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

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.