Pneumolysin (PLY) is a pore-forming, cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main bacterial cause for community-acquired pneumonia. Liberation of PLY during host infection leads to strong immune activation and cytolytic cell death. Thus, inhibition of PLY could be a valuable approach to attenuate detrimental effects of hyper-inflammatory immune reactions during pneumococcal lung infection. Here, we report discovery, development, and validation of small molecule inhibitors of PLY, denominated as pore-blockers (PB). PB-1 was identified by combined screening inhibiting PLY-mediated hemolysis. PB-2 blocked pore formation with greatly improved potency as demonstrated by cryo-electron tomography. Scaffold-hopping delivered PB-3 with superior chemical stability, solubility, and a specific mode of action, characterized by an elongated residence time. It prevented human lung epithelial cells from PLY-mediated cytolysis and cell death, also during infection with Streptococcus pneumoniae. In conclusion, druglike PLY-inhibitors such as PB-3 might become valuable adjuvant options in the treatment of severe pneumococcal infections.
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