Initial leads to combat streptogramin resistance generated from X-ray fragment screening against VatD.

Streptogramins are potent antibiotics targeting bacterial ribosome. The synergistic binding of group A and B streptogramins to 50S-ribosome yields bactericidal effects. However, their efficacy is compromised by resistance mechanisms, including enzymatic acetylation of group A streptogramins by virginiamycin acetyltransferase (Vat) enzymes, which reduces their affinity for ribosomes. Using fragment-based drug discovery we identified starting points for development of VatD inhibitors. X-ray crystallography screening revealed three primary fragment-binding sites on VatD. In the acetyl-binding subsite, fragments stabilized distinct conformational states in critical residues, His82 and Trp121. In the antibiotic-binding site, two fragments formed interactions that could be leveraged for competitive inhibition. Elaborations of these fragments showed weak inhibition of VatD activity, indicating potential for further optimization. These findings establish initial hits that could restore streptogramin efficacy by targeting VatD directly, providing a structural foundation for inhibitor development against resistant bacterial strains.