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.