Malaria is a devastating disease that continues to affect millions of people worldwide every year. Specifically, Plasmodium falciparum is the most common human malaria parasite, particularly in sub-Saharan Africa. P. falciparum causes the most malignant and debilitating symptoms with the highest mortality and complication rates. Even with the worldwide efforts of many researchers and organizations, the road to discovering a vaccine has been difficult and challenging. Due do to the improvements in in vitro liver stage assays as well as rodent models of mammalian malaria, pre-erythrocytic stages of malaria have become a more accessible target for experimental studies. These vaccine candidates target Plasmodium sporozoites in the liver and liver stages to prevent development to the blood-stage forms, which is responsible for the debilitating symptoms of the disease. Scanning electron microscopy has been used for decades to provide insight on the morphology and topography of specimens, which cannot be seen through a light microscope. The purpose of this study was to analyze the morphology of sporozoites with some target antibodies. Sporozoites have previously shown uncharacterized appearances and development in an immunofluorescent stain at different concentrations of particular antibodies. With this further understanding on the morphological impact few of the target antibodies have on sporozoites through scanning electron microscopy, further grasp can be acquired.

Human malaria caused by Plasmodium falciparum is a major global burden killing between 700,000 and 2.7 million people every year. Africa bears the greatest portion of this burden, with over three quarters of deaths occurring in African children, accounting for 18% of all child deaths in sub-Saharan Africa. Synthesis of tetrahydrofolate through the folate biosynthetic pathway is vital for the survival of P. falciparum parasites and is lacking in the human host. As such, enzymes of this pathway have long presented attractive targets for drug therapy and although increasingly being compromised by resistance, anti-folates such as pyrimethamine and sulfadoxine are still very valuable drugs in many malaria-endemic regions.In this project, further investigation of the enzymes of the folate biosynthetic pathway has been attempted by developing protocols to quantify these proteins and others through proteomic techniques. Two quantification techniques were pursued. The first was quantification using whole, heterologously expressed, stable-isotope labelled forms of P. falciparum proteins for use as heavy standards in mass spectrometry. Great difficulty was experienced in the effort to express and purify P. falciparum enzymes in E. coli expression systems, with only one enzyme successfully expressed and purified in a 13C-labelled form. This one protein was taken forward into quantification experiments. The second quantification technique used a stable-isotope labelled ‘QConcat’ protein, consisting of a number of peptides from 12 P. falciparum proteins of interest, as a heavy standard in mass spectrometry. This was successfully expressed and purified in a 13C-labelled form from an artificial gene using an E. coli expression system. This too was taken forward into quantification experiments.Quantification experiments using the QConcat-based quantification technique were successfully performed on whole P. falciparum extract. Among the proteins quantified were SHMT and DHFR, two proteins of great interest from the folate biosynthetic pathway. Consistent with results from different expression analysis techniques in the literature, the folate enzymes were found to be of lower abundance than housekeeping enzymes and SHMT was found to be more abundant than DHFR.For deep quantitative analysis of the P. falciparum proteome, it was found that fractionation was necessary. Fractionation in this project was performed using a ZOOM™ IEF fractionator (Invitrogen), an OFFGEL™ IEF fractionator (Agilent) and 1D SDS-PAGE. It was found that by using these fractionation techniques, more proteins could be identified within the P. falciparum proteome, with all but one of the enzymes of the folate biosynthetic pathway being identified. Significant advances in the sensitivity of mass spectrometers during this project have also greatly facilitated the investigation of the proteome. In some cases, this meant that proteins which were only previously accessible by prefractionation of the proteome could be seen in whole P. falciparum extract. Unfortunately, QConcat-based quantification using both fractionation and sensitive mass spectrometry could not be successfully achieved in the time available. However, the promising results obtained suggest that, after careful optimisation, such an approach will be valuable.

