Exploring advanced methodologies for NS5 methyltransferase targeted drug discovery.

Emerging viral pathogens highlight the need for targeted antiviral strategies, particularly against mechanistically validated enzymes like the Dengue virus NS5 methyltransferase - an essential component of viral RNA capping and immune evasion. Despite its therapeutic relevance, the discovery of potent small-molecule inhibitors has been constrained by the absence of robust complementary assays. In this study, we address this gap through a comparative evaluation of three orthogonal methodologies that enable comprehensive identification and characterization of inhibitors targeting this enzyme. We synthesized a focused library of thirty S-adenosylhomocysteine analogs via copper-catalyzed azide-alkyne cycloaddition and screened them using three orthogonal methods: (i) fluorescence polarization to monitor ligand displacement from the methyl donor site; (ii) NMR titration to resolve residue-specific interactions; and (iii) a label-free enzymatic assay employing acoustic mass spectrometry to quantify the inhibition of methyl group transfer. Consistent potency rankings across all platforms validated this approach. The fluorescence assay enabled high-throughput triaging, NMR provided structural insight into ligand binding, and enzymatic profiling confirmed functional inhibition. This integrated, modular screening strategy enables the rational development of NS5 methyltransferase inhibitors and is adaptable to related RNA-capping enzymes of viral or other origin.