Identification and evaluation of small molecules targeting NS5 methyltransferase activity of Dengue Virus

Abstract

Dengue virus (DENV) is a major global health threat, causing endemic diseases with approximately 2 lakh cases annually in India alone. DENV replication is complex and relies on the host cellular machinery, making it a challenging target for antiviral intervention. The NS5 protein, a nonstructural protein of DENV, has methyltransferase (MTase) activity that catalyzes methylation of the viral RNA cap using S-Adenosyl-L-methionine (SAM) as a substrate. This step is crucial for viral translation and replication. The conservation of the SAM binding pocket in the MTase domain of NS5 across flaviviruses makes it a promising target for the development of small molecule inhibitors with broad spectrum antiviral activity. In this study, the NS5 MTase protein was expressed, purified, and crystallized to facilitate structural studies. Two compounds with reported inhibitory activity against the MTase of other viruses were selected for evaluation, and their binding affinity was determined using fluorescence spectroscopy. Site-directed mutagenesis was also performed to generate negative control mutants for comparative analysis. Co-crystallization and soaking techniques were employed to elucidate the protein-ligand complex structure. The findings of this project have significant implications for the development of effective antiviral therapies against DENV. The identification of small molecule inhibitors against the NS5 MTase domain of DENV can disrupt viral replication and translation, and potentially lead to the development of novel antiviral strategies. Moreover, the insights gained from this research can also aid in the development of inhibitors against other flaviviruses that share similar MTase structures and functions. Overall, this study contributes to the field of antiviral drug discovery and addresses the unmet medical need for the management of flavivirus infections, including DENV, providing a valuable foundation for future research in this area.