br Methods br Results and discussion br
Results and discussion
Conclusions In this work, 100-ns MDSs were applied on the WT and on the R155K and D168A single point mutations of the NS3/4A protease in the apo form and in complex with ASV, a current drug in phase III clinical trials. According to the PCA, these two mutations dramatically converted the direction of motion of the key binding residues (123, 155 and 168) to move outwards from ASV, resulting in a loss of SR 2595 and H-bond formation. The QM/MM-GBSA binding free energy calculations at different levels of QM theory (AM1, RM1, PM3 and PM6) suggested that both mutations caused a significant reduction in the binding affinity, ranked in the order of WT < R155K < D168A, which agreed with the observed experimental EC50 values. In addition, it can be postulated that other related mutations might have a similar pattern in drug binding and the loss of interactions like in our cases present here. Consequently, our findings can allow us to predict the effect of mutations on the binding affinity at least without simulations. Taken together, the theoretically obtained information can be used as a rational guide for antiviral drug design and development towards the HCV genotype 1, which is the most prevalent genotype worldwide.
Acknowledgements This research was supported by the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University (CU). We also thank the Research Chair Grant, the National Science and Technology Development Agency (NSTDA), Thailand, and the Structural and Computational Biology Research Unit, CU. This research work was partially supported by Chiang Mai University, Thailand. JK is thankful for the Scholarship from CU to develop research potential for the Department of Biochemistry, Faculty of Science, CU (Ratchadaphiseksomphot Endowment Fund), the 90th Anniversary of CU Scholarship, and the Overseas Presentations of Graduate Level Academic Thesis from Graduate School. PM thanks the Science Achievement Scholarship of Thailand. We would like to thank Mr. Phakawat Chusuth for helpful suggestions and advices.
Introduction Hepatitis C Virus (HCV) is the causative agent of liver hepatitis C. If it remains untreated, it may develop to liver cirrhosis, fibrosis and hepatocellular carcinoma. Around 180 million people are infected with HCV worldwide [1,2]. HCV replication is very fast and error prone due to the low fidelity of NS5B RNA polymerase. As a consequence, the virus is present as a large population of viral variants in patients . There are seven genotypes and several subtypes of HCV that are identified up till now . They differ in their sequences, geographic distribution and response to available treatments [1,4]. Genotype 1 is prevalent in Europe and North America . It is the most widely studied genotype and most of the designed drugs are tailored based on it. For long time, the standard of care was Pegylated-interferon α and ribavirin, but it wasn't so effective against genotype 1 and had several side effects [3,5,6]. So, there was an urgent medical need to discover novel HCV treatments. Direct Acting Antivirals (DAAs) are the best solutions to this problem [5,6]. They target key components of the virus life cycle and shut it down. NS3/4A protease, NS5A and NS5B proteins are the best targets for designed DAAs . Food and Drug Administration (FDA) approved many drugs from 2011 up till now [5,6]. These drugs are mainly NS3/4A protease inhibitors such as boceprevir, telaprevir, simeprevir, grazoprevir, paritaprevir and recently glecaprevir. These drugs are now part of all-oral, interferon-free therapies and show high success rates [5,6]. NS3/4A is a bifunctional protein [3,, , ]. It contains N-terminal serine protease domain and C-terminal DExH/D helicase and NTPase functionalities. The serine protease domain belongs to the chymotrypsin superfamily [, , ]. It contains H57, D81 and S139 as the catalytic triad that catalyze the peptide hydrolysis at four downstream junctions of the large HCV polyprotein [3,7]. It also attacks two important signaling proteins, MAVS and TRIF, which activate the immune system against the viral infection [, , ]. The inhibition of NS3/4A protease has two effects: it prevents the maturation of viral proteins and restores the host immune response [, , ].