Identification and Experimental Validation of Peptide-Based Inhibitors Targeting Toxin A.

infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three discovered peptides, the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, the reference peptide (RP). These peptides may serve as potential inhibitors against infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in human jejunum cells. Both phage-display peptide RP and peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the peptide NPB demonstrates a relatively low efficacy against TcdA.