Molecular Simulation Study On The Binding Mode Between HCV NS5B Polymerase And Benzofuran Nuclear Pan-genotypic Inhibitors

BackgroundHepatitis C virus（HCV）is the causative agent of hepatitis C.HCV infection can lead to chronic hepatitis C,cirrhosis,hepatocellular carcinoma and other liver diseases,which are very dangerous to patients’life and health.NS5B polymerase is a key enzyme in the replication process of HCV,and there is a lack of homologous enzyme in human’s body,so it can be an important target for anti-HCV.It has been found that benzofuran nuclear inhibitors inhibit NS5B polymerase of various genotypes,but their binding modes are unclear.Therefore,it is of great significance to carry out research on the development of novel pan-genotypic inhibitors against HCV based on the pan-genotypic binding modes of benzofuran NS5B polymerase inhibitors.Objectives1.Constructing complex structures of benzofuran nuclear inhibitors with different genotypes of NS5B polymerase（GT1a,GT1b,GT2a and GT2b）.2.Exploring the binding modes of benzofuran nuclear NS5B polymerase inhibitors with NS5B polymerase and analyzing the detailed interaction between them.3.To investigate the reason for the pan-genotypic inhibitory activity of benzofuran nuclear NS5B polymerase inhibitors.Methods1.To optimize the crystal structures of the four genotypes HCV NS5B polymerase obtained from the RCSB PDB protein database,and to construct and optimize the 3D structures of small molecule inhibitors.2.To construct complex structures of inhibitors with NS5B polymerase using semi-flexible docking method.3.Performing 100 ns molecular dynamics simulations for each complex systems and analysing the binding modes were analyzed in detail.4.Calculating the steady state’s binding free energy and binding energy decomposition analysis of each system with MM/GBSA.Results1.A total of twenty complex structures of five benzofuran nuclear NS5B polymerase inhibitors docked with NS5B polymerase of four genotypes（GT1a,GT1b,GT2a and GT2b）,respectively,were constructed.2.The results of molecular dynamics simulations show that each system reached steady state after 60 ns MD.3.Combined with the results of free energy calculations,it was verified that the five inhibitors had different degrees of inhibition on the NS5B polymerase of four genotypes.4.The analysis of binding modes showed that after binding to the NS5B polymerase of four genotypes,the 2-phenylbenzofuran nuclear of the five inhibitors were all in the Palm II region of NS5B polymerase,and their 5-position cyclic side chains could all extend to the Palm I region.In addition,the methyl amide group at the 3-position of2-phenylbenzofuran nuclear could form hydrogen bonds with NS5B polymerase in each complex system,which was essential for the binding of the benzofuran nuclear inhibitor to NS5B polymerase.Interestingly,our study showed that the binding modes of inhibitors with a long chain group at the 5-position of the 2-phenylbenzofuran core with NS5B polymerase of different genotypes was divided into two characteristic types:upon binding to NS5B^GT1a and NS5B^GT2b polymerase,the long chain group at the 5-position faced to one side;whereas upon binding to NS5B^GT1b and NS5B^GT2a polymerase,the long chain group at the 5-position faced to the other side.ConclusionMolecular modeling simulation studies of the binding modes of five benzofuran nuclear pan-genotypic inhibitors with four different genotypes of HCV NS5B polymerase（GT1a,GT1b,GT2a and GT2b）showed that the 3-position methylamide group of these inhibitors’2-phenylbenzofuran nuclear of these inhibitors can form hydrogen bonds with residues of NS5B polymerase.And there are two binding types when benzofuran nuclear inhibitors with long chain group at the 5-position bind to NS5B polymerase of different genotypes.The relevant research results can provide valuable information for the further development of novel benzofuran nuclear anti-HCV pan-geneotypic inhibitors.