Malaria still remains one of the world’s biggest killers with more than two million people dying from the disease each year. Present drugs have become ineffective because parasites are developing resistance to most of them. Efforts are now being directed in obtaining drugs with different structural features. Plants have provided most of the antimalarial drugs so far and efforts now concentrate on screening plants for new antimalarial drugs. South Africa with its rich floral resources and ethnobotanical history is an ideal place to screen plants for antimalarial activity. The antimalarial activity of 20 extracts from 14 ethnobotanically selected South African plants was screened for antimalarial activity in vitro. Results obtained showed that most of the plants had strong antimalarial activity. IC50 values obtained with the flow cytometric method were between 0.9 and 2 µg/ml for 9 of the 10 selected extracts. This represents a very high number of extracts with very good antimalarial activity. Cytotoxicity of the most active extracts were determined against monkey kidney cells as well as a luminescent bacteria method. Results obtained had a ID50 between 35 and 100 µg/ml with the monkey kidney cell test and between 100 and 2000 µg/ml with the bacteria test. Therapeutic values ranged between 35 and 100. Extracts therefore have a poor selectivity towards Plasmodium. The dichloromethane extract from Ozoroa engleri was further purified with silica gel column chromatography, Sephadex column chromatography and HPLC. Results obtained showed at least five or six compounds responsible for the antimalarial activity of the extract, all with moderate antimalarial activity and no further efforts were undertaken to identify them. The acetone extract of Croton pseudopulchellus was then selected for isolation of active principles and was purified by silica gel column chromatography, Sephadex column chromatography and PTLC. Kaurenoic acid was isolated as one of the active principles and identified by NMR and GC-MS. Kaurenoic acid was found to have an antimalarial IC50 of 38 µg/ml, while its cytotoxicity ID50 was 35 µg/ml. Kaurenoic acid was responsible for only some of the activity found in the purified fraction and other compound(s) in the extract might have much better antimalarial activity. / Dissertation (MSc (Plant Physiology))--University of Pretoria, 2008. / Plant Science / unrestricted

Malaria remains a debilitating disease, especially in developing countries of the tropics and sub-tropics. Increasing drug resistance and the rising cost of drug development calls for methods that can cost-effectively identify new drugs. The proteins of the malaria causing Plasmodium parasites often exhibit unique features compared to their mammalian counterparts. Such features invite discovery of parasite-specific drugs. In this study computational methods were applied to understand unique structural features of enzymes from the Plasmodium polyamine biosynthesis pathways. Molecular modeling of P. falciparum arginase was used to explore the structural metal dependency between enzyme activity and trimer formation. This dependency is not observed in the mammalian host. A novel inter-monomer salt-bridge was discovered between Glu 295 and Arg 404 that helps mediate the structural metal dependency. Removal of the active site metal atoms promoted breaking of the Glu 295á::Arg 404b interaction during simulation. The involvement of this salt-bridge was further confirmed by site-directed mutagenesis of the recombinantly expressed enzyme and subsequent simulation of the mutants in silico. Mutations designed to break the salt-bridge resulted in decreased enzyme activity and oligomerisation. Furthermore, simulation of the mutants indicated potential loss of metal co-ordination within the active site. The interface around Glu 295á::Arg 404b could thus serve as a novel therapeutic target. In Plasmodium the usually separate activities S-adenosylmethionine decarboxylase and ornithine decarboxylase occur in a single bifunctional enzyme. Previous studies have established the importance of complex formation and protein-protein interactions for correct enzyme functioning. Disturbing these interactions within the complex may therefore have inhibitory potential. In the second aspect of this study the potential quarternary structure of AdoMetDC/ODC was studied by homology modeling of the domains followed by protein-protein docking. The results from five Plasmodium species suggest that one face of each domain is favoured for complex formation. The predicted faces concur with existing experimental results, suggesting the direct involvement of Plasmodium-specific inserts in maintaining complex formation. Further fine-grained analysis revealed potentially conserved residue pairs between AdoMetDC/ODC that can be targeted during experimental follow-up. In both aspects of this study computational methods yielded useful insights into the parasite-specific features of polyamine biosynthesis enzymes from Plasmodium. Exploitation of these features may lead to novel parasite-specific drugs. Furthermore, this study highlights the importance of simulation and computational methods in the current and future practice of Science. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

