Cell Death and Unprecedent Cytoskeletal Changes in Leishmania amazonensis Parasites Evidenced by 3D Reconstruction

Leishmaniasis remains a neglected tropical disease with limited and highly toxic therapeutic options. In search of safer and more effective alternatives, bithiophene derivatives have emerged as promising scaffolds. In this study, we evaluated the antileishmanial potential of BT-Ac, a bithiophene previously shown to exert antitrypanosomal activity. BT-Ac displayed selective antiproliferative activity with IC₅₀ for 28.9 and 50 μM against promastigotes and amastigotes of Leishmania amazonensis at nontoxic concentrations for host cells, achieving a selectivity index of 4.69 against intracellular amastigotes. Electron microscopy revealed early morphological damage in promastigotes, including unprecedented changes at the plasma membrane and cytoskeletal interface, mitochondrial swelling, lipid body accumulation, and autophagic vacuole formation. Three-dimensional reconstructions further validated these findings, confirming that the ultrastructural alterations were intrinsic to BT-Ac treatment rather than technical artifacts. Biochemical analyses demonstrated reactive oxygen and nitrogen species overproduction, lipid peroxidation, and ATP fluctuations, indicating oxidative stress as a central mechanism. These events culminated in apoptosis-like and autophagic death in promastigotes, while amastigote death was primarily mediated by macrophage activation, evidenced by nitric oxide release. Together, our findings identify BT-Ac as a selective leishmanicidal agent that disrupts parasite homeostasis through oxidative damage, highlighting its potential as a lead compound for therapeutic strategies against leishmaniasis.