[Lys3, Lys4] M-PONTX-Dq3a [1-15]: a selective dinoponeratoxin-derived peptide that disrupts parasite membranes and inhibits TcGAPDH activity in Trypanosoma cruzi.

Chagas disease remains a neglected tropical disease and a major public health concern, particularly due to the limitations of current treatments with benznidazole and nifurtimox, which are associated with significant adverse effects and limited efficacy during the chronic phase of infection. In this context, antimicrobial peptides (AMPs) have emerged as promising scaffolds for the development of antitrypanosomal drugs, because to their broad-spectrum activity and multiple mechanisms of action. Previous studies demonstrated the potent anti-Trypanosoma cruzi activity of M-PONTX-Dq3a, an AMP derived from the venom of Dinoponera quadríceps. Subsequent structural optimization yielded the truncated peptide M-PONTX-Dq3a [1-15] and the analogue [Lys3]M-PONTX-Dq3a [1-15], both exhibiting enhanced trypanocidal activity and selectivity compared with the original peptide. Based on these findings, the present study aimed to further optimize [Lys3]M-PONTX-Dq3a [1-15] through rational amino acid substitutions designed to generate peptides capable of promoting membrane disruption while simultaneously inhibiting enzymes essential for parasite survival. Among the analogues evaluated, [Lys3, Lys4] M-PONTX-Dq3a [1-15] exhibited potent trypanocidal activity against multiple developmental forms of T. cruzi, reduced cytotoxicity toward mammalian cells, low hemolytic activity at biologically active concentrations, and an improved selectivity index. Mechanism of action studies showed that this analogue promoted membrane permeabilization, the accumulation of reactive oxygen species, and mitochondrial membrane depolarization. In addition, molecular docking predicted distinct binding modes for TcGAPDH and hGAPDH, consistent with preferential recognition of the parasite enzyme, while enzymatic assays demonstrated significant inhibition of TcGAPDH activity. Taken together, these findings support [Lys3, Lys4] M-PONTX-Dq3a [1-15] as a promising scaffold for the development of new anti-Chagas agents and highlight the potential of rational peptide engineering for the development of novel antiparasitic agents.