Evelline Araújo Edson¹, Emanuel de Paula Magalhães¹, Bruna Viana Barroso Martins¹, Mariza Pereira dos Reis¹, Márcia Machado Marinho², Emmanuel Silva Marinho², Ramon Róseo Paula Pessoa Bezerra de Menezes¹, Alice Maria Costa Martins Nunes¹

Introduction: Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered a neglected disease and a global public health concern. Furthermore, the available drugs used for treatment show relevant adverse effects. Antimicrobial peptides (AMPs) act in the natural defense of many organisms and have potential application in biotechnology, being promising as molecular models for the development of new therapeutic approaches. The AMP [Lys³, Lys⁴]M-PONTX-Dq3a[1-15], a synthetic analogue of the dinoponeratoxin M-PONTX-Dq3a, shows promising action against T. cruzi. Given the need to develop new effective and less toxic anti-Chagasic therapies, and since trypanosomid enzymes constitute strategic therapeutic targets in the development of these drugs, the objective of this study was to evaluate whether the analogue [Lys³, Lys⁴]M-PONTX-Dq3a[1-15] has activity on the enzyme TcGAPDH, which acts in the glycolytic pathway of T.cruzi. Methodology: For this purpose, the analogue [Lys³, Lys⁴]M-PONTX-Dq3a[1-15] was tested for its toxicity in epimastigote forms of T. cruzi in a concentration range of 0.75–50 µM for 24 hours of incubation. After this period, the epimastigotes were counted in a Neubauer chamber to obtain the mean inhibitory concentration (IC₅₀) of the peptide. Subsequently, molecular docking was performed using the AutoDock Vina program to simulate the coupling of the peptide with the binding site of the TcGAPDH enzyme. Benznidazole (BZN), a reference drug for Chagas disease, and chalepin, a natural inhibitor of TcGAPDH, were also used in the simulations to obtain comparative data. The inhibition of TcGAPDH enzymatic activity was evaluated by a GAPDH assay kit (Abcam), using the peptide according to the IC₅₀ and twice this concentration, obtained in the cytotoxicity assay. Results: Regarding cytotoxicity, the peptide [Lys³, Lys⁴]M-PONTX-Dq3a[1-15] showed an IC₅₀ of 4 µM in 24 hours of treatment. The molecular docking result revealed that the peptide binds to the active site of the TcGAPDH enzyme, interacting with essential residues such as Arg12, Glu336 and Gln317 through hydrogen bonds and hydrophobic interactions. The overlap with the Chalepin binding site suggests a possible competitive inhibition mechanism. Subsequently, the IC₅₀ and twice this concentration (4 µM and 8 µM, respectively) were selected to evaluate the effect of the analogue [Lys³, Lys⁴]M-PONTX-Dq3a[1-15] on the TcGAPDH enzyme. The enzymatic activity of TcGAPDH was significantly (p < 0.05) reduced after incubation with [Lys³,Lys⁴]M-PONTX-Dq3a[1-15], since compared to the control group, incubation with 4 µM or 8 µM of the peptide for 60 minutes resulted in an almost total inhibition of the enzymatic activity (units/mL ≈ 0.000), suggesting strong inhibitory potential. Conclusion: Given the results obtained, it is possible to conclude that the peptide [Lys³, Lys⁴]M-PONTX-Dq3a[1-15] is capable of reaching multiple targets, such as the parasite membrane and inhibiting key enzymes in the development of T. cruzi, opening prospects for the identification of candidates for new anti-Chagasic drugs from modified dinoponeratoxins. 

Agradecimentos: We acknowledge the financial support from the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES).