An In Silico and In Vitro Approach to Identify Potential Phytocompounds as Inhibitors of Pneumolysin, a Secretory Toxin of Streptococcus pneumoniae

Abstract

Pneumolysin (PLY), a cholesterol-dependent cytolysin produced by Streptococcus pneumoniae, plays a key role in the pathogenesis of bacterial pneumonia. The present study aimed to identify potential phytocompound inhibitors of pneumolysin and evaluate their protective effects against toxin-induced cytotoxicity using combined in silico and in vitro approaches. A ligand library comprising 200 phytocompounds was constructed using three-dimensional structures obtained from the PubChem database, while the PLY protein structure was retrieved from the Protein Data Bank. Molecular docking was performed to analyse protein-ligand interactions, followed by visualization using BIOVIA Discovery Studio Visualizer. Top-ranked phytocompounds from docking were further screened by computational ADME analysis, followed by molecular dynamics (MD) simulations for stability analysis. Selected compounds were then validated using RAW 264.7 macrophages, and cytotoxicity was assessed by flow cytometry. Among the screened compounds, three showed high binding affinity, with oridonin exhibiting the most favourable interaction profile (−7.906 kcal/mol). MD simulation confirmed the stability of the pneumolysin–oridonin complex. In vitro results demonstrated that pneumolysin induced significant, concentration-dependent cytotoxicity, whereas pre-incubation with oridonin significantly reduced cell death. To provide direct functional evidence of pneumolysin inhibition, haemolysis inhibition assay using sheep erythrocytes was performed where reduction of pneumolysin-mediated haemolysis by oridonin in a concentration-dependent manner was demonstrated. These findings suggest that oridonin may serve as a potential inhibitor of pneumolysin-mediated cytotoxicity and pneumococcal virulence.