Chemical Physics Letters 619 (2015) 97–102 Contents lists available at ScienceDirect Chemical Physics Letters jou rn al hom epage: www.elsevier.com/locate/cplett Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease José Ángel Martínez-González a,1 , Alex Rodríguez b , María Pilar Puyuelo a , Miguel González c,∗ , Rodrigo Martínez a,∗ a Departamento de Química, Universidad de La Rioja, C/ Madre de Dios, 51, 26006 Logro˜ no, Spain b Scuola Internazionale Superiore di Studi Avanzati (SISSA), Via Bonomea 265, I-34136 Trieste, Italy c Departament de Química Física i IQTC, Universitat de Barcelona, C/ Martí i Franquès, 1, 08028 Barcelona, Spain a r t i c l e i n f o Article history: Received 6 August 2014 In final form 20 November 2014 Available online 2 December 2014 a b s t r a c t The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second- order Møller–Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different sub- strates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a con- certed mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The hepatitis C virus (HCV) has infected an estimated 170 million people, with almost 3–4 million newly infected worldwide every year [1,2]. Long-term carriers of this pathogen [3] are under the risk of developing a liver disease such as cirrhosis or liver cancer. Most antiviral compounds that act directly against the HCV infection are inhibitors of three viral proteins: NS3/NS4A protease, NS5B polymerase, and NS5A protein (whose function is still not fully clear) [4]. Computational chemistry has contributed to the development of many of these antiviral compounds. Thus, tech- niques such as QSAR (Quantitative Structure–Activity Relationship) and molecular dynamics have been successfully employed in the development of HCV NS3/NS4A and NS5B inhibitors [5,6]. The last antivirals approved in Europe, simeprevir (an NS3/NS4A protease inhibitor) and sofosbuvir (an NS5B polymerase inhibitor), consti- tute a very promising treatment since they can reduce the duration of antiviral treatment and interferon is not needed for some HCV genotypes [7]. ∗ Corresponding authors. E-mail addresses: miguel.gonzalez@ub.edu (M. González), rodrigo.martinez@unirioja.es (R. Martínez). 1 Present address: Unitat de Química Física, Departament de Química, Edifici Cn, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Despite this, the study of the reaction mechanism of NS3/NS4A protease is still interesting in order to obtain deeper theoretical insight into the proteolytic reactions it is involved in. Such informa- tion, determined at a rather higher theoretical level than in previous studies, can provide in-depth knowledge of the mechanism of this viral serine protease. Moreover, it could also help in the develop- ment of future generations of antivirals using transition state (TS) analogs [8]. The NS3 enzyme is produced by the infected cell (see, e.g., Refs. [2,3,9–11], and references therein) and is active as protease once it binds to the NS4A cofactor. The NS3/NS4A protease acts on its main natural substrates: the HCV polyprotein peptide junction regions NS5A/5B (sequence EDVVCCSMSY), NS4B/5A (ECTTPCSGSW), and NS4A/4B (DEMEECSQHL) (substrates 1–3, respectively). The three reactions involved are sequential and as a result of them the NS5A, NS5B, NS4A, and NS4B proteins are liberated. These proteins are implied in different key processes related to the illness [2]. By analogy with other serine proteases, the proposed catalytic mechanism of NS3/NS4A protease begins with the formation of the NS3/NS4A-substrate Michaelis complex (MC). The acylation process then follows, which produces the acylenzyme interme- diate once the N-terminal fragment of the substrate is released. The whole reaction ends with the deacylation of the acylenzyme intermediate and the release of the C-terminal fragment. The study proposed here focuses on the acylation process which, in turn, takes place in two steps: the tetrahedral intermediate formation and peptide bond breakage steps. http://dx.doi.org/10.1016/j.cplett.2014.11.041 0009-2614/© 2014 Elsevier B.V. All rights reserved.