Engineering of a Novel Amphibian Skin Peptide Isolated from Agua Rica Leaf Frog (Callimedusa ecuatoriana) into Active Antimicrobial Agents

<b>Background/Objectives</b>: The increasing antimicrobial resistance is a current human health threat, which has stimulated research on new biologically active molecules against infections caused by microorganisms resistant to conventional therapies. Antimicrobial peptides (AMPs) from amphibian skin secretions have generated great interest in tackling this problem due to their antibacterial, antifungal, antiprotozoal, wound-healing, and even anticancer properties. In Ecuador, there are still unexplored endemic amphibian species as a source of new AMPs, such as <i>Callimedusa ecuatoriana</i>. In this study, we report a novel peptide derived from the skin secretion of <i>Callimedusa ecuatoriana</i> identified by molecular cloning of the mRNA precursor. The functional analysis demonstrated that it lacks antimicrobial activity due to its alpha-helix kink structure. <b>Methods</b>: Inspired by the native structure of PTR-CE1, we designed and synthesized two analogs (PTR-CE1a and PTR-CE1b) to adopt a complete α-helix secondary structure, a conformation often associated with antimicrobial activity. In silico tools were used to predict the peptide activity, which was confirmed by experimental findings. <b>Results</b>: Both analogs displayed higher activity than the native peptide, even against the ampicillin-resistant bacterial strain. While PTR-CE1b showed Minimum Inhibitory Concentration (MIC) values of 26.62-212.99 μM and 24.36% of hemolytic activity at 26.62 μM, PTR-CE1a displayed a more potent broad-spectrum activity against all the microorganisms, with MIC values of 3.02-12.06 μM and hemolytic activity of 7.5% at 3.02 μM. <b>Conclusions</b>: This study demonstrates the importance of the α-helix structure for antimicrobial activity in <i>C. ecuatoriana</i> PTR-CE1 analogs and highlights the potential of unexplored biological and molecular diversity in endemic species of Ecuador to provide novel templates for peptide design.