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

Abstract

Streptogramins, a potent antibiotic class targeting bacterial ribosomes, are effective against severe infections such as those caused by vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococcus faecium (VRE). The synergistic binding of group A and B streptogramins to the peptidyl transferase center (PTC) and nascent peptide exit tunnel (NPET), respectively, yields a bactericidal effect. However, the efficacy of streptogramins is compromised by resistance mechanisms, including the enzymatic acetylation of group A streptogramins by Virginiamycin Acetyl Transferase (Vat) enzymes, which reduce antibiotic affinity for the bacterial ribosome. Here, we applied fragment-based drug discovery (FBDD) to identify starting points for the development of inhibitors of VatD. X-ray crystallography screening revealed three primary fragment-binding sites on VatD. In the acetyl-CoA binding site, fragments bound to stabilize distinct conformational states in critical residues, His82 and Trp121, within the acetyl subsite. In the antibiotic binding site, two fragments were identified that form unique hydrogen/hydrophobic bonds, suggesting specific interactions that could be leveraged for competitive inhibition by disrupting streptogramin modification. Elaborations of these fragments showed weak inhibition of VatDs acetyltransferase activity, indicating the 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.