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International audience; The limited success of recent phenotypic anti-leishmanial drug screening campaigns calls for new screening strategies for the discovery of clinically relevant hits. Here we present such a novel strategy based on physiologically relevant, ex vivo biology. We established high content phenotypic assays that combine primary murine macrophages and lesion-derived, virulent L. donovani and L. amazonensis amastigotes, which we applied to validate previously identified, anti-leishmanial hit compounds referred to as 'GSK Leish-Box'. Together with secondary screens using cultured promastigotes, our pipeline distinguished stage-and/or species-specific compounds, including 20 hits with broad activity at 10 µM against intracellular amastigotes of both viscerotropic and dermotropic Leishmania. Even though the GSK Leish-Box hits were identified by phenotypic screening using THP-1 macrophage-like cells hosting culture-derived L. donovani LdBob parasites, our ex vivo assays only validated anti-leishmanial activity at 10 µM on intra-macrophagic L. donovani for 23 out of the 188 GSK Leish-Box hits. In conclusion, our comparative approach allowed the identification of hits with broad anti-leishmanial activity that represent interesting novel candidates to be tested in animal models. Physiologically more relevant screening approaches such as described here may reduce the very high attrition rate observed during pre-clinical and clinical phases of the drug development process. Leishmaniases are neglected diseases caused by protozoan parasites of the genus Leishmania that are transmitted by the bite of female Phlebotomine sandflies. Almost 1 billion people are at risk of infection in close to 100 endemic countries throughout the tropical and subtropical regions of Africa, Asia, the Mediterranean countries and South and Central America 1. Leishmaniasis clinical manifestations range from self-healing cutaneous lesions with possible mucosal dissemination to severe visceral forms, causing death if untreated. In the absence of efficient reservoir and vector control strategies, and of preventive or therapeutic human vaccines , the mainstay of current intervention strategy to limit the disease is chemotherapy. Leishmaniases remain the only trypanosomatid diseases for which therapeutics are largely based on repurposed drugs, including anti-fungal (amphotericin B), anticancer (miltefosine), antibiotic (paromomycin) and antimalarial (sitamaquine) molecules 2,3. However, all current treatments are limited and show serious drawbacks, including high cost that are prohibitory for resource-limited countries, poor compliance due to constraining mode of administration 4 , poor safety with important adverse effects due to toxicity 5 , treatment failure and drug resistance 6. Similarly, current efforts to overcome the drug resistance by developing drug combination therapies 7,8 may fail in light of the identification of an L. infantum strain that gained resistance against antimony and amphotericin B 9. Multi-drug resistance was also documented in Indian field isolates 10 , and was confirmed experimentally by the in vitro selection of L. donovani parasites showing resistance to different drug combinations and even cross-resistance to unrelated drugs 11. Combination therapy therefore may be of only limited use, raising important concerns on the current

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