Screening Of Anti-HCV Drugs From Natural Products

Hepatitis C virus(HCV)infection is a major cause of chronic liver disease,often leading to liver fibrosis,liver cirrhosis and hepatocellular carcinoma.HCV infected approximately 185 million persons in worldwide.During the replication of HCV,the polyprotein is cleaved by NS3/4A serine protease at four junctions in the nonstructural part of this protein.This central role played by NS3/4A protease in the maturation of HCV makes it an attractive target for drug development.In recent years,the direct antiviral drugs against NS3/4A protease have been used in clinical treatment of HCV infection.However the drug resistance and side effects of DAAs is significant.Therefore,it is important to screen novel NS3/4A protease inhibitors.In this study,a high throughput screening model was used to screen active compounds from natural products,providing lead compounds for the development of new anti-HCV drugs.The main contents are as follows:1)The gene sequence of the virus strains HCV-1b/US/BID-V130/1994(GenBank: EU155330.2)with the maximum similarity was selected as the research object which is the most representative of HCV 1b type virus.The gene sequences encoding NS3/4A protease were selected and optimized for total synthesis.The recombinant NS3/4A protease was expressed and purified by E.coli expression system.Using FRET principle,the high throughput screening model of NS3/4A protease was constructed.Furthermore,the reaction of pH,the amount of enzyme,and the amount of substrate were optimized.The high throughput screening model of NS3/4A protease constructed in this study will contribute to the further screening of active samples from natural products.2)We screened the soil microbial fermentation sample library which was established early in the laboratory.7 bacterial strains were finally obtained through the first screening and rescreening from the collected strains.SIPIFW-F4581,F4065,F4004,F4479 were used in the following studies.F4581 was identified to be Aspergillus terreus,F4065 was identified to be Penicillium meleagrinum,F4004 was identified to be Fusarium oxysporum,F4479 was identified to be Meyerozyma guilliermondii.Using the screening model of NS3/4A protease inhibitor,bioassay-guided fractionation were carried out in the fermentation extract of the active strains.26 active compounds were obtained from the four strains.Compounds 4479-8 and 4479-10 are new naphthyridinone,which contain a parent nucleus of 1,2-dimethyl-2,3-dihydro-2,7-naphtyridin-4(1H)-one and a triaziridin-2-yl side chain group in the structure.This is the first report of the cyclic dipeptide,ergosterol and naphthyridinone compounds with NS3/4A protease inhibitory activity.The present results may be used to design and development lead compounds for the development of anti-HCV agents.3)To screen natural inhibitors of NS3/4A protease,20 ethanol extracts of Chinese medicinal herbs,which have been reported with antiviral activity were investigated in this study.Chinese herbs Taxillus sutchuenensis(Lecomte)Danser and Maytenus hookeri Loes were used in the following studies.We traced the efficient constitution of the extract through bioassay-guided fractionation,11 active compounds were obtained from Taxillus sutchuenensis(Lecomte)Danser,among them 9 compounds are found for the first time in the genus Taxillus.8 active compounds were obtained from Maytenus hookeri Loes,among them 2 compounds are found for the first time in the genus Maytenus.During the research,flavonoid,diarylheptanoid,oleanane-type triterpene and symmetries polyphenol were responsible for anti-HCV constitution of these two Chinese herbs.And diarylheptanoid and symmetries polyphenols' inhibition to HCV NS3/4A protease was reported for the first time,which may be used to design and development lead compounds for the development of anti-HCV agents.