Virtual Screening and Multistage Computational Profiling of Small-Molecule Inhibitors Targeting Clostridioides difficile Toxin B.

Abstract

Clostridioides difficile infection (CDI), a leading cause of antibiotic-associated diarrhea, is driven by the virulence factor toxin B (TcdB), whose receptor-binding interfaces (RBIs) exhibit evolutionary divergence into α and β subtypes with distinct host-receptor specificities. Leveraging this insight, we developed a multistage computational pipeline to repurpose FDA-approved drugs as pan-RBI, pan-subtype TcdB inhibitors. Virtual screening of 10,027 compounds against AlphaFold3-predicted TcdB1-8 structures, integrated with molecular docking (AutoDock Vina/LeDock), MM/GBSA binding free energy calculations, and molecular dynamics simulations, prioritized three candidates. Dirlotapide (DB11399) emerged as the lead compound, demonstrating high-affinity binding to TcdB2 via surface plasmon resonance (SPR) and triggering partial unfolding of its α-helical structure as measured by circular dichroism (CD). In vitro, Dirlotapide rescued Caco-2 cells from cytotoxicity induced by all four major TcdB subtypes. Following rectal administration in mice, it protected against TcdB2-induced colonic damage, restored epithelial integrity, and significantly reduced proinflammatory cytokines (IL-6, TNF-α). Validation of gut-restricted pharmacokinetics, intestinal permeability, CYP450 interactions, and low nephrotoxicity supports its translational potential. Dirlotapide represents a rapidly repositionable anti-TcdB agent that neutralizes clinically relevant subtypes through dual RBI blockade.