WP1 involves two partner institutions, the Institut Pasteur in Paris (IP) and the Institut Pasteur of South Korea (IPK). The principal investigators of this work package are Dr. Spencer Shorte, head of the IP imaging facility, Dr. Geneviève Milon, head of the Immunophsyiology and Parasitology Unit at IP, and Dr. Lucio Freitas-Junior, head of the Systems Biology of Pathogens Group at IPK. WP1 aims to establish a high-content screening approach drawing upon multi-disciplinary expertise in bio-imaging technologies, host-cell parasite propagation, and chemical library screening to discover compounds capable to kill intracellular Leishmania amastigotes without deteriorating the natural (macrophage) host cell. While the entire consortium (including this WP) is focused on the clinically relevant L. donovani species, we will use L. amazonensis expressing fluorescent DsRed2 [20] for the in situ screens for technical reasons and feasibility, including (i) availability of infective fluorescent and bio-luminescent amastigotes, (ii) low biohazard requirements compatible with the screening approaches (virulent L.donovani needs to be handled in P3 facilities, which are not available for medium/high tp screens), (iii) lack of interfering extracellular parasite contamination (L.donovani shows robust extracellular growth in macrophage cultures interfering with the screen), and (iv) close to 100% infection efficiency. The challenge here is to develop the already validated cell-based model assays into a throughput efficient format allowing significant numbers of compounds to be tested, while retaining their biological relevance to parasite-host interactions and therefore potential clinical efficacy. This will be achieved through three stages, including (i) optimization of the primary host cell-based screening assay using selected compounds or peptides having kinase inhibitory activity (provided by partners 8, 10, 12 and 13), (ii) applying targeted strategies based on the screening of compounds from small molecule libraries including kinase inhibitor specific libraries (partners 2 and 10), and (iii) hit-to-lead optimization using secondary screens based upon compound effects on isolated luciferase-transfected parasites in a plate reader-based assay [20] and target identification by chromatographic and proteomics approaches (partners 1, 10 and 11).