Characterization of Novel Peptides with Antimicrobial and Immunomodulatory Potential

In this study, we designed six peptides based on the characteristics of natural antimicrobial peptides (AMPs), including small size, cationicity, amphipathicity, and -helix structure. The peptides were evaluated for their antimicrobial activity, hemolytic potential, binding affinity to TLR4/MD-2 receptors, and immunomodulatory effects. Five out of the six designed peptides were classified as AMPs, while one peptide was predicted to be a non-antimicrobial peptide (NAMP) by the AxPEP server. The designed peptides exhibited varying degrees of antimicrobial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with SK1217 and SK1260 demonstrating stronger antimicrobial potential. Hemolytic assays revealed minimal hemolytic activity for SK1203, SK1217, SK1260, and SK1281, indicating a low propensity to cause red blood cell (RBC) lysis. However, SK1286 and SK1283 exhibited significant hemolytic effects at higher concentrations, with SK1286 causing complete hemolysis at 100 g/mL. Molecular docking analysis showed favourable binding affinities of SK1260, SK1217, and SK1286 with TLR4/MD-2 receptors. Furthermore, SK1260 displayed potent immunomodulatory activity, reducing the release of pro-inflammatory cytokines IL-6 and TNF- in LPS-stimulated macrophage cells. Cell viability assays demonstrated minimal cytotoxicity of SK1260. In vivo studies on infected mice showed that SK1260 treatment increased leukocyte recruitment and cytokine levels (GM-CSF, INF-g, and MCP-1) in the peritoneal lavage fluid. Overall, these findings highlight the promising potential of these cationic AMPs as antimicrobial and immunomodulatory agents for further therapeutic exploration.