Recherche de composés à visée thérapeutique et développement de nouveaux tests in vitro pour l'optimisation de la thérapie antipaludique – Add-Mal

A major goal of malaria research is to find drugs with novel targets and a novel mechanism of action. In parallel, more research is needed to elucidate mechanisms of drug resistance, since new drugs should not only be effective, but should also evade mechanisms that contribute to parasite resistance. Plasmodium falciparum, the most pathogenic human malaria parasite, becoming pharmacoresistant to conventional as well as newly discovered drugs the need of new antimalaria strategies involving novel targets and new drugs appear crucial as ever. The proposal aims to present an innovative approach combining target caracterisation to design new bioactive compounds and evaluation of antimalaria candidate drug resistance. Two main objectives are pursue, both related to the medication of malaria in case of drug resistant Plasmodium infections. Enzymes of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) pathways have been shown to be essential for the parasite survival, thereby designated them as crucial targets for the development of antimalaria therapy. The biochemical and structural characterization of these novel antimalaria targets, namely the rate-limiting enzymes of the PC and PE pathways: CTP:phosphocholine and CTP:phosphoethanolamine cytidylytransferases, will be performed. The aim is to determine X-ray 3D structures of apo-enzymes and complexes enzymes-substrates. Based on these data, new drug scaffolds will be indentified by in silico potential hit-compounds. The transmembrane choline/ethanolamine phosphotransferase will be produced and characterize using specific techniques as insect cell for production and membrane assays. Also, we will characterize the interaction of candidate and validated antimalarial agents with key human transporters influencing ADMET properties to eventually screen oraly-available compounds. The substrate and inhibitor profiles of the human and Plasmodium transporters may differ, testing whether a compound interacts with a human homologue of the transporter of interest contribute to the understanding of the mode of action of the compounds. The Plasmodium transporter Pgh-1 is responsible for certain antimalarial drug resistances. Biochemical and functional characterization of the transporter will be performed by several in vitro membrane assays. The catalytic activity of Pgh-1 will be evaluated by the quantification of ATPase activity (liberation of inorganic phosphate). A measurement of substrate translocation and its modulation will be achieved by the quantification of the intravesicularly trapped substrates in vesicular transport assays. The functional assay will enable the characterization of Pgh-1; testing drug-transporter interactions; and testing the susceptibility of novel antimalarials for Pgh-1-mediated transport. Based on the Pg-1 transporter activity assays, the development of an in vitro diagnostic kit (using fluorescent substrates) to detect Plasmodium and predict its drug resistance profile will be explored. The project exploits the complementary expertise of three academic centers (two French, one Hungarian) and one mid-size Hungarian biotechnology company. The scaffold required for a successful collaborative project will be build up from the unique expertise in the biology of phospholipid synthesis of the malarial parasites of partner 1, the know-how of partner 2 in structure-based design of bioactive molecules and combined knowledge of Partners 3 and 4 in enzymology/structural biology and the biology of pharmacologically-relevant membrane transport mechanisms.