Leishmaniasis is a Neglected Tropical Disease caused by protozoa of the Leishmania genus. No human vaccine is available, and current treatments are limited by toxicity and drug resistance. Thus, the identification of new active molecules remains an urgent priority. However, drug discovery is severely hampered by the lack of fast, robust, and high-throughput screening assays, particularly for the clinically relevant intracellular amastigote stage. Here, a transgenic Leishmania infantum line stably co-expressing the bioluminescent marker PpyRE9H fused to the fluorescent reporter tdTomato was generated and used to develop a novel dual-reporter microplate-based platform to identify compounds active against promastigotes or intracellular parasite stages. The engineered strain exhibited comparable growth and infectivity to the wild-type strain, validating its suitability for drug screening assays. The activity of reference drugs was evaluated, and results were compared to those obtained using standard methods, such as MTT assay for promastigotes and Giemsa staining with microscopic quantification for amastigotes. Comparable IC₅₀ values were obtained using fluorescent/bioluminescent assays and conventional methods. These proposed assays provide a sensitive and reproducible alternative to conventional methods. The microplate format enables higher throughput screening, while the dual bioluminescence and fluorescence readouts enable cross-validation of results and reduce the risk of compound-specific signal interference.

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.