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  • Introduction Hepatitis C virus HCV is a small

    2021-10-21

    Introduction Hepatitis C virus (HCV) is a small (50–80 nm in size) enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family [1]. The HCV particle consists of a nucleocapsid, which contains the positive single-stranded RNA genome covered by a host cell-derived lipid envelope [2]. Currently, HCV infects over 80.2 million people and long-term HCV-infected patients are under an increased risk of developing liver diseases, such as cirrhosis and hepatocellular carcinoma. Genotype 1 is the most common HCV genotype worldwide, accounting for 46% of all HCV infections followed by 30% from genotype 3. The highest monensin sale of genotypes is observed in China and South-East Asia, while only genotype 4 is found in Egypt and Mongolia [[3], [4], [5]]. The currently used anti-HCV agents are targeted inhibitors against the important NS3/NS4A protease, NS5B polymerase and NS5A viral proteins [6]. For the treatment of HCV patients, the combination of PEGylated-interferon-α and ribavirin monensin sale is extremely expensive but only effective for ∼50% of HCV patients [7,8]. Moreover, this therapy causes several adverse side effects, including alopecia, rash/itching, nausea, thrombocytopenia and anaemia. In the HCV life cycle, after protein translation, the large polypeptide encoding the nonstructural proteins is processed by the viral NS2/3 and NS3/4A proteases. The NS3/4A protease cleaves the NS3A-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS4B junctions and so is essential in the viral replication process. Consequentially, the NS3/4A protease has become one of the attractive targets for anti-HCV drug design and development [9]. Importantly, the NS3 proteolytic activity and the acceleration of the cleavage rate are associated with the NS4A cofactor [10]. The HCV therapy has been improved by the use of boceprevir [[11], [12], [13]] and telaprevir [14,15], which specifically inhibit the NS3/4A protease enzyme. Although this treatment leads to an increased rate of sustained virologic response, such therapeutic drugs have a low success rate [16]. Recent drugs, such as simeprevir [17,18] and vaniprevir [19,20], were approved for the treatment of HCV-infected patients in 2013 and 2014, respectively. In addition, other inhibitors are currently used in clinical studies such as danoprevir [21,22], grazoprevir [23], faldaprevir [24], vedroprevir [8] and asunaprevir (ASV; [25,26]). Because HCV lacks a proofreading activity of its RNA polymerase enzyme, high mutation rates in response to the treatments have been widely reported, resulting in resistance to drugs/inhibitors [[27], [28], [29], [30]]. Asunaprevir (ASV) is a second-generation NS3/4A protease inhibitor that was approved for use in combination with daclatasvir in Japan for the treatment of HCV genotype 1-infected patients [31]. However, ASV has not yet been approved in the USA and Europe, where it is currently in phase III clinical trials [32]. This compound shows a strong antiviral activity against genotypes 1 and 4 of HCV [25,26]. From in vitro studies, the acquired resistance against ASV caused by the two single point mutations of R155K and D168A in the NS3/4A protease results in a lower susceptibility to ASV, with an ∼21- and 23-fold reduced half maximal effective concentration (EC50), respectively [28]. These two mutated residues are located near the protease active site (Fig. 1) and were also found to reduce the binding affinities of some other anti-HCV inhibitors, such as danoprevir [21,22], simeprevir [17,18] and grazoprevir [23], by changing the conformation of the active site [[33], [34], [35]]. For instance, the absence of a Nε, which is the nitrogen atom at epsilon position, of residue K155 prevented the interaction between residues 155 and 168, which disrupted the interaction of ASV with the target protein [36]. To investigate the effect of such mutations on the ASV binding to NS3/4A, in terms of the inhibitor-protein interactions and binding efficiency towards the HCV NS3/4A protease, all-atom molecular dynamics simulations (MDSs) and binding free energy calculations based on QM/MM-GBSA and MM/GB(PB)SA methods were applied on the ASV in complex with the wild type (WT) and the R155K and D168A mutations of the HCV genotype 1 NS3/4A protease (Fig. 1). In addition, the crucial motion of amino acids located in the active site of the apo (APO) and complex (CPX) forms was characterized using principal component analysis (PCA).