This thesis is divided into two distinct sections which can be combined under the broad umbrella of structural bioinformatics studies related to drug discovery. The first section involves the establishment of an online South African natural products database. Natural products (NPs) are chemical entities synthesised in nature and are unrivalled in their structural complexity, chemical diversity, and biological specificity, which has long made them crucial to the drug discovery process. South Africa is rich in both plant and marine biodiversity and a great deal of research has gone into isolating compounds from organisms found in this country. However, there is no official database containing this information, making it difficult to access for research purposes. This information was extracted manually from literature to create a database of South African natural products. In order to make the information accessible to the general research community, a website, named “SANCDB”, was built to enable compounds to be quickly and easily searched for and downloaded in a number of different chemical formats. The content of the database was assessed and compared to other established natural product databases. Currently, SANCDB is the only database of natural products in Africa with an online interface. The second section of the thesis was aimed at performing structural characterisation of proteins with the potential to be targeted for antimalarial drug therapy. This looked specifically at 1) The interactions between an exported heat shock protein (Hsp) from Plasmodium falciparum (P. falciparum), PfHsp70-x and various host and exported parasite J proteins, as well as 2) The interface between PfHsp90 and the heat shock organising protein (PfHop). The PfHsp70-x:J protein study provided additional insight into how these two proteins potentially interact. Analysis of the PfHsp90:PfHop also provided a structural insight into the interaction interface between these two proteins and identified residues that could be targeted due to their contribution to the stability of the Hsp90:Hop binding complex and differences between parasite and human proteins. These studies inspired the development of a homology modelling tool, which can be used to assist researchers with homology modelling, while providing them with step-by-step control over the entire process. This thesis presents the establishment of a South African NP database and the development of a homology modelling tool, inspired by protein structural studies. When combined, these two applications have the potential to contribute greatly towards in silico drug discovery research.

Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.

No description available.

Malaria is a major public health problem that affects millions of lives globally. The increased burden of malaria requires new interventions that will address the eradication of the disease. Current interventions include vector control by using insecticide-treated bed nets and indoor residual spraying, and antimalarial drugs to control the parasite. Parasite resistance has been reported for the currently used effective antimalarial drugs. To pre-empt the impact of parasite resistance a continued development of new antimalarial drugs that have novel mechanisms of action should be pursued. Antimalarial drug discovery requires that potential antimalarial drugs should have different drug targets to those already targeted, to lower the chances of resistance. Potential antimalarial drugs should preferably provide a single radical cure to prevent reproduction at all life cycle stages. This study tested the effects of in silico designed compounds targeting plasmodial Ca2+- dependent protein kinases (CDPK) 1 & 4, FIKK kinases and bromodomain proteins on the Plasmodium parasite. These enzymes are involved in gene regulation and are important factors during gene transcription. In P. falciparum the gatekeeper kinases contain small hydrophobic pockets near the ATP-binding site. These hydrophobic pockets allow for selective inhibition of these proteins at the ATP-binding site. The compounds were tested in vitro to determine their antiplasmodial activity. These compounds are shown to be potential inhibitors of the intra-erythrocytic P. falciparum parasites as three of the compounds showed selective cytotoxic activity at less than 1 μM against the chloroquine sensitive laboratory strains (3D7 and NF54). Even though the proteins targeted by these compounds have been previously indicated to play a role at specific stages during the parasite’s life cycle, the compounds tested here were not able to target the sexual gametocyte stages of the Plasmodium parasite. Further optimisation of these compounds should be performed to improve activity against both the asexual and sexual stages of the parasites. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

PhD (Biochemistry) / Department of Biochemistry / Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.