Propone un modelo matemático que describe la dinámica de la malaria, formado por ecuaciones diferenciales ordinarias(ODEs). Los resultados muestran que si el número de reproducción R0 es menor que 1, entonces el punto de equilibrio libre de enfermedad es estable, por lo tanto la enfermedad desaparece. Si R0 es mayor que 1, entonces el punto de equilibrio libre de enfermedad es inestable, por lo tanto la enfermedad se propaga. Se realiza simulaciones numéricas con el software matemático Matlab. Estas simulaciones muestran el comportamiento de las poblaciones en el tiempo y la estabilidad de los puntos de equilibrio libre de enfermedad y endémicos. / Trabajo de suficiencia profesional

The primary aim of this study was to use a computational structure-based ligand design strategy in finding novel ligands that could act as inhibitors of PfGST as basis for future antimalarial drug development. Since there is only one PfGST isoenzyme present in the parasite and the architecture of the binding site differs significantly from its human counter part, PfGST is considered a highly attractive drug target. Inhibition of PfGST is expected to interfere at more than one metabolic site in synergy: it is likely to disrupt the glutathione-dependent detoxification process, which will lead to an increase in the cytotoxic peroxide concentration and most likely lead to an increase in the levels of ferriprotoporphyrin IX and hemin as well. S-hexyl glutathione was co-crystallized with PfGST (Harwaldt et al., 2004), consequently it was seen as one of the most important lead compounds in the development of PfGST inhibitors. The first step in the rational drug design strategy was to modify GTX, concentrating on its ability to bind competitively to the G site and the hydrocarbon chain protrudes into the H site as well. Considering the 3D structure of the enzyme, modifications to GTX were made by LUDI and NEWLEAD, resulting in a library of active site binding ligands ranked by AutoDock according to their ability to optimally bind to PfGST. Additionally, the ligands were ranked according to their affinity for binding to PfGST produced by AutoDock, LUDI and XScore. Once all the compounds were ranked by these in silico methods they were screened for acquisition or synthetic accessibility and those available were experimentally screened for activity against recombinantly expressed PfGST. Based on in silico predictions NDA was the best inhibitor followed by LAP and EDP. From the biological assay and Lineweaver-Burk analysis the order of inhibition was NDA as the best inhibitor tested, followed by LAP and EDP. EDP and LAP showed competitive inhibition but the inhibition constant values were signi_cantly lower than GTX. With respect to GSH and CDNB, NDA was found to be a non-competitive inhibitor. It was suggested therefore that NDA binds to a non-substrate Summary 93 binding site that may lead to conformational change of the enzyme and hence lead to a loss in enzyme activity. This data leads to the conclusion that the H site should be better exploited in order to find more potent inhibitors or non-substrate binding sites. It was concluded that the experimental results add confidence to the discriminative power of the structure-based ligand design strategy and that these inhibitors could form scaffolds for future antimalarial drug development. / Dissertation (MSc (Bioinformatics))--University of Pretoria, 2008. / Biochemistry / unrestricted

