Characterization of novel peptides with antimicrobial and immunomodulatory potential

In the present study, we designed six peptides based on key characteristics of natural antimicrobial peptides (AMPs), including small size, cationicity, amphipathicity, and α-helical structure. The peptides were evaluated for their antimicrobial activity, hemolytic potential, binding affinity to TLR4/MD-2 receptors, and immunomodulatory effects. Using the AxPEP server, five of the six peptides were classified as AMPs, while one was identified as a non-antimicrobial peptide (NAMP). The antimicrobial activity of the designed peptides varied against Escherichia coli (E.coli), Staphylococcus aureus (S.aureus), Klebsiella pneumoniae (K.pneumoniae), and Pseudomonas aeruginosa (P.aeruginosa), with SK1217 and SK1260 being the most potent. Hemolytic assays revealed that SK1203, SK1217, SK1260, and SK1281 caused minimal red blood cell (RBC) lysis, indicating low hemolytic potential. In contrast, SK1286 and SK1283 displayed significant hemolysis at higher concentrations, with SK1286 inducing complete hemolysis at 100 μg/mL. Molecular docking analysis demonstrated strong binding of SK1260, SK1217, and SK1286 to TLR4/MD-2 receptors. Notably, SK1260 exhibited robust immunomodulatory activity, suppressing the release of pro-inflammatory cytokines IL-6 and TNF-α in LPS-activated macrophages. Cytotoxicity assays showed minimal toxicity for SK1260. Treatment with SK1260 enhanced the white blood cell count and increased cytokine levels (GM-CSF, INF-γ, and MCP-1) in the peritoneal lavage fluid of infected mice. Collectively, these findings underscore the potential of these cationic AMPs, particularly SK1260, as promising candidates for antimicrobial and immunomodulatory therapeutic applications.