domain 1 and structural domain 2, our molecular dynamics simulation is found, the catalytic activity district is made up of 23 amino-acid residues, and they are: Cys-22, Gly-23, Thr-24, Thr-25, Leu-27, His-41, Val-42, Cys-44, Thr-45, Ala-46, Glu-47, Asp-48, Met-49, Leu-50, Asn-51, Pro-52, Tyr-54, Cys-145, His-164, Met-165, Asp-187, Arg-188, Gln-189.
2. the computer simulation of molecular dynamics butt joint
The computer simulation that the sars coronavirus main protease docks with Wogu polypeptide VLQSGFR is to finish with medicine design software bag MOE (Molecular Operate Environment, Chemical Comuting Ltd.).Lytic enzyme cutting polypeptide or proteinic catalysis sensitive part are formed by holding 8 amino acid whose 8 sites usually, and as usual, 8 amino-acid residue notes are made P
4, P
3, P
2, P
1, P
1 ', P
2 ', P
3 ', P
4 ', and cut point is at site P
1And P
1 'Between, be labeled as P
1↓ P
1 '(K.-C.Chou.Review prediction of human immunodeficiency virus protease cleavagesites in proteins, Analy.Biochem.1996,233,1-14; G.E.Schulz, R.H.Schirmer.Principles ofProtein Structure, Chap.2, pp.17-18, Springer-Verlag, New York, 1985).Although the reactive site of the lytic enzyme that has once in a while can hold 7-9 amino acid, 8 amino acid are the most general situations.Our research only limits to the severability of seven peptides.
3.SARS the binding mechanism of coronavirus proteolytic and Wogu polypeptide
The Wogu polypeptide closely is combined in the pocket-like position between the structural domain 1 and 2 of proteolytic enzyme, closely is combined in the catalytic activity zone of sars coronavirus main protease by 6 hydrogen bonds, if polypeptide P
1The amino-acid residue Q (glutamine) of position replaces with E (L-glutamic acid), and the hydrogen bond between main protease and the polypeptide reduces to 3, and bonding force is reduced greatly.The shearing point of the main protease of sars coronavirus main protease and other coronavirus has tangible specificity, P
1The site can only occupy for Gln, P
1 'The site can be Ser or other amino-acid residue, is expressed as Gln ↓ (Ser, Ala and Gly).Computer simulation has confirmed the binding mechanism of sars coronavirus main protease and polypeptide and the specificity of shearing point.These find further to have supported Anand (K.Anand, J.Ziebuhr, P.Wadhwani, J.R.Mesters, R.Hilgenfeld.Coronavirus main proteinase[3CLpro] structure:basis for design of anti-SARS drugs.Science 2003,300,1763-1767) about P in the octapeptide
1The Gln of position has also pointed out direction for the chemically modified of polypeptide to the saying that the specificity of sars coronavirus main protease plays a crucial role.
4. the analysis of the chemically modified of " key of distortion " model and Wogu polypeptide
According to the relation of " key and lock " in the zymetology, the polypeptide that makes a variation out from the sars coronavirus main protease must have good combination with avtive spot.Yet,, can lose its riving property fully, but it can combine still with avtive spot if after a polypeptide is modified the key of easy fracture with some conventional methods.In fact, the molecule that this method is modified similar and " key of distortion ", it can insert in the hole of lock, can not pull out key but can not open lock, and this is exactly the inhibitor of the competitive anti-SARS proteolytic enzyme of ideal naturally.Therefore the polypeptide separated from enzyme itself of research and the bonding mechanism of SARS proteolytic enzyme provide Useful Information can for our medicinal design.Shown in Figure 1 is one of chemically modified scheme of Wogu polypeptide (embodiment 3).
5.Wogu the chemosynthesis of polypeptide A VLQSGFR is synthetic according to a conventional method with Peptide synthesizer, synthesizes to begin to extend to aminoterminal from carboxyl terminal.
