The stakes of this work are considerable. Leishmaniasis and Chagas disease, caused by trypanosomatid parasites transmitted by sand flies and triatomine bugs respectively, affect millions of people across more than 90 countries, with Brazil among the six most burdened nations. According to the Pan American Health Organization, an estimated 6 to 7 million people are infected with Chagas disease worldwide, with roughly 30,000 new cases and 14,000 deaths each year. Leishmaniasis manifests in clinical forms ranging from disfiguring cutaneous lesions to fatal visceral disease, while chronic Chagas cardiomyopathy can silently progress to heart failure and sudden death decades after the initial infection.

Current therapies fall far short of what clinicians need. Pentavalent antimonials, amphotericin B, miltefosine, benznidazole, and nifurtimox all suffer from significant toxicity, lengthy treatment regimens, variable efficacy depending on disease stage, and rising resistance. This therapeutic gap motivated the research team, led by scientists at the State University of Maringá in Brazil, to screen a panel of four synthetic azobenzenoid compounds—PH011669, S981796, L170224, and CaCS2—against multiple life stages of both parasites using a combination of laboratory and computational approaches.

The in vitro results revealed a striking hierarchy of activity. While PH011669 and S981796 showed no meaningful effect on proliferating promastigotes or epimastigotes, both displayed potent activity against intracellular amastigotes of L. amazonensis, the clinically relevant stage that hides inside host macrophages, with half-inhibitory concentrations below 10 micromolar and selectivity indices exceeding 44. L170224 showed a narrower profile, active against Leishmania but inert against T. cruzi. Only CaCS2 demonstrated broad-spectrum activity across every stage tested: promastigotes and amastigotes of L. amazonensis, and both epimastigotes and bloodstream trypomastigotes of T. cruzi, with inhibitory concentrations ranging from roughly 9 to 22 micromolar.

Just as important as potency was safety. CaCS2 showed remarkably low toxicity toward mammalian cells, with half-cytotoxic concentrations of 428 micromolar against J774A.1 macrophages and 673.5 micromolar against LLC-MK2 epithelial cells, yielding selectivity indices between 30 and 60 depending on the parasite stage. By comparison, the reference drugs miltefosine and benznidazole, though potent in some assays, displayed substantially lower selectivity margins. This favorable therapeutic window prompted the team to focus all subsequent mechanistic work on CaCS2 alone.

The mystery deepened when the researchers considered the compound’s history. CaCS2 was previously identified as an inhibitor of chorismate synthase, an enzyme found in fungi, mycobacteria, plants, and apicomplexan parasites—but not in trypanosomatids. Since T. cruzi and Leishmania lack the chorismate synthase gene entirely, the observed antiparasitic activity could not be explained by the compound’s known mechanism. Something else inside these parasites had to be the target.

To find it, the team turned to a reverse screening strategy called the Similarity Ensemble Approach, which compares the chemical structure of a query molecule against the known ligands of thousands of annotated proteins. The analysis flagged RNA editing ligase 1, or REL1, as a high-confidence candidate, with a statistical significance value of 1.76 × 10⁻¹². REL1 is a compelling target for several reasons: it is indispensable for the uridine insertion and deletion reactions that edit mitochondrial mRNA in trypanosomatids, it cannot be functionally replaced by its partner enzyme REL2, and it has no close homolog in mammalian cells, minimizing the risk of host toxicity.

Using AlphaFold-predicted structures of REL1 from both L. braziliensis and T. cruzi, superimposed onto the crystal structure of the T. brucei enzyme bound to ATP and magnesium, the researchers mapped the enzyme’s binding cavities. Blind docking across 100 simulations revealed that CaCS2 preferentially occupied two pockets: the orthosteric site, where the ATP cofactor normally binds, and an alternative cavity on the opposite face of the protein. Guided docking with three independent programs—AutoDock Vina, AutoDock 4.2.3, and Molegro—consistently ranked CaCS2’s binding at the orthosteric site second only to ATP itself, with the ligand forming hydrogen bonds and electrostatic contacts with key catalytic residues such as Lys42, Arg63, and Phe162 in the Leishmania enzyme, and Ile55, Lys81, and Phe203 in the Trypanosoma version.

Molecular dynamics simulations running 100 nanoseconds for each protein-ligand complex provided the decisive test. The systems reached equilibrium after about 40 nanoseconds, and CaCS2 bound at the orthosteric site remained stably anchored throughout the simulation in both parasite enzymes, mirroring the behavior of ATP. In contrast, the ligand at the alternative site proved transient: it diffused away from the pocket early in the Leishmania simulation, and in the Trypanosoma system it detached before returning after 35 nanoseconds. Analyses of the radius of gyration and per-residue fluctuations confirmed that the protein never unfolded in any complex, and contact-frequency calculations showed that CaCS2 at the orthosteric site shared nine key residues with ATP in both species—far more than the six or seven residues engaged at the alternative site.

Taken together, the evidence suggests that CaCS2 inhibits REL1 by competing with ATP at the catalytic site rather than by acting as an allosteric effector, a conclusion consistent with the compound’s negative net charge, which resembles the charged phosphate backbone that anchors ATP in the pocket. The authors caution that direct enzyme inhibition assays will be needed to confirm the mechanism, but the convergence of broad-spectrum antiparasitic activity, low mammalian cytotoxicity, and stable predicted binding to a parasite-essential, human-absent enzyme makes CaCS2 a promising lead scaffold. As drug resistance continues to erode the value of existing therapies for Chagas disease and leishmaniasis, molecules that exploit the unique biochemistry of RNA editing may offer a much-needed path forward.

Subject of Research: Azobenzenoid derivatives as inhibitors of RNA editing ligase 1 in Trypanosoma cruzi and Leishmania amazonensis parasites

Article Title: Integrated Experimental and Computational Investigation of Azobenzenoid Derivatives Against Trypanosomatid: RNA Editing Ligase 1 as a Potential Molecular Target

Article References: Khan, A., Balbinot, R. B., Lazarin-Bidóia, D., Paetzold, M. G., Seixas, Í. N., Ramos Silva, G. N., Fernandez, M. A., Nakamura, C. V., & Seixas, F. A. V. (2026). Integrated Experimental and Computational Investigation of Azobenzenoid Derivatives Against Trypanosomatid: RNA Editing Ligase 1 as a Potential Molecular Target. Acta Parasitologica, 71(5), Article 201. https://doi.org/10.1007/s11686-026-01391-1

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01391-1

Keywords: Trypanosoma cruzi, Leishmania amazonensis, RNA editing ligase 1, azobenzenoid derivatives, Chagas disease, leishmaniasis, molecular docking, molecular dynamics simulation, drug discovery, neglected tropical diseases, cytotoxicity, CaCS2