Structure‐based development of a covalent inhibitor targeting Streptococcus pyogenes over Staphylococcus aureus sortase A

Sortase A (SrtA), a cysteine transpeptidase critical for surface protein anchoring in Gram‐positive pathogens, represents an attractive antivirulence target. While covalent SrtA inhibitors show therapeutic potential, existing compounds lack species selectivity. Through structure‐guided design, we developed T10, a covalent inhibitor selectively targeting Streptococcus pyogenes SrtA (SpSrtA) over Staphylococcus aureus SrtA (SaSrtA). Molecular docking revealed that shortening a "C=C" bond in lead compound ML346 eliminated SaSrtA inhibition due to steric hindrance from W194, while maintaining SpSrtA binding. X‐ray crystallography confirmed T10's covalent modification of Cys208 in SpSrtA. T10 demonstrated two fold enhanced inhibitory potency and species‐specific disruption of M‐protein anchoring and biofilm formation in Streptococcus pyogenes, without affecting Staphylococcus aureus viability. In a Galleria mellonella infection model, T10 conferred potent protection against lethal infection. This work demonstrates the development of narrow‐spectrum antivirulence agents through a structure‐based rational strategy.