Chikungunya virus (CHIKV) is an arbovirus belonging to the Alphavirus genus and the Togaviridae family, responsible for a clinical syndrome characterized by debilitating polyarthralgia, high fever, and skin rash [[1], [2], [3]]. Its (+)ssRNA genome encodes both non-structural (nsP) and structural viral proteins [[4], [5], [6], [7]]. nsP1 facilitates viral mRNA capping [[8], [9], [10]], nsP2 exhibits multiple enzymatic activities, including helicase and protease functions [[11], [12], [13]], nsP3 supports RNA replication [2,14], and nsP4 functions as an RNA polymerase [15]. The envelope glycoproteins E1 and E2 mediate the adhesion and fusion of the virus to susceptible host cells [2], while the 6K protein is essential for virus assembly [2,16]. Lastly, the capsid protein (C) contributes to the release of structural proteins during viral assembly [[17], [18], [19]].

Typically, proteases are known for hydrolyzing peptides and proteins, and they are classified based on their nucleophilic residue, such as serine, aspartate, cysteine, glutamate, threonine, and metalloproteases [[20], [21], [22]]. Among these, cysteine proteases have been extensively studied, with their catalytic mechanism involving an initial proton transfer (Brønsted-Lowry mechanism) from a Cys residue to a His residue, thereby activating the protease [23,24].

In CHIKV, the cleavage/proteolysis of the P1234 polyprotein into nsP and structural proteins is facilitated by the protease activity of nsP2 [25,26]. nsP2 also exhibits additional enzymatic functions, including RNA helicase, nucleoside triphosphatase (NTPase), and RNA 5′-triphosphatase activities, which are localized within its N-terminal domain, whereas its proteolytic activity resides in the C-terminal region [2,26]. The proteolytic activity of CHIKV nsP2 was previously demonstrated by Pastorino et al. (2008) using fluorophore-containing peptides as substrates, such as Boc-AGG-MCA. Optimal conditions for the proteolytic activity of CHIKV nsP2, functioning as a cysteine protease, were identified as pH 9.5 and 10–50 mM NaCl, involving the catalytic dyad Cys478 and His548 [27]. CHIKV nsP2 is classified as a papain-like protease [13,28]; however, Saisawang et al. (2015) proposed that it does not behave typically as previously believed. Their findings suggested that the Ser482 residue might also contribute to the enzyme's activity, which was confirmed by partial inhibition of its activity following the use of serine protease inhibitors [26]. Although definitive proof of its classification remains elusive, CHIKV nsP2 is conventionally regarded as a cysteine protease. In its X-ray structure, Ser482 is relatively distant from the catalytic dyad, implying that a conformational change may be required to bring the catalytic residues into proximity to execute proteolysis [29]. In a study by Rausalu et al. (2016), CHIKV nsP2 demonstrated that Cys478 is critical for proteolysis, as its mutation to Ala (C478A) abolished protease activity, viral replication, and consequently, virus release [30]. In the nsP2 proteases of Sindbis (SINV) and Semliki Forest (SFV) viruses, a conserved tryptophan residue is essential for proteolytic activity. However, in CHIKV nsP2, Trp549 prevents direct participation in proteolysis [29]. Instead, it engages in hydrophobic and van der Waals interactions with other residues, stabilizing the loop containing His548 [26], though mutation of this residue does not affect the enzyme's proteolytic activity [29].

As aforementioned, cysteine proteases have an important role in several physiological processes, in different organisms. The acid-base catalytic residues (Cys and His) are found to be conserved in different alphaviruses, such as Venezuelan equine encephalitis (VEEV), SINV, and CHIKV (Fig. 1A). Since CHIKV nsP2 is considered as a cysteine protease in the most studies, it must behavior then as a nucleophilic protein, by attacking electrophile substrates, as reported for other cysteine proteases in computational studies, such as cruzain of Trypanosoma cruzi [31,32], cathepsin-L-like protease of T. brucei [33], falcipain-2 of Plasmodium falciparum [34]. Typically, cysteine protease activation involves a mechanism within two steps, being (1) acylation and, then (2) deacylation. Initially, an activated thiol, Cys(S−), attacks an electrophilic substrate (acylation step), resulting in a tetrahedral intermediate, which is stabilized into the oxyanion hole (via an auxiliary residue), releasing an amine as a product. Then, acylated subproduct is hydrolyzed by an water molecule (activated by an imidazole residue, His), releasing a carboxylic acid derivative as product and regenerating the catalytic residues (Fig. 1B) [23,24]. In 2010, a computational study was performed by Russo et al. investigated the substrate specificity of nsP2 protease and the importance of the catalytic dyad for several different alphaviruses, except for CHIKV [35]. Surprisingly, there are no studies elucidating how the CHIKV nsP2 protease is catalytically activated.

In this study, we used a cluster approach comprising molecular dynamics (MD) simulations and Density Functional Theory (DFT) calculations to answer three specific questions: (1) If CHIKV nsP2 is a truly cysteine protease, it should be activated to become a nucleophilic protein. Is it indeed? (2) Would it be a water-catalyzed mechanism or one that occurs in the absence of water (into a hydrophobic environment)? (3) Would be a concerted mechanism or not? Thusly, MD simulations using OPLS_2005 molecular mechanics force field (MMFF) and DFT calculations, with different functionals (ωB97X-D, B3LYP, M06, TPSSh, and BHandLYP), and basis sets (SVP, QZVP, TZVP, and TZVPP) were used elucidate how CHIKV nsP2 catalytically activated. Furthermore, this study represents an open-avenue for research groups working on novel antiviral agents targeting CHIKV, by interfering with this protease, which could be used for developing electrophilic inhibitors.