Gurunathan Rubha Shri, Rajaram Abhirami, Chandrabose Selvaraj, Singh Sanjeev Kumar
Computer Aided Drug Design and Molecular Modeling Lab, Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu, 630002, India.
CSRDD Lab, Bioinformatics Division, Department of Marine Biotechnology, AMET University (Deemed to Be University), Tamil Nadu, East Coast Road, Kanathur, Chennai, 603112, India.
Mol Divers. 2025 Sep 16. doi: 10.1007/s11030-025-11343-y.
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.
基孔肯雅病毒（CHIKV）是一种主要通过蚊子传播的媒介传播疾病，可引起发热，其致病性与非结构蛋白2（nsP2）的功能密切相关，nsP2在病毒复制和宿主免疫调节中起关键作用。病毒蛋白酶的酶促效率和结构稳定性受到环境pH的强烈影响，环境pH可调节活性位点的可及性和抑制剂结合效率。这种非结构蛋白2（nsP2）包含一个通过柔性区域连接的N端RNA解旋酶和C端半胱氨酸蛋白酶。因此，本研究利用广泛的分子动力学（MD）模拟和分子对接，研究了不同pH条件对CHIKV nsP2蛋白酶的无配体形式和全配体形式结构灵活性的影响。MD叠加后发现活性位点从位点2转移到位点1，表明结合口袋发生了构象重组。本研究还评估了其对CHIKV nsP2蛋白酶与半胱氨酸蛋白酶抑制剂E-64和亮抑酶肽相互作用的影响。在各种pH条件下进行的模拟显示出显著的变化，特别是在催化二聚体残基Cys 1013和His 1083处。均方根偏差（RMSD）、均方根波动（RMSF）、回转半径和氢键分析表明，两种抑制剂均表现出可变的结合稳定性，在环和β链区域有明显波动。值得注意的是，在pH 7和8时，β2链会转变为环，这可能会影响底物识别和催化活性。因此，这些计算机模拟结果为CHIKV nsP2蛋白酶在不同pH条件下的动态行为提供了关键见解，并为合理设计在各种生理条件下保持效率的耐pH抗病毒抑制剂提供了策略。
