Nicotinamide-Based Covalent Inhibitors of tRNA (m ¹ G37) Methyltransferase (TrmD) to Increase Antibacterial Activity

High Resolution Image Download MS PowerPoint Slide Bacterial tRNA (m 1 G37) methyltransferase (TrmD) is an essential enzyme required for accurate translation and represents an attractive antibacterial target distinct from its eukaryotic counterpart, Trm5. Here, we describe the development of nicotinamide-based TrmD inhibitors incorporating electrophilic Michael-acceptor motifs to enhance cellular antibacterial activity. Mass spectrometry-based peptide mapping shows covalent modification of TrmD at Cys112, a residue proximal to the AdoMet ( S -adenosylmethionine) binding region, providing a mechanistic basis for covalent target engagement. Consistent with this mechanism, electrophile-containing analogues showed improved antibacterial activity compared with the parent scaffold. In bacterial growth inhibition assays, selected compounds displayed greater functional specificity for TrmD-driven growth suppression relative to human Trm5, supporting preferential bacterial target engagement. Collectively, these results establish Cys112 as a chemically addressable site for covalent inhibition of TrmD and provide a foundation for the development of TrmD-directed antibacterial agents.