Malaria is considered the most prevailing human parasitic disease. Despite various chemotherapeutic interventions being available, the parasite responsible for the most lethal form of malaria, Plasmodium falciparum, is continuously developing resistance towards drugs targeted against it. This, therefore, necessitates the need for validation of new antimalarial development. Polyamine biosynthetic enzymes, particularly S-adenosylmethionine-L-decarboxylase (PfAdoMetDC), has been identified as a suitable drug target for protozoan parasitic diseases due to its essential role in cell proliferation. Furthermore, in Plasmodium polyamine biosynthesis, PfAdoMetDC is organised into a unique bifunctional complex with ornithine decarboxylase (PfAdoMetDC/ODC) covalently linked by a hinge region, distinguishing this enzyme as unique a drug target. However, inhibitors targeting this pathway have not been successful in clinical assessment, creating the need for further research in identifying novel inhibitors. This study focused on the structural and functional characterisation of protein-specific properties of the AdoMetDC domain in P. falciparum parasites, as well as identifying novel inhibitors targeting this enzyme as a potential antimalarial therapeutic intervention. In order to develop novel inhibitors specifically targeting PfAdoMetDC through a structure-based drug discovery approach, the three-dimensional structure is required. However, due to a lack of structural and functional characterisation, determination of the crystal structure has been challenging. Heterologous expression of monofunctional PfAdoMetDC was achieved from a wild-type construct of the PfAdoMetDC domain including the covalently linked hinge region. In chapter 2, deletion of a large non-homologous, low-complexity parasite-specific insert (A3) in monofunctional PfAdoMetDC resulted in an increased yield, purity and sample homogeneity, whilst maintaining protein functionality and structural integrity. However, truncation of the proposed non-essential hinge region resulted in low-level expression of insoluble protein aggregates and a complete loss of protein activity, indicating that the hinge region is essential for monofunctional PfAdoMetDC. However, in the absence of the three-dimensional PfAdoMetDC crystal structure, novel derivatives of a well-known AdoMetDC inhibitor, MDL73811, were tested for their activity against heterologous PfAdoMetDC, as well as their potency against P. falciparum parasites, in chapter 3. The compound Genz-644131 was identified as a lead inhibitor of PfAdoMetDC, however, the poor membrane permeability of the compound resulted in low in vitro activity. Drug permeability of Genz-644131 into P. falciparum infected erythrocytes and its potency was significantly improved by its encapsulation into a novel immunoliposome based drug delivery system. The results presented here provide essential information for development of a unique strategy in obtaining suffiecient levels of fully active recombinant PfAdoMetDC of sufficient purity for crystallisation studies and subsequent structure-based drug design efforts. The combination of Genz-644131 with the novel drug delivery system, which markedly improved its potency against PfAdoMetDC may proof to be a viable antimalarial chemotherapeutic strategy for future investigations. / Thesis (PhD)--University of Pretoria, 2014. / tm2015 / Biochemistry / PhD / Unrestricted

Malaria presents a global health risk that is becoming increasingly difficult to treat due to increased resistance of both the parasite and mosquito to all known drugs. Identification of novel drug targets are therefore essential in the fight against malaria. Polyamines are small flexible polycations that are represented by three basic polyamines. The interaction of polyamines with various macromolecules may lead to stabilisation of DNA, regulation of transcription, replication, and also have an important role in cellular differentiation, proliferation, growth and division. Therefore, disruption of polyamine biosynthesis presents a unique drug target worth exploiting. Polyamine biosynthesis in P. falciparum is regulated by a unique bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) complex, which is unique to P. falciparum and differs completely from human polyamine biosyntehsis. The inhibition of AdoMetDC induces spermidine and subsequent spermine depletion within the parasite that ultimately results in cell cycle arrest. A functional genomics approach was used within this study to identify a global response of the parasite due to the inhibition of AdoMetDC with the irreversible inhibitor, MDL73811. The proteomics approach was optimised for conditions specific to our laboratory with regard to protein extraction, Plasmodial protein quantification, spot detection and finally protein identification by mass spectrometry (MS). This methodology resulted in reliable spot detection and achieved a 95% success rate in MS/MS identification of protein spots. Application of this methodology to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots from the Plasmodial ring and trophozoite stages, which also confirmed stage specific protein production. Various protein isoforms were present which may be of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle. Subsequent application of the 2-DE methodology to the proteome of AdoMetDC inhibited parasites resulted in the identification of 61 unique Plasmodial protein groups that were differentially affected by the inhibition of AdoMetDC in 2 time points. The transcriptome of AdoMetDC inhibited parasites were also investigated at 3 time points. Investigation into the transcriptome revealed the differential regulation of 549 transcripts, which included the differential regulation of polyamine specific transcripts. Inhibition of AdoMetDC provided a unique polyamine specific transcriptomic signature profile that demonstrated unique interactions between AdoMetDC inhibition and folate biosynthesis, redox metabolism and cytoskeleton biogenesis. The results presented provide evidence that the parasite responds to AdoMetDC inhibition by the regulation of the transcriptome and proteome in an attempt to alleviate the effects of AdoMetDC inhibition. Further analyses of the metabolome also provided evidence for the tight regulation of the AdoMet cycle. Overall, this study demonstrated important functional consequences as a result of AdoMetDC inhibition. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

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. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted