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Virtual Screening and Multistage Computational Profiling of Small-Molecule Inhibitors Targeting Clostridioides
Runze Wang1,2, Qiuyu Zhang1, Yi Lin1
1Hangzhou Medical College, School of Laboratory Medicine and Bioengineering, Hangzhou, Zhejiang CN 310053, China.
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.
Insights
A repurposed drug, Dirlotapide, effectively inhibits Clostridioides difficile toxin B (TcdB) by blocking its receptor-binding interfaces. This pan-subtype inhibitor shows promise in preclinical models for treating C. difficile infection (CDI).
Area of Science:
- Microbiology
- Pharmacology
- Computational Biology
Background:
- Clostridioides difficile infection (CDI) is a major cause of antibiotic-associated diarrhea.
- Toxin B (TcdB) is a key virulence factor in CDI, with diverse receptor-binding interfaces (RBIs).
Purpose of the Study:
- To computationally identify and repurpose FDA-approved drugs as broad-spectrum inhibitors of TcdB.
- To develop a pan-RBI, pan-subtype TcdB inhibitor for potential CDI treatment.
Main Methods:
- Multistage computational pipeline including virtual screening, molecular docking, binding free energy calculations, and molecular dynamics simulations.
- Experimental validation using surface plasmon resonance (SPR), circular dichroism (CD), in vitro cell-based assays, and in vivo mouse models.
Main Results:
- Dirlotapide was identified as a lead compound with high-affinity binding to TcdB.
- Dirlotapide protected Caco-2 cells from TcdB-induced cytotoxicity and demonstrated efficacy in a mouse model of CDI.
- Preclinical validation confirmed gut-restricted pharmacokinetics, low toxicity, and translational potential.
Conclusions:
- Dirlotapide is a promising, rapidly repositionable therapeutic agent against clinically relevant TcdB subtypes.
- This study highlights the potential of computational drug repurposing for infectious diseases.
