Head-to-Tail Cyclization and D-Amino Acid Substitution Redesign the Biological Activities of a Naturally Occurring Amphibian Peptide.

Peptide engineering has emerged as a powerful strategy to optimize naturally occurring peptides. Here, the amphibian skin peptide Hp-1891 from Boana pulchella was selected as a model scaffold to investigate the effects of two complementary engineering approaches, namely site-specific D-amino acid substitution and head-to-tail cyclization. A library of twelve analogues was synthesized by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis and evaluated for inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), together with antioxidant and hemolytic activities. Circular dichroism spectroscopy and molecular modeling were performed to investigate the structural basis of the observed biological effects. Head-to-tail cyclization consistently enhanced inhibition of AChE, BChE, and Mpro, whereas D-amino acid substitution exerted a greater influence on antioxidant activity and hemolysis. Among the analogue library, c-Hp-d2 emerged as the most promising multifunctional peptide, displaying enhanced inhibition of all three enzymes while maintaining reduced hemolytic activity compared with the native peptide. Structural analyses indicated that cyclization promoted conformational organization, whereas D-amino acid incorporation reduced α-helical propensity. These findings demonstrate that rational peptide engineering effectively reshapes the biological profile of amphibian peptides and highlight head-to-tail cyclization as a versatile strategy for generating multifunctional peptide scaffolds with therapeutic potential.