Rational engineering of a novel cationic LL-37-temporin SHa hybrid peptide with anti-parasitic activity and effects on mitochondrial membrane potential in protozoan parasites.

The emergence of drug resistance and host toxicity is associated with conventional antiprotozoal therapies which necessitates development of novel and selective antiparasitic agents. In the present study, novel hybrid peptide which is HP-AP-1 (KRIVQRIDKFLRNFLPLLAKK) was rationally designed and evaluated through combination of LL-37 membrane-active core with a truncated temporin-derived hydrophobic motif followed by C-terminal cationic optimization. Physicochemical analysis revealed 21-amino-acid peptide with molecular weight of 2597.23 Da, high theoretical pI (11.74), +6 net charge, moderate hydrophobicity (GRAVY -0.257) and strong α-helical propensity (>95%). Helical wheel projection confirmed well-defined amphipathic structure. Analytical RP-HPLC verified approximately 97.25% purity and an accurate molecular mass was confirmed through MALDI-TOF MS. Biological evaluation also demonstrated negligible hemolytic activity of <5% at 64 μM and low cytotoxicity toward RAW264.7 macrophages of >75-85% viability. HP-AP-1 has exhibited dose-dependent antiparasitic activity against Tritrichomonas foetus and Leishmania infantum with ICvalues approximately 8-9 μM. Notably, the peptide was significantly reduced intracellular amastigote burden in the infected macrophages. Mechanistic studies revealed concentration-dependent mitochondrial membrane depolarization by suggesting potential membrane-associated effects. Collectively, these findings have demonstrated that rational hybridization of LL-37 and temporin motifs with C-terminal cationic reinforcement yields a selective, amphipathic and mechanistically versatile antiparasitic peptide. HP-AP-1 represents a promising in vitro anti-parasitic peptide candidate requiring further in vivo validation and serves as a rationally designed scaffold for future optimization studies.