Transcriptomic and ultrastructural responses to Amiodarone–Itraconazole in naturally benznidazole-resistant and -susceptible Trypanosoma cruzi strains

Chagas disease (CD), caused by Trypanosoma cruzi, remains a major therapeutic challenge, primarily due to the limited efficacy of benznidazole and the emergence of naturally resistant strains. In this context, drug repurposing offers a promising strategy to identify compounds with trypanocidal activity. In this study, we evaluated the effect of Amiodarone-Itraconazole (Amiozole) against two T. cruzi strains belonging to DTU-TcI: one benznidazole-sensitive (MG) and one naturally resistant to benznidazol (DA). We employed an integrated approach combining transcriptomic and ultrastructural analyses to elucidate the compound's mechanisms of action. Trypanocidal activity was assessed through cell viability assays (MTT), and IC50 values were determined using epimastigotes cultured in LIT medium. Subsequently, RNA sequencing was performed on treated samples, with reads mapped against the T. cruzi Dm28c reference genome. Differential gene expression was analyzed using DESeq2, followed by Gene Ontology enrichment analysis and metabolic pathway reconstruction via KAAS. In parallel, transmission electron microscopy (TEM) was used to evaluate ultrastructural alterations induced by treatment. Our results revealed susceptibility to Amiozole in both strains, although they exhibited markedly distinct transcriptomic responses. In the DA strain, 35 genes were upregulated and 87 downregulated, with notable activation of purine metabolism and inhibition of surface renewal pathways. In contrast, the MG strain showed 57 upregulated and 412 downregulated genes, including enhanced sphingolipid metabolism-potentially linked to membrane repair-and widespread suppression of energy and nucleotide biosynthesis pathways. At the subcellular level, both strains displayed severe damage, including mitochondrial disruption, nuclear disorganization, formation of autophagosomes, and extensive membrane vesiculation, reflecting multifocal cellular stress. Collectively, these findings provide a comprehensive view of Amiozole's effects on T. cruzi, supporting a multifaceted mode of action that disrupts key biological processes essential for parasite viability. Our study underscores the potential of Amiozole as a combinatorial therapy against T. cruzi strains with distinct resistance profiles. Nevertheless, further research using infective forms, variable dosages, and diverse intra-DTU lineages is essential to validate its clinical applicability for Chagas disease.