Pneumolysin (PLY) is a pore-forming toxin belonging to the family of cholesterol-dependent cytolysins (CDC). The 53 kDa protein is a major virulence factor of Streptococcus pneumoniae (S.pn.), which is the leading bacterial cause of community-acquired pneumonia, meningitis, and otitis media. Despite of the broad availability of antibiotics against S.pn., every year around two million patients die from pneumonia worldwide, primarily infants and elderly people. The virulence factor PLY is considered widely as a main trigger of morbidity and mortality in invasive S.pn. The protein is expressed within nearly all known serotypes and released from the bacteria as soon as the cells are ruptured due to auto-or antibiotic-mediated lysis. As a consequence, the toxin can exert active function inside the host, even in the case the antibiotics successfully killed the bacteria. Therefore, there is a crucial medical need for an anti-virulence strategy able to neutralize PLY. PLY comprises 471 amino acids organized in four domains. C-terminal domain 4 attaches to cholesterol molecules embedded in mammalian cell membranes via amino acids T459 and L460. Once bound to the cell membrane, monomers of PLY oligomerize via the noncontiguous N-terminal domains 1-3 to form a pre-pore complex that subsequently undergoes a sequence of structural transitions to form a membrane-infiltrating β-barrel pore. Pore formation results subsequently in cell death by cytosolic and mitochondrial calcium overload. A typical pore is assembled from up to 50 PLY monomers and has an average diameter of 350 Å. Pathogenesis associated with PLY, however, is not limited to cell death. PLY significantly enhances bacterial colonization throughout the host and activates the immune system by triggering the inflammasome and may also contribute to organ damage and septic shock. Despite of the medical need currently no clinically admitted drugs are available for the targeted treatment or prevention of PLY-mediated pathogenesis. Unspecific phytochemicals with reported polypharmacology, including flavonoids such as quercetin or epigallocatechin, plant steroids, and other natural products have been found to interfere with PLYactivity, however, these molecules have been reported active toward numerous proteins and against many diseases without adequate biological or clinical evidence. In addition, lipoproteins and liposomes loaded with cholesterol or sitosterol have been shown to scavenge PLY. The therapeutic value of such lipid-based approaches to remove PLY is, however, not clear, yet, and interference with lipid homeostasis might create additional problems. In summary, no drug-like and directly acting PLY inhibitors have been reported so far and PLY can be considered constituting a "difficult drug target".