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

Malaria is a serious disease that can lead to fatal consequences if not treated. It is mainly spread via Plasmodium falciparum, a parasite carried by mosquitoes as host organisms. As a potential way of treating malaria, research is being done on possible inhibitors of calmodulin (CaM) in the parasite. CaM is a highly conserved protein found in all eukaryotes, and is important in many essential biochemical reactions. The potential inhibitor analysed in this study is calmidazolium (CZM). This study aims to characterise structure and stability differences between the C-lobes of human and P. falciparum CaM, while analysing the effect of the presence of CZM.  Previous studies have proven that CZM acts as an inhibitor to human CaM by binding to the C-lobe, with a dissociation constant in the nano molar range. In other studies, thermal stability measurements have shown that the secondary structure of P. falciparum CaM is more stable than that of human CaM.  In this study, the stability measurements showed that for the ANS binding site and around tyrosines, the C-lobe of human CaM was more stable than the C-lobe of P. falciparum CaM, knowledge which was previously unknown. When studying the entire secondary structure, the C-lobe of P. falciparum CaM was found to be more stable, which is in agreement with previous studies for the secondary structure of the complete CaM variants. For binding, the dissociation constants for both the C-lobe of human CaM and for the C-lobe of P. falciparum CaM were proven to be at a lower range than micro molar, most likely in the nano molar range. This is in agreement with earlier findings regarding the entire human CaM. Furthermore, CaM and CZM were proven to have their absorbance at the same wavelengths. Finally, several amino acid differences between the C-lobes of human and P. falciparum CaM were found that could play a role in binding and stability. One specific amino acid that was suggested to contribute to the stabilisation of the C-lobe of P. falciparum CaM was isoleucine. In the C-lobe of human CaM, these isoleucines were exchanged to threonine and arginine. Another amino acid difference that could potentially play a key role was the valine versus isoleucine, where valine might contribute to the stabilisation of the ANS binding site of the C-lobe of human CaM. To perform this study, the methods fluorescence spectroscopy, UV spectroscopy and circular dichroism were used, as well as several bioinformatic tools.  Overall, both stability and structure analyses have helped determine several differences between the two CaM variants, opening up possibilities to find an inhibitor that targets only the CaM of P. falciparum. CZM still remains as an interesting potential inhibitor, and can hopefully be a part of future research in malaria treatment.

No description available.

Natural products are acknowledged sources of novel compounds for use in the treatment of diseases such as cancer, malaria, and human African trypanosomiasis. However, health burdens of such diseases still remain high, with drug resistance leading to failure of current medication. Therefore, there is a need for new treatments, and this project considers the potential of Nigerian ethno-medicinal plants and their products. Firstly, the aims were to isolate cytotoxic compounds through bio-guided evaluation and fractionation from 3 medicinal plants; Vitellaria paradoxa, Cyperus articulatus and Securidaca longepedunculata used traditionally in the treatment of cancer in North-East Nigeria. Extracts from S. longepedunculata were the most active when assessed in a panel of cancer cell lines, with IC50 values below 10 µg/ml, whilst fractions isolated from V. paradoxa and C. articulatus were moderately cytotoxic and able to overcome drug resistance mechanisms in drug resistant cell lines. In the second part of the thesis, novel cryptolepine analogues were semi-synthesized using environmentally friendly methods and evaluated for cytotoxic, anti-plasmodial and anti-trypanosomal activity. The compounds were found to be highly cytotoxic in cancer cell lines with the ability to overcome drug resistant mechanisms, with sub-µM IC50 values, and were also active against drug resistant strains of Plasmodium parasites in addition to Trypanosoma brucei, with IC50 values below 500 nM, and 300 pM respectively. / Schlumberger Faculty for the Future Foundation

