Fluorescence polarization-based high-throughput screening identifies SAM-competitive inhibitors of monkeypox virus N7-methyltransferase.

Monkeypox virus (MPXV) encodes its own RNA-capping machinery that is essential for viral RNA maturation, productive replication, and evasion of host innate immunity. However, the antiviral potential of targeting MPXV RNA capping has remained largely unexplored, in part due to the lack of robust biochemical assays and validated inhibitors. Here, we report the development of a fluorescence polarization (FP) based high-throughput screening (HTS) assay that enables direct measurement of S-adenosyl-L-methionine (SAM)-competitive binding to the MPXV N7-methyltransferase (N7-MTase) complex. Using a fluorescent S-adenosyl-L-methionine (SAM) analogue, FL-NAH, we optimized assay conditions and demonstrated high signal-to-noise ratios, strong tolerance to dimethyl sulphoxide (DMSO) and detergent, and reliable competitive displacement by known methyltransferase inhibitors. Application of this assay to a pilot screen of an FDA-approved drug library identified several SAM-competitive inhibitors with low-micromolar potency. Subsequent screening of a structurally diverse 10,000-compound library yielded multiple hit compounds that directly inhibited N7-methylation catalyzed by both MPXV and vaccinia virus (VACV) N7-MTases in biochemical assays. Selected compounds suppressed VACV yield in cell-based assays at non-cytotoxic concentrations. Time-of-addition analyses and replication-defective MPXV system further indicated that these inhibitors act at the post-entry stage and impair viral gene expression. In summary, this study establishes a robust FP-based HTS platform for discovering SAM-competitive inhibitors of the MPXV N7-MTase and provides chemical probes and lead compounds for investigating poxvirus RNA capping as a target for antiviral intervention.