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 is a global pandemic that affects millions of people each year. It is a parasitic infection caused by the Plasmodium family, with Plasmodium falciparum being the most virulent strain. Malaria is transmitted to humans by the female Anopheles mosquito. The parasite undergoes two different cycles of its life cycle within the human host: the liver and intraerythrocytic life cycle. The latter consists of an asexual and sexual cycle. The intraerythrocytic cycle is perhaps the most important stage of the parasite's life cycle as it promotes the spread of the disease within and between hosts. The focus of this investigation was aimed at the invasion process of the merozoites into the erythrocytes. The Plasmodium merozoite utilises a cascade of proteins during the erythrocyte invasion process, which is a swift action that takes place in approximately 30 seconds. A number of surface proteins are expressed during merozoite development and are distributed along the merozoite surfaces to assist with attachment and invasion, the most crucial being MSP-1, AMA-1 and RON-2. MSP-1 and AMA-1 are vital targets for the development of malaria vaccines. AMA-1 is the central target protein of this investigation as it plays an essential role in the invasion process. AMA-1 commits the merozoite to invade the erythrocyte, as it assists the RON proteins in the formation of an irreversible tight-junction with the membrane of the erythrocyte. Antibodies, specific to AMA-1, bind to the protein, which prevents the formation of the tight junction and inhibits the invasion of the merozoite into the erythrocyte, therefore preventing the spread of the disease. However, before invasion, AMA-1 undergoes a number of proteolytic processes. It is synthesized as an 83 kDa (AMA-183) precursor protein in the apical organelle of the merozoite. This is then cleaved at the N-terminus to give rise to a 66 kDa (AMA-166) fragment, which is secreted onto the surface of the merozoite. The AMA-166 fragment is then cleaved into either a 48 kDa (AMA-148) or 44 kDa (AMA-144) fragment. One of these three fragments is then used by the merozoite for erythrocyte invasion. The aim of this investigation was to isolate and characterise each of the fragments of the Plasmodium falciparum AMA-1 (PfAMA-1) protein using the 3D7 lab strain of P. falciparum and to visualise the merozoite-erythrocyte invasion process, to possibly identify which of the AMA-1 fragments are involved in the invasion process. In order to achieve this large clusters of merozoites from sorbitol-synchronised cultures were isolated. Schizonts were isolated from culture by magnetic separation and incubated with E64 to prevent the release of merozoites. Merozoites that were required for the isolation of PfAMA-1 were harvested from the schizonts by saponin lysis, then homogenised, separated by SDS-PAGE and digested for LC-MS/MS analysis. Merozoites that were required for the visualisation procedures were not incubated with E64, to allow natural egression from the erythrocyte. The transmission electron microscopy results produced clear images of the merozoiteerythrocyte invasion process and the positioning of PfAMA-1 on the merozoite, before and after schizont rupture, was visualised from results obtained from confocal microscopy. Then PfAMA-1 was identified in isolated merozoite samples by LC-MS/MS analysis. However, due to its low abundance, isolation of high enough concentrations of PfAMA-1 to characterise its different fragments was not achieved. Further investigation into the development of the culturing and isolating methods could help in future projects aimed at isolating higher concentrations of merozoite proteins from synchronised cultures with a lower merozoite egression window period, in order to accomplish detailed analysis on invading proteins for the future development of treatments against malaria. / Dissertation (MSc)--University of Pretoria, 2016. / Pharmacology / MSc / Unrestricted

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models. / Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) / Revisión por pares

Magister Pharmaceuticae - MPharm / Malaria is an infectious disease which continues to kill more than one million people every year and the African continent accounts for most of the malaria death worldwide. New classes of medicine to combat malaria are urgently needed due to the surge in resistance of the Plasmodium falciparum (the parasite that causes malaria in humans) to existing antimalarial drugs. One approach to circumvent the problem of P. falciparum resistance to antimalarial drugs could be the discovery of novel compounds with unique scaffolds and possibly new mechanisms of action. Natural products (NP) provide a wide diversity of compounds with unique scaffolds, as such, a library of virtual compounds (VC) designed from natural products with antiplasmodial activities (NAA) can be a worthy starting point.