Experimental example 1 Wogu polypeptide A VLQSGFR is to the toxicity test of African green monkey kidney passage cell (VERO)
Dilute the Wogu polypeptide by a certain percentage, totally 9 extent of dilution are added in 96 orifice plates that contain the VERO cell, and the nutrient solution in every hole is 100 μ l, and the final concentration of polypeptide is 0.1mg/ml～7mg/ml, 37 ℃ of following 5%CO
2Cultivated 48 hours, every hole adds 20 μ l MTS/PMS, continues to cultivate 3 hours, adds 50 μ l 10%SDS, detects OD
490, be contrast with the VERO cell that does not add seven peptides, the OD of laboratory sample
490Value greater than the control cells value 90% as the nontoxicity index.Experimental result is the OD of experimental cell
490Value is 1.87～2.06, the OD of control cells
490Value is 2.01, and its ratio shows that all greater than 0.9 the Wogu polypeptide does not have toxicity to the VERO cell.
Experimental example 2 Wogu polypeptide A VLQSGFR are to the restraining effect of SARS virus
10000 VERO cells of every hole inoculation in 96 orifice plates, the nutrient solution volume is 100 μ l, 37 ℃ of following 5%CO
2Cultivated 24 hours, and added the polypeptide (with embodiment 1) of different concns, every hole adds 2 μ l SARS-BJ-01 viruses, continues to cultivate 24～96 hours, and acellular contrast, cell contrast and virus control are set, and when in the virus control group during cell mass mortality, measures OD
490Value.Experimental result is the OD of experimental cell
490Value is 1.01～1.72, the OD of control cells
490Value is 2.11, and its ratio shows that all less than 0.9 this peptide has certain restraining effect to the infection of SARS-BJ-01 virus.
Experimental example 3 Wogu polypeptide A VLQSGFR and other medicines are to the inhibiting comparison of SARS virus
As stated above, Wogu polypeptide A VLQSGFR and other 5 kinds of viral inhibitors are measured the inhibition effect of SARS-BJ-01 virus, the results are shown in following table.The result shows the medium effective concentration minimum (2.7 * 10 of Wogu polypeptide in the table
-5Mg/ml), selectivity is best.
5 kinds of medicines are to the restraining effect of SARS-BJ-01 virus
Medicine medium effective concentration (mg/ml) is selected coefficient
Wogu polypeptide 2.7 * 10
-5＞3704
6-azauracil nucleosides 16.8 6
Pyrazofurin 4.2 12
Mycophenolic acid＞50 can not be calculated
Ribavirin＞1000 can not be calculated
Glycyrrhizin 300＞67
Claims (8)
1. polypeptide that suppresses coronavirus, it is characterized in that: its amino acid consists of X-Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine-Z, and described Val-Leu-glutamine-Serine-glycine-phenylalanine-arginine is represented with sequence table SEQ ID No1.
2. a kind of polypeptide that suppresses coronavirus according to claim 1, described X are an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
3. a kind of polypeptide that suppresses coronavirus according to claim 1, described Z are an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
4. a kind of polypeptide that suppresses coronavirus of one of claim 1 to 3, the application in preparation anti-coronavirus medicine.
5. polypeptide derivative that suppresses coronavirus, it is characterized in that: it is by shown in the following chemical expression
6. a kind of polypeptide derivative that suppresses coronavirus according to claim 5, described X are an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
7. a kind of polypeptide derivative that suppresses coronavirus according to claim 5, described Z are an amino acid arbitrarily of 1 to 10, or amino, or carboxyl.
8. a kind of polypeptide derivative that suppresses coronavirus of one of claim 5 to 7, the application in preparation anti-coronavirus medicine.
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| CN111471088A (en) * | 2020-04-21 | 2020-07-31 | 北京中科微盾生物科技有限责任公司 | Polypeptide for inhibiting SARS-COV-2 infection, composition and use thereof |
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| CN111471088A (en) * | 2020-04-21 | 2020-07-31 | 北京中科微盾生物科技有限责任公司 | Polypeptide for inhibiting SARS-COV-2 infection, composition and use thereof |
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