Clostridioides difficile (C. diff) infection is a major health issue which causes severe colitis and kills 30,000 out of the 500,000 people it affects every year in the United States. Current antibiotic treatments for C. diff are not always effective due to antibiotic resistance. Toxin B (TcdB) is one of the two major toxins secreted by C. diff. It contributes significantly to tissue damage and inflammation in the colon. Peptides are being considered as potential drug candidates to prevent and treat C. diff infection by inhibiting the biocatalytic activity and receptor-mediated internalization of TcdB, the primary virulence factor of C. diff. In this work, we computationally designed three types of peptide inhibitors to target TcdB: (1) 9-mer peptides for broad-spectrum activity against the TcdB1 and TcdB3 glucosyltransferase domain; (2) 30-mer α-helical peptides to target to the TcdB1 glucosyltransferase domain; and (3) 20-mer α-helical peptides to bind the TcdB1 delivery domain and prevent toxin internalization mediated by Frizzled proteins on the surface of the cell membrane. These designs were generated using a Monte Carlo–based algorithm, Peptide Binding Design (PepBD), and evaluated via explicit-solvent molecular dynamics simulations. Thus far, preliminary in vivo assays indicate that some of the 9-mer peptide candidates from method (1) not only neutralize TcdB with greater potency than previous peptide candidates but also exhibit broad-spectrum activity against both TcdB1 and TcdB3. In summary, we have applied a computational strategy that combines the PepBD algorithm and explicit-solvent atomistic molecular dynamics simulations, to design novel peptide inhibitors for TcdB. The therapeutic approaches in this work may pave the way of development of new options for treating C. diff infections.
