Desenvolvimento de peptídeos tripanocidas otimizados a partir da dinoponeratoxina M-PONTX-Dq3a

• Ano de defesa: 2022
• Autor(a) principal: Monteiro, Marilia Lopes
• Orientador(a): Martins, Alice Maria Costa Nunes
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Trypanosoma cruzi; Doença de Chagas; Peptídeos Antimicrobianos; Lisina; Venenos de Artrópodes
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/69794

Chagas disease affects millions of people around the world. In Brazil, the available drug has limited efficacy and severe adverse effects, so there is a need to seek substances with therapeutic potential. The antimicrobial peptide M-PONTX-Dq3a, isolated from the venom of the ant Dinoponera quadriceps, showed better trypanocidal activity (in vitro) than current therapies, but the length of the peptide may limit its possibilities of achieving clinical application. In this work, the antichagasic effect of two fragments and eight analogues of M-PONTX-Dq3a was investigated in order to improve the antichagasic activity and reduce production costs. The M- PONTX-Dq3a fragments and analogues had their toxicity evaluated in LLC-MK2 host cells, with a higher EC50 being observed for the M-PONTX-Dq3a[3-15] fragment (188.86 μM) and for the analogues substituted by lysine with EC50 varying between (60 μM and 97 μM), the effect on the trypomastigote forms, lower EC50 values were observed for the M-PONTX- Dq3a[1-15] fragment (0.59 μM) and for the analogs substituted by lysine varying between (0.86μM and 1.09μM), the selectivity index (SI) was determined, and by means of the WHO criteria, the most promising peptides were selected, the fragment M-PONTX-Dq3a[1-15 ] (IS=72.9) and the analog [Lys]3-M-PONTX-Dq3a[3-15] (IS=85.3). Subsequently, the effect on epimastigote forms was analyzed, M-PONTX-Dq3a [1-15] showed the lowest EC50 at 24 hours (7.2 μM) and did not show a significant change between the times of 48 and 72 hours, [Lys] 3M-PONTX-Dq3a[1-15] showed significant differences between the EC50 of the 3 times, with the 24-hour EC50 being the lowest found (13.5 μM) and under the amastigote forms a promising effect was observed with the two peptides tested in the times of 24h and 48h, it was obtained for M-PONTX-Dq3a [1-15] EC50 24h 1.32 μM and IS/24h 32.61; EC50 48h 0.37 μM and IS/48h 96.7 and for [Lys]3M-PONTX-Dq3a[1-15] it was obtained EC50 24h 0.91 μM and IS/24h 80.65; EC50 48h 0.28 μM and IS/48h 156.6). Results were expressed as mean ± SEM and considered p < 0.05. In the search for the mechanism of action of the selected peptides, alterations were observed by scanning electron microscopy (SEM) and flow cytometry markings suggesting necrosis as the main mechanism of action, an increase of around 50% of reactive species was also observed. of oxygen in both peptides tested and mitochondrial damage by about 50% with the treatment of [Lys]3M-PONTX-Dq3a[1-15]. Molecular docking was performed with the enzymes cruzain, trypanothione reductase and TcGAPDH, demonstrating that M-PONTX-Dq3a[1-15] was coupled at different catalytic sites of each of the enzymes tested, while [Lys]3-M-PONTX- Dq3a[3-15] is coupled to the catalytic triad of cruzin. In conclusion, the studies led to the identification of two smaller peptides, the M-PONTX-Dq3a[1-815] fragment and the analog fragment [Lys]3-M-PONTX-Dq3a[3-15] with similar and superior antichagasic activity. respectively, when compared to the parental peptide selectivity index.