Bioluminescence-based high-throughput screening and identification of direct-acting dengue virus NS5 methyltransferase inhibitors.

Dengue virus (DENV) is a mosquito-borne orthoflavivirus that infects millions of people annually across tropical and subtropical regions, posing a severe and growing threat to global public health. Currently there are no antiviral drugs available for DENV infection. The methyltransferase domain of non-structural protein 5 (NS5 MTase) catalyzes two essential methylation reactions in synthesis of the 5' cap-1 structure of viral mRNA, making it a promising yet under-validated target for anti-DENV drug development. Herein, we optimized a bioluminescence-based enzymatic MTase-Glo assay using purified recombinant DENV NS5 MTase and performed high-throughput screening of the focused Methylation Compound Library. Four compounds-SGC0946, AMI-1, salirasib, and gambogenic acid-were identified as potent DENV methylation inhibitors effective against all four serotypes (DENV-1 to -4). Surface plasmon resonance (SPR) analysis confirmed the direct binding between inhibitors and NS5 MTase. Among them, SGC0946 exhibited the strongest inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.05-0.11 μM against DENV-1 to -4. Molecular docking predicted that SGC0946 binds to the S-adenosylmethionine (SAM)-binding pocket of NS5 MTase. In addition, SGC0946 suppressed DENV replication in C6/36 and BHK-21 cells with EC50 values of 6.67 and 0.60 μM, respectively. Collectively, these findings identify novel DENV MTase inhibitors with diverse scaffolds and provide valuable molecular insights for the rational design of more potent NS5 MTase-targeting inhibitors in the future.