Strathclyde Minor Groove Binders Bearing Amidine Tail Groups are Effective against Gram-Positive Bacterial Pathogens.

The rise of multidrug-resistant Gram-positive pathogens necessitates new antibacterial agents with mechanisms that are distinct from those of existing therapies. Here, we report the antibacterial activity and mechanistic characterization of two Strathclyde minor groove binders (S-MGBs), S-MGB-234 and S-MGB-235, synthetic DNA binding molecules designed to disrupt essential bacterial processes. These compounds displayed potent in vitro activity against clinically relevant Gram-positive pathogens, including Staphylococcus aureus and Enterococcusspp. However, potency was less pronounced against Enterococcus faecalis. Activity was retained against drug-resistant strains, and reduced susceptibility emerged more slowly during serial passaging than that observed for gentamicin under the conditions tested. Whole-genome sequencing of reduced susceptibility mutants did not identify mutations in canonical DNA targets but instead revealed recurring changes in genes associated with the cell envelope, including norA, fmtA, and cozEb, suggesting that envelope-mediated effects influence compound access rather than direct target modification. Biophysical assays demonstrate strong interactions with AT-rich oligonucleotides and gDNA, consistent with the DNA binding properties previously reported for the S-MGB class. Together, these findings demonstrate that S-MGBs represent a promising class of DNA-targeting antibacterials and provide initial evidence that reduced susceptibility emerges through heterogeneous mechanisms that do not involve obvious modifications of DNA targets.