Inadequate membrane permeability often undermines the effectiveness of pharmacotherapy by preventing drugs from reaching their intracellular targets. Peptide-based delivery systems have been gaining traction, but designing an applicable peptide using traditional methods is costly and time-consuming. Herein, we employed an in-silico approach to developing a novel peptide from a marine source with cell-penetrating and reduced hemolytic activity. Template-based designing was conducted by relating activity to a series of physicochemical properties, followed by predicting the cell-penetrating activity via different prediction servers. Based on the available literature, knowledge-based modifications such as shortening of peptide sequence and amino acid substitutions have been made in the sequence.
Furthermore, MD simulation studies were employed to prioritize peptides for experimental validation and to gain mechanistic insights. The umbrella sampling technique accelerated the dynamics and sampling of rare events. In this direction, a novel peptide (PMSN) has been developed by modifying Polyphemusin, a well-known antimicrobial peptide from Limulus polyphemus. The designed peptide exhibited high penetration efficiency with a nuclear and cytoplasmic localization. Furthermore, PMSN acquired an alpha-helical structure in a membrane-mimicking environment. Interestingly, low cytotoxicity and nucleic acid binding ability have opened up various avenues for PMSN to serve as a potential nanocarrier to deliver nucleic acid cargo for therapeutic use. Moreover, beta-galactosidase as a cargo, coupled with peptide via electrostatic interactions, was also successfully loaded into MDA-MB-231 cells.