Biochemical and structural characterization of novel drug targets regulating polyamine biosynthesis in the human malaria parasite, Plasmodium falciparum

Abstract

Malaria is prevalent in over 100 countries which is populated by half of the world’s population and culminates in approximately one million deaths per annum, 85% of which occurs in sub-Saharan Africa. The combined resistance of the mosquitoes and parasites to the currently available pesticides and antimalarial chemotherapeutic agents requires the concerted effort of scientists in the malaria field to identify and develop novel mechanisms to curb this deadly disease. In this study, a thorough understanding of the role players in the polyamine pathway of the parasite was obtained, which could aid future studies in the development of novel inhibitory compounds against these validated drug targets. The uniquely bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) of Plasmodium falciparum forms an important controlling node between the polyamine and methionine metabolic pathways. It has been speculated that the unique bifunctional association of the rate-limiting enzymes allows for the concerted regulation of the respective enzyme activities resulting in polyamine synthesis as per requirement for the rapidly proliferating parasite while the methionine levels are strictly controlled for their role in the methylation status. The results of this study showed that the enzyme activities of the bifunctional complex are indeed coordinated and subtle conformational changes induced by complex formation is suggested to result in these altered kinetics of the individual AdoMetDC and ODC domains. Studies also showed that the identification of the interaction sites between the domains, which allows for communication across the complex, may be targeted for specific interference with the enzyme activities. Furthermore, these studies showed that the current knowledge on the different subclasses of the AdoMetDC family should be re-evaluated since P. falciparum AdoMetDC shows diverse properties from orthologues and therefore points towards a novel grouping of the plasmodial protein. The extensive biochemical and biophysical studies on AdoMetDC has also provided important avenues for the crystallisation and solving of this protein’s 3D structure for subsequent structure-based identification of drug-like lead compounds against AdoMetDC activity. The application of structure-based drug design on malarial proteins was additionally investigated and consequently proved that the rational design of lead inhibitory compounds can provide important scaffold structures for the identification of the key aspects that are required for the successful inhibition of a specific drug target. Spermidine synthase, with its intricate catalytic mechanism involving two substrate binding sites for the products of the reactions catalysed by AdoMetDC/ODC, was used to computationally identify compounds that could bind within its active site. Subsequent testing of the compounds identified with a dynamic receptor-based pharmacophore model showed promising inhibitory results on both recombinant protein and in vitro parasite levels. The confirmation of the predicted interaction sites and identification of aspects to improve inhibitor interaction was subsequently investigated at atomic resolution with X-ray protein crystallography. The outcome of this doctoral study shows the benefit in applying a multidisciplinary and multinational approach for studying drug targets within the malaria parasite, which has led to a thorough understanding of the targets on both biochemical and structural levels for future drug design studies.