Abstract
The causative agent of malaria is an intracellular protozoa parasite from the genus Plasmodium. In fact, Plasmodium falciparum is the stain responsible for the majority of the deaths. The symptomatic phase of malaria is due to the intra-erythrocytic cycle. The asexual multiplication in the intra-erythrocytic cycle requires a considerable quantity of cytoplasmic membrane synthesis in order to ensure the formation of new organelles and of the membranes of new daughter cells. To meet this need, the parasite uses its own metabolic machinery to produce the phospholipids (PLs) essential for its multiplication. Of the various PL present, phosphatidylcholine (PC) is the most abundant. The main pathway for the synthesis of PC by P. falciparum is the de novo Kennedy pathway. The most important step in this pathway is the second enzymatic step catalysed by CTP:phosphocholine cytidylyltransferase (CCT). This enzyme constitutes the limiting step of the pathway; it regulates the flow and therefore the rate of PC synthesis. Previous work in the laboratory (Deschamps et al.) has shown that CCT is essential for the survival of the murine parasite P. berghei. The first part of this thesis concerns the generation and characterisation of the conditional knockout of CCT in P. falciparum. We have shown that PfCCT is essential for the survival of the parasite at the blood stage. The three-dimensional structure of the C-terminal catalytic domain of PfCCT(581-775) solved in the laboratory (Guca et al.) shows very low enzymatic activity.In the second part of this thesis work, we produced different constructs of the C-terminal catalytic domain of PfCCT encompassing the amino acids: 581-795, 581-787, 581-779 and 581-772 with the aim of optimising both the crystallization and the activity of the protein fragments. After the production and purification steps of these different constructs, activity studies allowed the identification of the amino acid deletion responsible for the loss of activity. This work will serve as a basis for the identification of new active and crystallizable constructs. In the third part, we performed different screens of low and very low molecular weight molecules in order to select ligands for PfCCT. Several molecules belonging to different chemical families and capable of binding to the phosphocholine site and/or inhibiting PfCCT were identified. The screening of very low molecular weight molecules by crystallography led to the resolution of 23 structures of PfCCT-ligand complexes whose coordinates were deposited in the Protein Data Bank. The overall screening results provide crucial insights for the rational design of the fragment approach and the validation of the phosphocholine pocket of the active site as druggable.