Computational engineering of EB peptide to unravel potential mpox entry blockers through in silico saturation mutagenesis

Abstract

Background : The emergence of Mpox highlights the lack of virus-specific antivirals, as current agents offer limited efficacy and pose toxicity risks. Entry blocker (EB) peptide blocks Orthopoxvirus entry but is effective only when co-administered with the virus and shows no prophylactic or therapeutic benefit against vaccinia virus in vivo . Thus, this study applied in silico saturation mutagenesis to produce EB-derived peptides with enhanced affinity and stability toward Mpox A29L, M1R, A35R, and B6R surface proteins. Methods : The tertiary structure models for the target Mpox surface proteins were generated and validated prior to molecular docking. In silico saturated mutagenesis was conducted to analyze for critical residues that can aid in peptide-protein binding enhancement. Potentially toxic peptides were also identified and excluded. The dissociation constant (K D ) and Gibbs free energy of binding (ΔG bind ) were calculated. The values of differential binding energy (ΔΔG) were also determined to uncover candidate peptides. Results : High-quality tertiary structure models enabled reliable docking, revealing fifteen EB-derived peptides with stronger binding affinity than the wild-type EB (WTEB) peptide. Peptides predicted to interact with potentially glycosylated sites were determined, refining the set to eleven candidate peptides, including six candidates which demonstrated large binding enhancement (ΔΔG > 1 kcal/mol). Results from molecular dynamics analysis further verify that A29L3W and A29L8W can form more stable, well-equilibrated complexes with Mpox A29L than the WTEB. In addition, candidate peptides M1R1E and B6RCom displayed favorable physicochemical stability. Conclusion : Overall, the computational workflow developed and applied in this research presents eleven EB-derived candidate peptides as promising Mpox entry inhibitors, pending subsequent in vitro and clinical studies.