Antimicrobial resistance is a global health threat, which has been worsened by the slow development of new antibiotics. The rational design of natural-derived antimicrobial peptides (AMPs) offers a promising alternative for enhancing the efficacy of AMPs and accelerating drug discovery. This paper describes the rational design of improved peptide derivatives starting from hylin-Pul3, a peptide previously isolated from the frog Boana pulchella, by optimizing its hydrophobicity, cationicity, and amphipathicity. In silico screening identified six promising candidates: dHP3-31, dHP3-50, dHP3-50.137, dHP3-50.190, dHP3-84, and dHP3-84.39. These derivatives exhibited enhanced activity against Gram-negative bacteria, emphasizing the role of cationicity and the strategic arginine incorporation. Hemolytic assays revealed the derivatives’ improved selectivity, particularly for the derivatives with “imperfect amphipathicity”. In fibroblast assays, dHP3-84 was well-tolerated, while dHP3-84.39 promoted cell proliferation. Antioxidant assays (ABTS assays) highlighted the Trp-containing derivatives’ (dHP3-50.137, dHP3-31) significant activity. The lipid membrane interaction studies showed that hylin-Pul3 disrupts membranes directly, while dHP3-84.39, dHP3-50, and dHP3-50.137 promote vesicle aggregation. Conversely, dHP3-84 did not induce membrane disruption or aggregation, suggesting an intracellular mode of action. Machine learning models were effective in predicting bioactivity, as these predicted AMPs showed enhanced selectivity and potency. Among them, dHP3-84 demonstrated broad-spectrum potential. These findings highlight the value of rational design, in silico screening, and structure–activity studies in optimizing AMPs for therapeutic applications.

Ocellatins are a family of antimicrobial peptides found exclusively in the Leptodactylus genus. To date, 10 species have been studied and more than 23 peptides described. Here we report the sequences of five new peptides from the skin of the frog Leptodactylus latrans (Anura: Leptodactylidae) determined by cDNA cloning of the complete prepro-peptide structures. The mature peptides were characterized with in silico tools and compared with those previously described. With 21 amino acid residues, this new set of peptides not previously described in the Leptodactylus genus share between 100 and 76.2% similarity to ocellatin antimicrobial peptides. These novel peptides are cationic and their three-dimensional (3D) structure holds the highly conserved residues G¹, D⁴, K⁷, and K¹¹ and a high theoretical amphipathic α-helix content. Furthermore, in silico analyses of these new peptides predicted antimicrobial activity. This study is framed in the context of previous work published about ocellatins, and therefore, provides a review of this intriguing family of peptides.