Visceral leishmaniasis caused by Leishmania donovani is one of the major neglected tropical diseases attributable to parasitic protozoa. In the absence of an effective vaccine, chemotherapy remains the only available therapeutic option. However, current treatments rely on a limited number of drugs that are largely obsolete, highly toxic or require intravenous administration, and their extensive use has led to the emergence of drug resistance. Consequently, the discovery of new antileishmanial agents is an urgent priority. In this study, a commercial library of 449 alkaloids in a high-throughput screening format was evaluated against both axenic bone marrow-derived amastigotes and intramacrophagic amastigotes from mice infected with L. donovani IRFP, a strain engineered to emit infrared fluorescence in its viable form. Six isoquinoline-type alkaloids showed the best antileishmanial efficacy against intramacrophagic amastigotes while exhibiting minimal cytotoxicity toward RAW 264.7 and HepG2 cell lines, with a promising selective index higher than four, and good mouse intestinal tolerance in mouse organoids. Among these compounds, the protoberberine scaffold emerged as the most promising candidate for further drug development.

Plant-derived secondary metabolites are a promising source of new anti-infective agents owing to their remarkable chemical diversity and broad spectrum of biological activities. Among them, halophytes are particularly promising because adaptation to saline environments promotes the accumulation of structurally diverse secondary metabolites. In the present study, organic extracts from four Tunisian halophytic species collected across different seasons were evaluated for their activity against Leishmania infantum axenic amastigotes, the etiological agent of visceral leishmaniasis in the Mediterranean Basin. Among the species tested, Suaeda vera was the only one to exhibit significant antileishmanial activity. Mass spectrometry-based phytochemical profiling of the bioactive extracts resulted in the putative annotation of 53 secondary metabolites and revealed qualitative differences in their annotated chemical composition among the seasonal samples. Three compounds putatively annotated in the active extracts—demethylzeylasteral, caffeic acid, and protocatechuic aldehyde—were subsequently evaluated individually using commercially available reference compounds and exhibited EC₅₀ values of 0.20, 1.09, and 14.56 μM, respectively. Caffeic acid displayed the most favorable overall profile, combining potent antileishmanial activity with a high selectivity index (SI > 91.7) in mouse intestinal organoids. In vitro assays revealed that none of the three compounds inhibited trypanothione reductase-associated activity. Furthermore, in silico drug-likeness and ADMET analyses suggested that caffeic acid may have a comparatively favorable predicted pharmacokinetic and toxicity profile among the three compounds evaluated. Collectively, these findings highlight S. vera as a promising source of antileishmanial metabolites and establish caffeic acid as the most promising hit compound for further investigation.