Design of 8-mer peptides that block *Clostridioides difficile* toxin A in intestinal cells

*Clostridioides difficile* (*C. diff*) is a bacterium causing significant intestinal infections, primarily impacting the colon. These infections, often resulting from antibiotic use, lead to diarrhea and colitis. Current treatments, such as antibiotics (metronidazole and vancomycin) and the monoclonal antibody Bezlotoxumab, have limitations including recurrence rates and high cost. Fecal Microbiota Transplant (FMT), while promising, has risks. The pathogenicity of *C. diff* stems from two major toxins, Toxin A (TcdA) and Toxin B (TcdB), which glucosylate Rho-family GTPases, disrupting cell function. This research explores the use of short peptides as a cost-effective and targeted therapeutic strategy. The study aims to identify peptide inhibitors that bind to the glucosyltransferase domain (GTD) of TcdA, leveraging computational design, simulations, and experimental validation. Prior research identified a 10-mer peptide, NPA, that showed toxin neutralizing activity in small intestinal cells, but not in colon cells. This is hypothesized to be due to protease activity in the small intestine, which cleaves the peptide into shorter, more active fragments. This study builds on this previous work by focusing on computationally designing shorter peptides (8-mers) that might exhibit greater potency, and which aren't reliant on protease activity in colon cells for their activity.

The literature review extensively covers the current understanding of *C. difficile* infection, highlighting the role of toxins A and B in pathogenesis. It also discusses existing treatment options, including antibiotics, Bezlotoxumab, and FMT, detailing their limitations in terms of efficacy, cost, and safety. The review emphasizes the need for novel therapeutic strategies and the potential of short peptides as cost-effective, targeted inhibitors. Existing research on peptide-based inhibitors of *C. difficile* toxins, such as the work by the Feig lab utilizing phage display to identify peptides that bind to the TcdA GTD, is incorporated to provide context for the current study. This prior work's limitations in achieving efficacy in colon cells are discussed, motivating the focus on shorter, 8-mer peptides in the present research.

The researchers employed a multi-faceted approach combining computational design, molecular-level simulations, and experimental validation. The study begins by using the PepBD algorithm, a computational peptide binding design algorithm developed by the research group. The algorithm screens for peptide binders to biomolecular targets. The 8-mer NPA was used as a reference peptide, generating sequences that can bind to the TcdA GTD with higher binding affinity than 8-mer NPA. Molecular dynamics (MD) simulations were performed on fragments of a previously identified 10-mer peptide (NPA) to determine the optimal length for effective TcdA GTD binding, which was determined to be 8-mers. The PepBD algorithm was then used to design 8-mer peptides using the 8-mer fragment of NPA as the reference. Explicit solvent atomistic MD simulations and binding free energy calculations were conducted to assess the binding affinity of these in silico-designed peptides. A microfluidic bead-based platform was used to quickly screen the peptides for TcdA binding and TcdA GTD binding, filtering out weak inhibitors. The remaining peptides were tested for efficacy using a trans-epithelial electrical resistance (TEER) assay in primary-derived human colon epithelial cells. Finally, the binding affinity of the top-performing peptide (SA1) to TcdA was characterized using surface plasmon resonance (SPR). The SPR analysis allowed for the determination of the equilibrium dissociation constant (K_D) and the kinetic parameters (association rate constant *k_a* and dissociation rate constant *k_d*). The methodology includes details on peptide synthesis, labeling of TcdA with Alexa Fluor 594, and the use of a fluorescent UDP-Glucose analog in the bead-based assay. The cell-based assay used primary human gut epithelial stem cells derived from the large intestine. The SPR experiments involved covalent attachment of SA1 to gold sensors using a mixed bioresistant thiol SAM for surface immobilization.

The computational design, using the PepBD algorithm and MD simulations, yielded seven candidate peptide inhibitors (SA1-SA7). A bead-based assay screened these peptides, identifying four (SA1-SA4) for further in vitro evaluation. SA1 emerged as the top performer in the subsequent TEER assay on primary human colon epithelial cells. SA1 demonstrated significant protection (~79%) against TcdA-induced toxicity at a clinically relevant TcdA concentration (30 pM). Surface plasmon resonance (SPR) measurements determined SA1's dissociation constant (K_D) to TcdA as 56.1 ± 29.8 nM, indicating a moderate-to-high affinity interaction. The SPR analysis revealed a high adsorption rate constant (k_a) of 7.0 ± 2.5 × 10⁻⁵ nM⁻¹ s⁻¹. Key interacting residues on SA1 were identified as Trp3, Trp4, Arg5, Arg6, His7, and Asn8, suggesting the importance of these amino acids for TcdA GTD binding. Notably, the PepBD algorithm successfully designed peptides that selectively bind to the TcdA GTD, indicating robustness of the computational approach. Analysis of the top-performing peptides revealed amino acid sequence signatures that could be further exploited for the design of even more effective inhibitors.

The findings directly address the research question by identifying and characterizing a novel 8-mer peptide (SA1) that effectively blocks TcdA activity in primary human colon epithelial cells. The success of SA1 highlights the potential of short peptides as a therapeutic approach for *C. difficile* infection. The moderate-to-high affinity binding of SA1, combined with its strong protective effects in the cell-based assay, suggests that it is a promising candidate for further development. The computational design strategy employed in this study provides a powerful tool for identifying new peptide inhibitors. The identification of amino acid sequence signatures associated with high-affinity TcdA binding also facilitates the rational design of improved inhibitors. The use of primary human colon epithelial cells enhances the physiological relevance of the findings compared to previous studies that utilized cancer cells or transformed cell lines.

This study successfully designed and validated an 8-mer peptide, SA1, as a potent inhibitor of *C. difficile* Toxin A in human colon cells. The combination of computational design and experimental validation demonstrates the power of this approach in drug discovery. Future research should focus on evaluating SA1's efficacy in vivo and exploring the possibility of developing it into a clinically useful therapeutic for *C. difficile* infection. Further optimization of peptide sequences based on identified amino acid signatures may lead to even more effective inhibitors. Investigation of the potential for combination therapy with SA1 and existing treatments should also be explored.

The study primarily focused on the inhibition of TcdA. While TcdA and TcdB share sequence homology, the efficacy of SA1 against TcdB needs further investigation. The in vitro nature of the study limits its direct translatability to the complexities of the human gut environment. Further studies are required to assess the in vivo efficacy, pharmacokinetics, and safety of SA1 before clinical application is considered. The sample size for the SPR assay was relatively small, though the consistent results across several measurements, including repeats, suggest that the results are reliable.