Minibinders targeting Staphylococcus aureus α-toxin exert protective effects in vivo.

Staphylococcus aureus is a leading bacterial pathogen worldwide. It secrets α-toxin, a pore-forming cytotoxin that significantly contributes to the pathogenesis of pneumonia, skin necrosis, and lethal infections. Targeting α-toxin has emerged as a promising strategy for developing therapeutics against S. aureus infections. In this study, we employed a de novo computational design pipeline to generate minibinder proteins specifically targeting to α-toxin. Three top-ranked designs were expressed in E. coli, purified, and characterized. The minibinders exhibited thermostable α-helical structures and specific binding to α-toxin, with Binder2 showing the highest affinity (KD = 40.2 ± 3.1 μM). All designs effectively inhibited α-toxin's hemolytic and cytotoxic activities in vitro. Notably, Binder2 attenuated α-toxin-induced cytokine expression in A549 and THP-1 cells. Mechanistically, binders neutralized α-toxin cytolytic activity by blocking its cell binding and heptamer formation. In a murine model, co-injection of minibinders with α-toxin significantly extended the survival times of mice challenged with α-toxin, and Binder2 exhibited the most potent protective effects. These findings highlight Binder2 as a promising lead molecule for further development as an α-toxin antagonist. Our study demonstrates the potential of computationally designed minibinders as a novel therapeutic approach against S. aureus infections.