Development of small molecule inhibitors of the bacterial toxin pneumolysin

Abstract

Pneumolysin (PLY) is a cholesterol-dependent cytolysin secreted by Streptococcus pneumoniae and a major virulence factor in invasive pneumococcal diseases, including pneumonia, meningitis, otitis media, and myocarditis. Importantly, PLY remains active even after bacterial lysis, and the increasing prevalence of antibiotic resistance highlights the urgent need for adjunct antivirulence therapies capable of directly neutralizing this toxin. This thesis focuses on the development and optimization of small-molecule PLY inhibitors, building upon the previously identified lead compound Pathoblocker 3 (PB.3). Although PB.3 exhibits potent PLY inhibition (IC₅₀ = 3.2 µM), its limited aqueous solubility and susceptibility to hydrolysis in phosphate-buffered saline constrain its therapeutic potential. The primary objective of this work was to improve the stability and solubility of PB.3-derived compounds while retaining or enhancing inhibitory activity. Using a scaffold-hopping approach, PB.3 was divided into three structural regions (A, B, and C) to enable systematic modification. A focused library of derivatives was synthesized using Knoevenagel, Henry, and Vilsmeier–Haack reactions, while Meerwein, CuAAC, and Suzuki reactions provided key intermediates. The resulting compounds were evaluated for aqueous stability and solubility, followed by assessment of PLY inhibition in a collaboratively performed hemolysis assay. Structure–activity relationship analysis identified thiophene as a superior replacement for the original furan ring, yielding compounds with complete aqueous stability and enhanced potency. Thiophene-containing derivatives, including compounds 9, 14, 23, and 40, displayed strong PLY inhibition with IC₅₀ values in the low micromolar range (~2–5 µM) and high stability. In contrast, pyrrole- and triazole-based modifications negatively affected stability. Alternative warheads, such as nitrovinyl and olefinic hydroxyl groups, improved stability but resulted in reduced inhibitory activity. Thiobarbituric acid derivatives exhibited excellent potency but showed impaired stability. Overall, this work identifies stable and potent PLY inhibitors and supports their further development as adjunct therapeutics to complement antibiotic treatment of pneumococcal infections.