Screening Of HCV NS3/4A Protease Inhibitors From Natural Products

Hepatitis c virus(HCV) infection which can lead to liver fibrosis, cirrhosis, liver cancer and even death, is a major public health burden. NS3/4A protease is an important target for the development of anti-HCV drugs. This study was aimed to screen active compounds from fungi SIPIFW-F4581 and Maytenus hookeri loes(Yunnan) using the screening model of NS3/4A protease inhibitor which had already been constructed in our previous study. Furthermore, the structure of active compounds was identified and their inhibitory activity of NS3/4A protease was quantitatively evaluated.Firstly, the optimal fermentation media for SIPIFW-F4581 was determined from six kinds of media(Y-1, Y-2, Y-3, Y-4, Y-5 and Y-6), and the result showed that when Y-1 and Y-6 were used, the metabolites of SIPIFW-F4581 showed higher inhibitory activity of NS3/4A protease. Next, using Y-1 and Y-6 as fermentation media, large-scale fermentation of SIPIFW-F4581 was carried out. And active compounds were separated from the metabolites of SIPIFW-F4581 and identified based on the screening model of NS3/4A protease inhibitor. As a result, two active compounds(J-1 and J-2) were obtained. J-1 which named(E)-2,4-dihydroxy-6-propenyl-benzaldehyde is a new compound, and its IC50 was 17.9 m M. J-2 was identified as(2S,3R)-2,3,4-trimethyl-5,7-diol-2,3-dihydrobenzofuran( IC50=312.0 μM), and its inhibitory activity of NS3/4A protease was reported for the first time.Based on the screening model of NS3/4A protease inhibitor, active compounds were separated from Maytenus hookeri loes and their structure was identified. Consequently, four active compounds(Z-1, Z-2, Z-3 and Z-4) were obtained. Z-1 was identified as(2R,3R)-3,5,7-triol-2-(3,5-dihydroxy-4-methoxyphenyl)-chromane, and its IC50 was 440.0 μM. Z-2 was identified as kaempferol glycoside, and its IC50 was 135.8 μM. Z-3 was identified as kaempferol-3-O-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside, and its IC50 was 49.2 μM. Z-4, whose molecular formula is C27H30O15, belongs to flavonol glycosides by structural analysis, IC50 was 110.3 μM. All active compounds’ NS3/4A protease inhibitory activity was discovered for the first time, furthermore, compound Z-1 and Z-3 were separated from Maytenus for the first time.In conclusion, six compounds with NS3/4A protease inhibitory activity were obtained from SIPIFW-F4581 and Maytenus hookeri Loes, furthermore J-1 is a new compound. These compounds could be used as lead compounds for further modification and preclinical study which can promote the development of anti-HCV drugs.