Nanoscale Direct-to-Biology Optimization and Structural Insights into Selective S. aureus TrmD Inhibitors

The tRNA m1G37 methyltransferase (TrmD) is considered essential in various bacteria, including Staphylococcus aureus, a pathogen responsible for a wide range of diseases. Here, we have performed a high-throughput nanomole-scale synthesis campaign (nanoSAR) by late-stage copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC)-functionalizing a library of structurally diverse azides (N = 320) to a pyrrolopyrimidone alkyne. We have identified selective S. aureus TrmD inhibitors with inhibitory activity in the nanomolar to low micromolar range using a direct-to-biology assay read-out. A carbamate-masked guanidine intermediate of the lead structure selectively inhibited S. aureus growth at low micromolar concentrations in cell-based assays, while Gram-negative bacteria and an off-target panel of methyltransferases were not affected. Subsequent cocrystallization resulted in a crystal structure of S. aureus TrmD bound to an inhibitor, providing detailed insights into its binding mode and enabling future structure-guided optimization.