Nicotinamide-Based Covalent Inhibitors of tRNA (m1G37) Methyltransferase (TrmD) to Increase Antibacterial Activity

Abstract Bacterial tRNA (m1G37) 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.