Molecular Simulation Study On The Interaction Between HCV Related Targets And Their Inhibitors

Discovery of specific and effective inhibitors that can inhibit the proteins related to hepatitis C virus(HCV) life cycle is of great importance in the process of anti-HCV drug development. These drug targets including NS3/4A, NS3 helicase and NS5 B play an important role in HCV replication and translation such as nucleotide unwinding and translocation. Currently, there are serials of inhibitors including NS3/4A inhibitors boceprevir, telaprevir and simeprevir were used in clinical. Unfortunately, the emergence of resistance to the antiviral HCV drug virus strains limits the use of these drugs. It is urgent to seek the novel and effective antiviral drugs. With the development of computational technology, molecular simulation, considered a strong tool for seeking novel inhibitor was widely used in target-drug interaction and resistance mechanism study. Various molecular simulation methods have been applied in study on the structure and function of HCV related targets, such as molecular dynamics(MD) simulation, steered molecular dynamics(SMD) simulation, adaptive biasing force(ABF) simulation and Metadynamics simulation. To gain insight into the structural features and energy characteristics of these protiens, we investigated the interaction and dissociation mechanism between NS5 B, NS3 helicase and NS3/4A targets and therelated drugs. These results will provide a useful guide for designing novel antiviral drugs.We first provide general introduction of the steps of HCV life cycle, the structure and function of HCV related drug targets and their inhibitors. We also reviewed the recent progress in the study on the function of HCV related targets and interaction mechanism between inhibitors and their targets. A brief introduction of various molecular simulation methods was also given, such as SMD, ABF and Metadynamics simulation.The first part of the thesis is to study the drug resistance mechanism of NS5 B mutants to BMS-791325. MD simulation combined with free energy calculation, free energy decomposition and ABF simulation was employed on the complex of wild type(WT), and A421 V, L392 I, P495 L mutant NS5 B with their inhibitor BMS-791325. MD simulation results show the hydrophobic interaction is the driving force for BMS-791325 binding. There are 8 amino acids of energy contribution over 1 kcal/mol, such as L392, A393, A396, T399, H428, V494, P495 and R503. ABF simulation proves that attenuation of the hydrophilic interaction between R503 and BMS-791325 is the first step for drug escaping from the binding site. Loss of the hydrophilic interaction makes drug move out of the hydrophobic pocket. The simulation results reveal that A421 V, L392 I and P495 L mutants reduce drug binding affinity. P495 L mutant makes the binding pocket more flexible and cannot anchor BMS-791325 better. The altered hydrophilic interactions of mutant residues are the essential reasons leading to drug resistance in A421 V and L392 I mutants.The second part of the thesis is to study the binding and unbinding mechanism of NS5 B with the active metabolite GS-461203 of NS5 B inhibitor sofosbuvir and the natural substrate UTP. We constructed the ternary complexes of NS5B-RNA-GS-461203 and NS5B-RNA-UTP based on the crystal structure. Our simulation results demonstrate that both polar and nonpolar interactions are favorable for GS-461203 or UTP binding. The 2’-fluoro-2’-C-methyl ribose of GS-461203 can form stronger polar and nonpolar interaction with residues S282 and I160 than UTP. The possible unbinding pathway of ligand was predicted using random acceleration molecular dynamics(RAMD) simulation method and is defined as the back of NS5 B palm domain. The dissociation process can be classed roughly into three steps: translation, reversal of base and ribose and complete divorce. In the S282 T mutant system, the binding affinity attenuation of UTP relative to wild type is less than that of GS-461203.In the third part, to explore the interaction mechanism between NS3 helicase and its indole derivatives inhibitors, we constructed the free energy surface(FES) of the ligand unbinding process using Metadynamics simulation. Three unbinding process steps including a hydrophobic moiety of 1-position indole scaffold stands up and escapes from hydrophobic pocket, the hydrogen bonds between the carboxylethyl group and G255 and T269 disappear, and the whole ligand adopts a favorable conformation for escaping from binding pocket through the cleft between NS3 helicase domainⅠand Ⅲ were defined. The indole ring has a great geometric matching with binding pocket, carboxylethyl group of 3-position indole scaffold can form strong hydrogen bonds with G255 and T269. When introducing large volume group in the 1-position and 6-position of indole scaffold can impede the ligand unbinding.In the fourth part of the thesis, we studied the drug resistance mechanism of NS3/4A mutants to BMS-650032. The nonpolar energy term was found to be a driving force for BMS-650032 binding based on MD simulation and free energy calculation, 11 key amino acids were determined using per-residue energy decomposition method. The dissociation process of BMS-650032 can be classed roughly into three steps: the P2’ moiety and P4 moiety of BMS-650032 escape from NS3/4A binding pocket, then the P1’ moiety and P1 moiety of BMS-650032 move out the binding site and complete divorce. The simulation results reveal that A156 T, R155 K and D168 A mutants lead to reducing of drug binding affinity and drug free energy well of escaping from binding site. For A156 V mutation, the occurrence of drug resistance is mainly from the changed binding pocket by a replacement of one bulky residue valine. R155 K and D168 A destroyed the salt bridges R123-D168-R155-D81 should be responsible for the large conformation changes of the binding pocket.