Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani is one of the neglected tropical diseases (NTDs) caused by trypanosomatids with treatment options limited to outdated drugs often causing adverse effects and promoting drug resistance. Previous antileishmanial drug discovery campaigns have identified nitroheterocyclic molecules with high efficacy and a high selectivity index. Therefore, we have evaluated on our screening platform of fluorescent L. donovani amastigotes, the antileishmanial activity of seven nitrofuran derivatives: furazolidone, nitrofurazone, nitrofurantoin, nifurtimox, 5-nitro-2-furaldehyde diacetate, PYZD-4409 and 5-nitro-2-furonitrile. These compounds showed good efficacy against axenic and intramacrophage amastigotes, most of them showing low cytotoxicity in mammalian cell lines. These nitrofuran derivatives induced reactive oxygen species production in axenic amastigotes and inhibited trypanothione reductase (TryR) either in uncompetitive or competitive manner, thus suggesting that their mechanism of action involves increased oxidative stress caused by an imbalance in redox metabolism. Furazolidone exhibited the most promising antileishmanial profile, and molecular docking analysis revealed consistency with the strongest TryR uncompetitive inhibitory effect, demonstrating its high affinity for an alternative binding site near the substrate (oxidized trypanothione) pocket. Docking results also highlighted PYZD-4409 as the compound with the highest binding affinity, and showed consistency with its competitive inhibition mechanism. Furthermore, similar binding modes identified across L. donovani TryR and other homologous proteins suggest the potential broad-spectrum activity of these nitrofuran derivatives, thus underscoring their importance as promising candidates for the development of novel antileishmanial therapies with broad-spectrum applications.

The development of new alternative models is essential to overcome the limitations of traditional two-dimensional (2D) cell cultures and animal models. Three-dimensional (3D) models, such as organoids, better mimic the structural and functional complexity of mammalian organs, thereby reducing the ethical and economic issues related to animal experimentation. These systems provide more physiologically relevant environments, improving the accuracy of disease modeling and drug response prediction. In this context, we have developed mouse hepatic organoids from livers of adult BALB/c mice and characterized them by microscopy and transcriptional analysis. This model was applied to a robust and reproducible high-throughput screening (HTS) platform for testing cytotoxicity at the preclinical stage of drug discovery. In addition, mouse hepatic organoids were co-cultured with amastigotes of Leishmania donovani parasites to establish a model of host–parasite interaction, which was characterized by RNA-seq linked to differential expression analysis and cytokine production by the hepatic organoids. The findings provided in this work establish mouse hepatic organoids as an alternative model for drug discovery and pathogenesis studies.

Leishmania infantum is the vector-borne trypanosomatid parasite causing visceral leishmaniasis in the Mediterranean basin. This neglected tropical disease is treated with a limited number of obsolete drugs that are not exempt from adverse effects and whose overuse has promoted the emergence of resistant pathogens. In the search for novel antitrypanosomatid molecules that help overcome these drawbacks, drug repurposing has emerged as a good strategy. Nitroaromatic compounds have been found in drug discovery campaigns as promising antileishmanial molecules. Fexinidazole (recently introduced for the treatment of stages 1 and 2 of African trypanosomiasis), and pretomanid, which share the nitroimidazole nitroaromatic structure, have provided antileishmanial activity in different studies. In this work, we have tested the in vitro efficacy of these two nitroimidazoles to validate our 384-well high-throughput screening (HTS) platform consisting of L. infantum parasites emitting the near-infrared fluorescent protein (iRFP) as a biomarker of cell viability. These molecules showed good efficacy in both axenic and intramacrophage amastigotes and were poorly cytotoxic in RAW 264.7 and HepG2 cultures. Fexinidazole and pretomanid induced the production of ROS in axenic amastigotes but were not able to inhibit trypanothione reductase (TryR), thus suggesting that these compounds may target thiol metabolism through a different mechanism of action.

Chemical diversity and binding-site landscape of reported trypanothione reductase inhibitors.

The emergence of drug resistance and the limited availability of effective therapeutics continue to hinder the treatment of protozoal diseases, highlighting the need for new chemotypes with broad-spectrum antiparasitic potential. In the present study, a series of nitro and trifluoromethyl-substituted thiochromene carboxamides, previously identified as antileishmanial agents, were further explored for their broad-spectrum antiparasitic potential. Biological screening identified several compounds with promising activity and favorable selectivity against Plasmodium falciparum (D10 and W2 strains) and Trypanosoma brucei (AnTat1.1E strain). As the synthesized analogues exhibited a more favorable antitrypanosomal activity and selectivity profile, representative analogues were subsequently evaluated for trypanothione reductase (TryR) inhibition to investigate their potential mechanism of action, as TryR is a validated and highly conserved drug target across kinetoplastid parasites. Among the synthesized molecules, compound 5p showed the highest antiplasmodial potency (D10 EC50 = 2.1 μM), whereas compounds 5c and 5r were the most potent antitrypanosomal analogues (EC50 = 1.8 μM). Structure–activity relationship analysis demonstrated that piperidine and piperazine-containing carboxamide is a key pharmacophoric feature for broad-spectrum activity, whereas incorporation of a nitro substituent enhanced the activity against T. brucei. To investigate the potential mode of killing, representative compounds were evaluated for inhibition of TryR, identifying compound 5v as the most potent enzyme inhibitor. Enzyme kinetic studies established an uncompetitive mode of inhibition (ki ≈ 29.5 μM), suggesting interaction at an allosteric site rather than the active catalytic site. Consistent with these findings, SiteMap analysis identified a druggable allosteric pocket at the dimer interface of substrate-bound TryR, where the active compounds exhibited favorable binding affinities and interaction profiles. Collectively, these results demonstrate that thiochromene carboxamides constitute a promising scaffold for the development of broad-spectrum antiprotozoal agents and support allosteric inhibition of TryR as a viable strategy for antitrypanosomal drug discovery.