Authors
O Leclercq2; N Rachidi2; F Dingli1; G Arras1; D Loew1; G F Späth2; 1 Institut Curie, France; 2 Institut Pasteur, France Discussion
Existing anti-leishmanial drugs are compromised by severe side effects and emergence of parasite resistances, calling for the discovery of new drug candidates to drive anti-parasitic chemotherapy. We recently identified hit compounds with anti-leishmanial activity by screening ATP-like compound libraries using target-based and phenotypic assays. Our current efforts are directed toward drug target deconvolution to reveal off-target effects of hit compounds identified by screening against recombinant Leishmania casein kinase CK1.2, and identify the unknown targets of hit compounds identified by screening against L. amazonensis infected primary macrophages. We use a proteomic strategy combining competition assays, successive ATP affinity chromatography, 2D-DiGE and MS analyses to evaluate the specificity of CK1.2 hit compounds, and discover novel targets based on the hits identified by phenotypic screening. As a first step towards the validation of this technology, we identified Leishmania ATP-binding proteins (ATPome). MS analysis of eluted fractions revealed 1405 putative ATP-binding proteins, including 241 proteins with a Gene Ontology annotation ATP-binding (GO: 0005524), including 96 protein kinases, thus establishing the first ATPome in any Trypanosomatid. We recovered 1164 proteins from our assay, including 456 hypothetical proteins that bear no overt homology to any ATP-binding protein. These proteins are either indirectly enriched through interaction with ATP-binding proteins, or directly enriched through yet uncharacterized, parasite-specific domains able to bind ATP. Our current efforts will be presented to (i) distinguish between these two possibilities, including enrichment analyses under high salt concentrations known to eliminate protein-protein interactions, (ii) identify novel domains by bio-informatics analyses, and (iii) apply our ATPome analysis for drug target discovery. 
