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

HCV NS3/4A protease is an irreplaceable nonstructural protein in HCV infecting and pathogenic process, thus it is an important antivirus target. Currently, drugs targeted NS3/4A protein have been sale on the market, such as Telaprevir, Boceprevir and Simeprevir. But these drugs must combine with PEG-IFNα/ribavirin and also have many disadvantages, such as susceptibility to drug resistance, side effects and low bioavailability. Hence, it is important to develop new NS3/4A protease inhibitor which has higher drug resistance barriers and bioavailability or individual usability.Using recombinant EGFP as substrate, a new screening model was constructed to reduce false positive in the screening process. NS5A/B sequence could be cleaved by NS3/4A protease and was inserted into EGFP after 173 rd amino acid to establish the recombinant EGFP/5AB. The intensity of the fluorescence signal is negative correlation with the amount of NS3/4A protease and is positive correlation with the strength of the inhibitor of NS3/4A protease, and then microplate reader could be used to detect the fluorescence signal.In this study, 575 g prepared ethanol extract, 104 g petroleum ether extract, 66 g ethyl acetate extract, 160 g normal butanol extract were obtained from 8.7 kg tree branches and leaves of Torreya grandis. Finally, 22 compounds were isolated from these organic solvent extract, furthermore 16 compounds are clear about the structure, and 1 new compound.NS3/4A protease model was used to screen 22 compounds obtained in this study, and the result shows that compounds YZ-2-5-2, YZ-3-1, YZ-3-2 have NS3/4A protease inhibitory activity. Compound YZ-2-5-2, molecular mass is 342 and molecular formula is C16H22O8, is identified as β-D-Glucopyranoside,3-(4-hydroxy-3-methoxyphenyl)-2-propen-1-yl. Compound YZ-3-1, molecular mass is 208 and molecular formula is C11H14O3, is identified as 2-methoxy-4-(3-methoxyprop-1-en-1-yl) phenol. Compound YZ-3-2, molecular mass is 194 and molecular formula is C10H12O3, is identified as 4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol. After preliminary bioactivity evaluation, compound YZ-3-2 shows higher activity and it’s IC50 on NS3/4A protease is 1 mM, and the IC50 of positive control VX950(Telaprevir) is 0.2 mM.In conclusion, 3 compounds obtained in this study could be used as leader compound of NS3/4A protease inhibitors, though which IC50 was significantly higher than the positive control VX950(Telaprevir).