| 摘要: | Hepatitis C virus (HCV) infection is a global health burden with over 180 million people infected worldwide and without vaccine available currently. HCV genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. The HCV NS3 protein is an essential protease for viral polyprotein processing. For full activity, the NS3 protease requires a NS4A protein as a cofactor. NS4A/NS3 protease cleaves the polyprotein at four specific regions NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B junctions. NS3 and its recombinant protease are regarded currently as a potential target for anti-viral drugs. Thus, specific inhibitor of its enzyme activity is highly important.
In this study, recombinant protein NS4A/NS3 was constructed by fused NS3 with NS4A peptide by different linkers (GSGS and GG). NS4A/NS3 with GSGS was found to have higher activity than NS4A/NS3 with GG and NS3con + Pep4A (wild type), however, wild type was more stable than fusion proteases. The best assay conditions were established for all tailor-made HCV proteases. To screen inhibitors against NS3 protease, software of molecular docking model (Autodock version 4.2) was used to evaluate the free energy after NS4A/NS3 protease was docked with ligands from chemical library (chemical compounds bank). After checking the docking score of 50,000 peptide bond-containing compounds, three compounds with free energy less than -10 Kcal/mol were obtained (Candidate 1, 2 and 3). A natural inhibitor, hexapeptide DEMEEC, was used to examine the inhibitory potency of three constructs. The kinetic and inhibitory parameters, Km, IC50 and Ki, of NS3con + Pep4A against hexapeptide were found to be 4.3 µM, 2.5 µM and 0.51 µM, respectively. That of NS4A-GSGS-NS3 against hexapeptide were 2.45 µM, 4.23 µM, 1.04 µM, respectively, and that of NS4A-GG-NS3 against hexapeptide were 3.71 µM, 4.9 µM, 2.54 µM, respectively. One of candidates, Candidate 1, was found to have high inhibition effect against NS4A/NS3 protease. The inhibitory parameters, IC50 and Ki, of NS3con + Pep4A against Candidate 1 were found to be 25.4 µM and 5.04 µM, respectively; that of NS4A-GSGS-NS3 against Candidate 1 were 61.1 µM and 7.88 µM, and that of NS4A-GG-NS3 against Candidate 1 were 87.9 µM and 6.89 µM, respectively. The Lineweaver–Burk plot of inhibitory kinetics showed that candidate 1 acted as a competitive manner, which suggested that it competes with substrate to bind into active sites. Interaction between Candidate 1 and NS3 protease domain simulated by PyRx software that showed binding affinity of candidate 1 with His57 and Ser139 (catalytic triad). The inhibition activity of Candidate 1 is not good enough, but it could be further modified on its chemical structure to improve the inhibitory activity and to be a better inhibitor. |
