Decoding pH-dependent structural dynamics of CHIKV nsP2 protease: insights from computational antiviral targeting.

Chikungunya virus (CHIKV) is a vector-borne disease transmitted primarily through mosquitoes and causes fever, and its pathogenicity is closely linked to the function of non-structural protein 2 (nsP2), which plays a pivotal role in viral replication and host immune modulation. The enzymatic efficiency and structural stability of viral proteases are intensely influenced by environmental pH, which can regulate the active site accessibility and inhibitor binding efficiency. This non-structural protein 2 (nsP2) encompasses an N-terminal RNA helicase and C-terminal cysteine protease linked by the flexible regions. Hence, this study investigates the influence of varying pH conditions on structural flexibility of apo form and holo forms of CHIKV nsP2 protease leveraging of extensive molecular dynamic (MD) simulation and molecular docking. Post-MD superimposition revealed that the active site shifted from Site 2 to Site 1, indicating a conformational reorganization of the binding pocket. This study also evaluated its influence on the interactions with a cysteine protease inhibitor, E-64 and Leupeptin of CHIKV nsP2 protease. Simulation conducted under various pH conditions revealed a notable shift, particularly in the catalytic dyad residues Cys 1013 and His 1083. RMSD, RMSF, radius of gyration, and number of hydrogen bond analyses indicated that both inhibitors exhibited variable binding stabilities, with pronounced fluctuation in loop and β-strand region. Notably, at pH 7 and 8, the β2 strand undergoes a conversion into a loop which could potentially influence the substrate recognition and catalytic activity. Thus, this in silico findings provides critical insights into the dynamic behavior of CHIKV nsP2 protease under various pH and suggests strategies for rational designing of pH-resilient antiviral inhibitors that maintain the efficiency under various physiological conditions.