An Imidazo[2,1‐b][1,3,4]thiadiazole Derivative Inhibits the Virulence Factor α‐Hemolysin by Blocking the Pullout of Its Stem Domain

Staphylococcus aureus is a major human pathogen responsible for severe infections that necessitate alternative therapeutic strategies. Its key virulence factor α-hemolysin (Hla) mediates host cell damage via pore formation, making it an attractive target for antivirulence interventions. Here, we report the development of a high-throughput cellular assay measuring toxin-induced calcium influx. Its application led to the identification of thiadiazole-based small molecule inhibitors of Hla. Structure-activity relationship studies with 18 analogs led to inhibitors with a cellular potency up to 5.4 µM. X-ray crystallography of Hla in complex with compound 1 revealed that the thiadiazole bound a hydrophobic pocket at the interface of the amino latch and prestem domains, exerting a dual mechanism that blocks stem loop unfolding as well as membrane attachment. These findings introduce thiadiazoles as a novel chemical class of antivirulence therapeutics against S. aureus infections.