INVESTIGATION OF THE REACTION MECHANISM OF THE CYS/HIS CATALYTIC DYAD OF CHIKUNGUNYA VIRUS (CHIKV) NSP2
Emanuelly Karla Araújo Padilha1, Midiane Correia Gomes1, Júlio Cosme da Silva1, Edeildo Ferreira da Silva-Júnior1
1 Post-Graduation Program in Chemistry and Biotechnology, Institute of Chemistry and Biotechnology, Federal University of Alagoas, CEP: 57072-970, Maceió-AL, Brazil.
INTRODUCTION: The Chikungunya virus (CHIKV) is an arthropod-borne virus belonging to the Alphavirus genus of the Togaviridae Family [1]. It is transmitted through the bite of female mosquitoes of the species Aedes aegypti, Ae. albopictus, Ae. furcifer, and Culex ssp. CHIKV represents a serious public health problem worldwide, particularly in emerging countries. It is considered a neglected tropical disease (NTD) with no vaccines or specific medications for Chikungunya fever (CHIKF) treatment. Therefore, the development of safe and effective antivirals against CHIKV is an urgent necessity to control the complications arising from potential epidemics [2]. One of the major challenges in the development of antivirals for CHIKV is due to its poorly understood physiopathological mechanism. In this context, the non-structural protein nsP2 of CHIKV plays a crucial role in viral replication and transcription during its infection cycle in the host. It has been suggested that the nsP2 protein cleaves peptide bonds as it is a cysteine protease, given that it contains a cysteine with a thiolate group activated through a Brønsted-Lowry acid-base mechanism involving an adjacent histidine, resulting in an ionic pair of residues (Cys(S-) - His(H+)) [3,4]. However, this catalytic mechanism has not yet been elucidated in the literature. In this regard, obtaining these essential pieces of information would be extremely relevant for the rational design of antiviral compounds aimed at combating CHIKV. AIMS: Investigating the activation mechanism of the catalytic dyad, Cys478-His548, in CHIKV's nsP2 using the quantum method Nudged Elastic Band (NEB) ,[5]; To determine the transition state (TS) for the formation of the ionic pair (Cys(S-) - His(H+)), and to identify the binding modes of potential inhibitors reported in the literature. METHODS: To investigate the mechanism of the Cys/His catalytic dyad formation, we employed the Nudged Elastic Band (NEB) method in conjunction with Density Functional Theory. All calculations were performed using the ORCA program, version 5.0. [6]. Initially, the structures of the reactant and product species were optimized using the B3LYP exchange-correlation functional with B3J dispersion correction and the def2-SVP Gaussian basis set. These structures were confirmed as local energy minima by calculating vibrational frequencies. Subsequently, the NEB-TS method implemented in ORCA was used to search for a transition state structure connecting the reactant and product in the proton transfer mechanism from the cysteine residue to histidine. Furthermore, similar methods were employed to study potential inhibitors of nsP2 described in the literature. RESULTS: Understanding the reaction mechanism of the Cys/His catalytic dyad and determining the activation energy has significant implications for virology and the development of antiviral therapies, given that nsP2 is a key protein in CHIKV replication. These insights can aid in comprehending the chemistry involved in viral replication, facilitating the development of therapeutic strategies aimed at inhibiting or interrupting this process. This, in turn, contributes to the development of new and effective drugs for the treatment of CHIKV. Following the investigation of the initial and final structures, a potential transition state was obtained. Thus, this transition state (TS) was identified as the highest-energy intermediate species on the potential energy surface. The results obtained revealed that the calculated activation energy for the Cys/His catalytic dyad of CHIKV's nsP2 is on the order of 13.0 kcal/mol, which is consistent with activation energies associated with enzymatic processes. This result suggests that such a state is plausible for the final products, in this case, the ionic pair (Cys(S-) - His(H+)). Additionally, some compounds described in the literature, belonging to the classes of acylhydrazones, hydrazones, and acrylamides, were also investigated against nsP2, including the acrylamides developed by our research group. In general, all these classes of potential inhibitors have an electrophilically favorable region for nucleophilic attack by the thiolate group, as they are Michael acceptors. As a result, the acrylamides represent an interesting group of inhibitors because they have relatively low energy values, indicating that these inhibitors indeed have a strong interaction with the target. Furthermore, the best of them was investigated for a potential covalent versus non-covalent inhibition mechanism to identify the chemical reactivity of its Michael acceptor group, thus gathering important information for drug design based on the inhibition mechanism. CONCLUSION: Our results provide significant kinetic and structural insights into the reaction mechanism of the Cys/His catalytic dyad in CHIKV's nsP2. The identification of the transition state and the determination of the activation energy contribute to a better understanding of the chemistry involved in viral replication, as well as the potential development of antiviral therapies. The use of the NEB method, combined with Density Functional Theory, has proven to be an efficient methodology for investigating and elucidating kinetic and structural aspects of the proton transfer reaction of the Cys/His catalytic dyad in CHIKV's nsP2. This methodology has allowed us to thoroughly examine the reaction mechanism of the Cys/His catalytic dyad in nsP2 of CHIKV, including the determination of the transition state and associated activation energy. The calculations conducted using the NEB method and specific basis sets have provided crucial insights for a molecular-level understanding of the chemistry involved in CHIKV replication, as well as for the development of research in therapeutic strategies. Furthermore, acrylamides represent the most promising chemical class for inhibiting nsP2, with their Michael acceptor group playing a significant role in the binding modes.
ACKNOWLEDGMENTS:
The authors thank to CAPES (finance code: 001), FAPEAL (grant number: E:60030.0000001720/2022, E: 60030.0000000161/2022), and UFAL.
REFERENCES
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Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
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Comissão Científica
Ricardo Olimpio de Moura
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