The polyamines putrescine, spermidine and spermine play essential roles in the proliferation and differentiation of most eukaryotic cells. Inhibition of the polyamine pathway is known to have antitumour and antiparasitic effects and á-difluoromethylornithine (DFMO), a polyamine biosynthesis inhibitor, is clinically used in the treatment of African sleeping sickness caused by Trypanosoma brucei gambiense. Ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC) are the rate-limiting enzymes in polyamine metabolism. Usually, these enzymes are individually regulated, however, in the malaria parasite, Plasmodium falciparum, these enzymes are part of a unique bifunctional PfAdoMetDC/ODC protein. In addition, compared to homologous proteins, this malarial protein contains six unique parasite-specific inserted regions, which can be targeted with novel drugs. A modified restriction enzyme-mediated inverse PCR method was developed to delete the largest parasite-specific insert (411 bp) from the large PfAdoMetDC/ODC gene (4257 bp). The method was compared to existing deletion mutagenesis PCR protocols and was shown to be the most effective method (80% mutagenesis efficiency) as opposed to the 40% positively mutated clones obtained with the overlapping primer method in deleting a >100 bp region. The independent removal of all three the PfAdoMetDC domain parasite-specific inserts and subsequent activity analysis thereof showed that these inserts are essential for the catalytic activities of both the decarboxylase domains. Plasmodia conserved secondary structures within these inserts were identified and were also shown to be very important for domain activities, possibly through protein-protein interactions across and within the domains of the bifunctional complex for the efficient regulation of intracellular polyamine levels. The N-terminally located O1 insert in the PfODC domain is a highly conserved and structurally distinct insert, which is essential for both domain activities. Previous studies showed that the deletion of this insert prevents dimerisation of the PfODC monomers and as a result influences association of PfODC with the PfAdoMetDC domain to form the bifunctional ~330 kDa complex. In addition, immobilisation of the insert via the mutagenesis of flanking Gly residues and the disruption of a single conserved α-helix within the insert severely affected both PfODC and PfAdoMetDC activities. It was thus hypothesised that the helix is involved in protein-protein interactions and the dimerisation of the PfODC domain. Size-exclusion chromatography of the monofunctional PfODC and bifunctional PfAdoMetDC/ODC proteins with disrupted helices resulted in the elution of only the monomeric (~85 kDa) and heterodimeric PfAdoMetDC/ODC (~160 kDa) proteins, respectively. The mono- and bifunctional wild type and immobile proteins eluted as both dimeric PfODC (~170 kDa) and heterotetrameric (~330 kDa) fractions as a result of intact protein-protein interactions. These results were subsequently exploited in the design and application of a parasite-specific, mechanistically novel, inhibitory peptide specific for this non-homologous insert in the bifunctional protein. A 1000x molar excess of a synthetic peptide, complementary to the α-helix within the O1 insert but opposite in charge, resulted in a ~40% inhibition of the PfODC enzyme. This study thus provides a proof-of-principle for the use of an inhibitory peptide targeting a parasite-specific insert in the dimerisation interface of a uniquely bifunctional malarial protein. / Dissertation (MSc)--University of Pretoria, 2008. / Biochemistry / unrestricted

Malaria is a global health threat that causes 300 – 500 million clinical cases annually, resulting in approximately 2 million deaths. Chemotherapy and prophylaxis are becoming less effective because of increasing drug resistance by the parasite. Resurgence of malaria calls for the development of mechanistically novel drugs. The bifunctional organization of the two rate-limiting enzymes, AdoMetDC and ODC, in the P. Falciparum polyamine pathway and the presence of six parasite-specific inserts, present potential target sites for novel Plasmodia-specific drugs. The inserts are species-specific, hydrophilic, low complexity segments and form non-globular domains. The inserts are involved in intra- and interdomain interactions, which are important for stability and activity of the bifunctional construct. This study investigated properties of the parasite-specific inserts, one being the mobility of the O1 insert and the other the secondary structures present in the parasitespecific inserts. It is postulated that the mobility of the O1 insert plays a role in either heterotetrameric complex formation of the bifunctional construct or that the O1 insert acts as a “lid” to the ODC active site, which is necessary for catalytic function. Successful mutagenesis of the O1 flanking Gly residues to Ala, rendered the O1 insert immobile. The probable immobility of the O1 insert had a detrimental effect on the activity of both the AdoMetDC and ODC domains of the bifunctional protein. Molecular dynamics studies showed that movement restriction of the insert caused a conformational change in the ODC monomers. The decrease of both domain activities upon movement restriction of the O1 insert suggests that the insert is involved in protein-protein interactions, which is communicated throughout the protein. In addition, the roles of selected, predicted secondary structures in the Hinge, O1 and O2 parasite-specific inserts were investigated. á-Helices were disrupted by the introduction of a Pro residue, â-plates were removed with deletion mutagenesis. The effects of the secondary structure alterations on protein activity were monitored in the bifunctional PfAdoMetDC/ODC protein. Both domain activities were affected by the disruptions, although the ODC domain was more sensitive to the small changes. The results obtained in this study showed that the secondary structures in the parasite-specific insert are important for activity of both the AdoMetDC and ODC domains of the bifunctional protein, possibly via interdomain protein-protein interactions. The delineation of essential intra- and interdomain protein-protein interactions presents possible interaction sites for disruptive molecules in the combat against malaria. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

