This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-AI-103: Develop drugs for neglected tropical diseases, with a special emphasis on malaria. New drugs are desperately needed to treat visceral leishmaniasis (VL), a tropical disease cause by the protozoan Leishmania donovani. This proposal is focused on the discovery of new compounds that will feed into the pipeline bringing new therapeutics to patients with VL. The Specific Aim of this proposal is to identify novel drug candidates for visceral leishmaniasis through optimization of 4-aminoquinoline lead compounds. A novel ex vivo splenic explant model, derived from hamsters infected with L. donovani, will be used to identify new 4-aminoquinoline derivatives with anti-leishmanial activity. This new model, which was recently developed in our lab, capitalizes on the pathogenic similarities between hamster and human VL and offers the unique opportunity to discover compounds that are active against the parasite within the context of the pathogenic mechanisms that contribute to progressive disease. We propose to identify candidate drugs for the treatment of VL by chemical optimization of the 4-aminoquinoline lead compounds we previously identified. We will do this through a systematic, parallel, and iterative process of repeated cycles of synthesis of small (40- 80 compound) iterative libraries of 4-aminoquinoline derivatives, screening of the libraries for anti-leishmanial activity, toxicity, and drug-likeness, and repeated SAR analysis to generate new compounds for testing. Quinoline molecules have several notable characteristics that favor drug development and the availability of extensive data related to structure-activity relationship (SAR) and the prior successful development of quinolines for malaria will greatly facilitate the proposed studies. The proposed approach is feasible because of the rapidity with which new compounds will be synthesized and tested. The in vitro assays to be used for compound toxicity and absorption and stability/metabolism are standard for the field. The ex vivo splenic explant model is an innovative approach because the anti-leishmanial activity of 4-aminoquinoline derivatives will be tested (1) in a medium- to high-throughput format, (2) within the cellular microenvironment of the spleen, which includes the cells that exert immunopathologic effects known to profoundly influence parasite replication or killing, (3) against intracellular amastigotes (mammalian stage) rather than axenically cultured promastigotes (vector stage), which show discordant responses to antileishmanial compounds, (4) against amastigotes in splenic macrophages, which are functionally distinct from the traditionally used peritoneal macrophages, and (5) will be identified as having anti-leishmanial activity whether it is through direct parasite killing and/or indirect immune-mediated mechanisms. Furthermore, the integration of the chemical optimization and SAR analysis by Dr. Frantz's laboratory with the biological testing by Dr. Melby's laboratory will enable the efficient discovery of new compounds suitable for future in vivo efficacy testing in the pre-clinical hamster model of progressive VL. PUBLIC HEALTH RELEVANCE: Visceral leishmaniasis (VL) is a major public health problem with millions of cases worldwide. This proposal is focused on the discovery of new compounds that will feed into the pipeline bringing new therapeutics to patients with VL.