Malaria continues to threaten human beings, causing a staggering number of more than 400,000 deaths each year. Although effective treatment and prevention methods are available, rapidly emerging resistance towards existing drugs is of great concern, and the need for novel antimalarial compounds are still urgent. The Malaria Box lead molecules MMV008138 and MMV665831 are promising in this regard, due to their apparently novel antimalarial mechanisms of action. The target of MMV008138 is the PfIspD enzyme in the MEP pathway, which is absent in humans. This difference makes the PfIspD a great target. However, while MMV008138 shows potency against Plasmodium falciparum-infected human erythrocytes in vitro, no efficacy was observed in a humanized mouse model or a P. berghei infected mouse in vivo. In order to block potential metabolic spots and to probe for steric demand, a series of analogous featuring C1-deuteration, methyl substitution on B- and C-ring, and an ethylene bridge were prepared. The effect of various substitution on the tetrahydro-β-carboline conformation and D-ring orientation was studied. In the course of preparing the C1-Me analog of MMV008138 featuring 2',4'- dichloro substitution, unexpected ring-expanded azepane products were isolated. Later it was found that the desired product could be isolated when the imine formed was treated with acid at lower temperature. Other intermediates possessing a 2ʹ- substituent were also isolated under the low temperature acid treatment protocol, which upon heating in acid gave the ring-expanded azepane we initially isolated. A mechanism was proposed to account for the formation of the azepane as well as other intermediates. The driving force of the expansion reaction was explored, and the hypothesis that the steric interaction between the 2ʹ-substituent and the C1-Me was supported via DFT calculation and conformational analysis. MMV665831 is another potent hit from the Malaria Box, and it appears to inhibit the hemoglobin endocytosis process of P. falciparum. The structure–activity relationship of MMV665831 was studied with analogues featuring modifications on C2-benzamide, C3-ester, C-7 phenol, as well as the phenolic Mannich base moiety. Modifications at phenolic Mannich base moiety leads to the discovery of an analogue that is twice as potent toward cultured P. falciparum compared to MMV665831. We were worried the phenolic Mannich base moiety might act as the precursor of toxic quinone methide intermediates, and designed two analogs to block this potential toxicophore. Although the modification resulted in reduced potency, this result proved that the potency of MMV665831 does not stem from the formation of quinone methides. Unfortunately, MMV665831 did not reduce parasitemia in P. berghei- infected mice. Fast hepatocyte metabolism was observed for MMV665831, and the loss of in vivo efficacy was discussed in comparison with other phenolic Mannich bases with similar hepatocyte stability. / Doctor of Philosophy / In the fight against malaria, one concerning issue is the rapidly emerging resistance towards existing drugs. The continuous development of antimalarials with novel mechanism of action is greatly needed. To accelerate the development of novel antimalarials, an open access ensemble of 400 compounds that are toxic to the malaria parasite known as the Malaria Box, has been made available. My work involves the optimization of two compounds from this ensemble, MMV008138 and MMV665831. MMV008138 kills the malaria parasite by inhibiting an enzyme named PfIspD, which is absent in human. In the parasite an enzyme called PfIspD is responsible for the biosynthesis of IPP and DMAPP, two chemical building blocks that are essential for all cells. It is unlikely that MMV008138 will interrupt with the biosynthesis of IPP and DMAPP in human, since we use another enzyme to synthesize them. Although MMV008138 shows great in vitro potency, but did not protect a live mouse from malaria infection. The lack of in vivo efficacy could stem from the rapid metabolism of MMV008138, and analogs aimed to prevent metabolism were designed and prepared. While preparing analogs featuring 2ʹ-substitution, the desired product was not found, but other unexpected by-products were isolated. The conditions that leads to both the desired products and the by-products were found, and the mechanistics detail of this unexpected reaction were studied. During the blood-stage, which causes malaria symptoms in human, the Plasmodium falciparum parasite invades and feeds on human red blood cells (erythrocytes). The parasite destroys human hemoglobin through a multistep process that begins by transporting the hemoglobin from the red blood cell into itself, a process called endocytosis. MMV665831 appears to interfere with this endocytosis process of P. falciparum, thus starving the parasite of its food. Analogs of MMV665831 were prepared to probe for the effect on potency, and one compound that is twice as potent in cultured parasites was found. The structure of MMV665831 contains a potentially unstable moiety, which might lead to toxicity in humans. Two analogs with the problematic moiety removed were designed and prepared, and one still shows antimalarial activity, showing that the reactivity of the potentially unstable moiety is not the reason for the antimalarial activity of MMV665831. However, MMV665831did not protect P. berghei-infected mice (murine malaria) in vivo, and the reason for the loss of efficacy was discussed.