Malaria remains the most serious and deadly parasitic disease, affecting millions of people mostly in the poorest countries in the world. With no vaccine likely in the foreseeable future, drugs remain the best means of controlling the disease. Plants have provided most of the antimalarial drugs so far and it is likely that more antimalarial drugs will be discovered in this way. A previous study on South African plants yielded very good results on the extract level. In this study Croton steenkampianus leaf extract was selected for isolation of active principles. Bio-guided fractionation of the extract was done on silica column chromatography and Sephadex column chromatography. Five compounds, two favonoids, a triterpene and two new diterpenes, with a novel skeleton were isolated. Compounds were identified with NMR, MS and X-ray crystallography. Antiplasmodial activity of the compounds varied from moderate to excellent, with crotrene A having excellent activity. Further studies on the antimalarial potential of this compound are planned. Cytotoxicity of compounds and extract were determined against human lymphocytes. Results obtained had an ID50 between >16,61 µg/ml. The therapeutic indexes were between 2.75 and 55.18, showing poor to moderate selectivity towards Plasmodium. Crotrene A had the best therapeutic index and more detailed studies on its cytotoxicity are necessary. Resistance to antimalarial drugs is a major problem in effective treatment of the disease. One way of overcoming this problem is combination drugs working synergistically. Chloroquine the most affordable antimalarial drug was combined with the isolated compounds. Two compounds showed synergistic activity with crotrene A having excellent activity, completely reversing chloroquine resistance. This combination of drugs showed no synergistic cytotoxic effects and its potential as a drug will be further investigated. The mode of action of antimalarial drugs can provide useful information about the long term potential and the likelihood of resistance development. Crotrene A was subjected to a basic test to determine a possible mode of action. Results showed a marked effect in the early phase of development (rings). The results suggest a very potent mode of action able to reduce the amount of parasites quickly and this holds promise for further development of this compound. / Thesis (DPhil)--University of Pretoria, 2008. / Plant Science / unrestricted

According to the World Health Organization, malaria has been classified as one of the three most important infectious diseases in Africa. The number of malaria cases is still on the increase in various countries, such as Rwanda and Zambia, which highlights the fragility of malaria control and the need to maintain and improve control programs. An innovative strategy for developing new antimalarial agents is through targeting epigenetic regulatory mechanisms in the malarial parasite, Plasmodium falciparum. Histone posttranslational modifications (PTMs) are factors contributing to epigenetic regulation in P. falciparum parasites. The epigenetic regulatory enzyme, Lysine-specific demethylase 1 (LSD1), has the ability to remove methyl groups from mono- and dimethylated lysine residues and is a regulator of gene expression through the modulation of chromatin structure. Polyamine analogues have been described as epi-drugs that target cell cycle development by blocking epigenetic control mechanisms in mammalian cells. A library of polyamine analogues were tested in cancer cells and found to specifically inhibit LSD1. In addition, these analogues were shown to have antiplasmodial activity against a drug-sensitive parasite strain, with IC50 values ranging from 88-100 nM but were metabolically unstable in vivo. In an attempt to overcome this in vivo hurdle, the leading compound was fluorinated at four different positions and tested for improved antiplasmodial activity and selectivity towards the parasites. Furthermore, the effect of the compounds on epigenetic regulatory mechanisms, through inhibition of LSD1 activity, was investigated. The analogues showed inhibition of parasite proliferation at low nanomolar concentrations and were very selective towards the parasites with low resistance indices. The leading compound showed reversible cytotoxicity towards parasite proliferation in addition to inhibitory activity against LSD1 and therefore, epigenetic regulatory changes. The approach taken in this dissertation is novel as none of the currently available antimalarials target LSD1 and as such, adds valuable information to future perspectives for drug design. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / Biochemistry / MSc / Unrestricted