Abstract
Cecropin A is a potent antimicrobial peptide with broad-spectrum activity; however, its clinical application is limited by poor stability, solubility, and bioavailability. In this study, an engineered analog of Cecropin A was developed by incorporating an M cell-targeting peptide (MTP) at the N-terminus, a self-assembling EAK16-II sequence at the C-terminus, and a rigid EAAAK linker to preserve domain integrity. This engineered peptide (Pep A) exhibited significantly improved physicochemical properties, including enhanced solubility (0.930), a higher extinction coefficient (8480 M(- 1)cm(- 1)), and a more favorable net positive charge (+ 7), all of which may support stronger interactions with microbial membranes and enhanced therapeutic potential. Despite a slightly elevated instability index, the peptide remained within the stable range. The engineered peptide was predicted to be non-toxic, in contrast to its native form, which was identified as toxic. Furthermore, its high solubility upon overexpression facilitates large-scale recombinant production in bacterial systems. Computational modeling using AlphaFold and Robetta confirmed the structural accuracy of the design, with Pep A outperforming other models in compactness and favorable folding. Molecular dynamics simulations further validated the improved stability of Pep A, demonstrating reduced flexibility, consistent hydrogen bonding, and lower solvent exposure. Point mutations at surface-accessible, flexible, and non-critical residues further enhanced its stability. Collectively, these modifications produced a multifunctional Cecropin A analog with improved structural stability, solubility, and bioavailability, establishing Pep A as a promising candidate for advanced antimicrobial and mucosal delivery applications. However, in vitro and in vivo validations are critical for confirming its final applications.