In meeting the high demand for monoclonal antibodies, the chicken immunoglobulin system was exploited to generate recombinant antibodies against multiple target antigens. Following simultaneous immunisation of two chickens with a mixture of Plasmodium falciparum recombinant lactate dehydrogenase (LDH), histidine rich protein II (HRPII) and aldolase (ALDO), recombinant trypanosome variable surface glycoprotein (VSG) and malignant catarrhal fever virus (MCFV) each chicken produced egg yolk antibodies (IgY) against four of the five antigens. Using phage display technology, two single-chain variable fragment (scFv) antibody libraries, one with the immunoglobulin VH and VL chain regions joined by a single amino acid (G) and the other with a 15 amino acid flexible linker [(G4S) 3] were constructed using pooled splenic RNA. The single amino acid-linked scFv repertoire was evaluated as a source of highly specific diagnostic antibodies by panning against each of the five different antigens. After two rounds of panning, polyclonal phage ELISA showed the presence of antigen-specific phage antibodies against three (LDH, HRPII and VSG) of the five antigens. Five different anti-LDH and six different anti-HRPII scFvs were identified by sequence analysis. Evidence of high levels of antigen-driven gene conversion events was found in the framework and complementary determining regions and the VL chain pseudogene donors were identified. Stability of the selected scFvs was determined by incubation at different times and at different temperatures. The specificity and potential use of an LDH-specific scFv as a diagnostic reagent was shown in sandwich and competitive inhibition ELISAs. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

Malaria claims millions of lives annually. This global killer causes approximately 2.7 million annual deaths worldwide; addressing this problem has become more and more crucial. Due to pathogen evolution no efficient vaccine for treatment of malaria currently exists. As infection has developed as a field of study, it became ever more clear that infections could only be understood within the context of the host-pathogen community. This project aims to predict possible drug targets based on host-pathogen interactions rather than just protein-protein interactions within a single organism. Similar to Lee et al. (2008) pathogen-host interaction predictions are based on orthology, these interactions are then analysed to identify potential drug targets. This could potentially aid researchers in their continuous battle against malaria and the larger scale battle against pathogen evolution. To predict in vitro host-pathogen interactions DISCOVERY uses an ortholog clustering method called ORTHOMCL. ORTHOMCL is very suitable for ortholog clustering of malaria data for two reasons. Firstly, it is capable of distinguishing between recent paralogs and ancient paralogs, which enables the inclusion of recent paralogs together with orthologs. Secondly, ORTHOMCL was initially developed for the use of malaria data. Identification of in vitro interactions is followed by scoring methods to determine the possible in vivo interactions that might occur between the Plasmodium parasite and the human and mosquito hosts. Scoring measures and weights were applied to 5 different factors to calculate a final score. These final scores allow user input to define the preferred stringency when viewing possible interactions with a single protein. These different factors are sequence similarity, PEXEL/VTS motif presence, microarray expression, metabolic map sharing and sub-cellular locations boundaries. DISCOVERY’S results and results from two other (Dyer et al. and Lee et al.) in silico prediction methods were compared with Vignali et al’s experimental interactions which are based on a yeast two-hybrid approach. Similar to results shown by Doolittle and Gomez these comparisons had poor results. The next step was to compare the in silico results with each other. Dyer et al’s and Lee et al‘s results compared poorly with each other. Although DISCOVERY did not compare well with Dyer et al’s results, comparisons with Lee et al. showed more promise. Poor comparisons with Dyer et al. may be due to their unique approach to predict in vitro host-pathogen interactions. This project identified the lack of enough valid and reliable experimental data to evaluate in silico prediction methods as a definite challenge for host-pathogen interaction predictors. Although this is a major problem, DISCOVERY improved on older prediction methods with the use of a more applicable ortholog clustering technique and the use of more assessment methods during in vivo interaction predictions. DISCOVERY also used scoring methods rather than exclusion methods during the identification of in vivo interactions. This allows a user to specify a threshold of sensitivity when viewing interactions. The true potential of host-pathogen interaction predictions would only be realized when the gap between predictions and evaluation data is bridged. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

The malaria-causing parasite Plasmodium falciparum 1s dependent on tightly regulated gene expression for its progression through the intra-erythrocytic life cycle, pathogenesis and establishment of persistent infection by evasion of the human host's immune system. Evidence points towards P. falciparum being unusually dependent on nuclear architecture and genomic organisation for the control of gene expression. Spatially defined nuclear regions of transcriptional activity have been detected and the spatial positioning of loci may determine their transcriptional potential. Additionally, a number of epigenetic markers have been shown to occupy spatially distinct subcompartments of the nuclear volume. Limitations of microscopic assays used until now have left us with a stereotyped and incomplete image of the organisation of the parasite nucleus and the transcriptional and epigenetic factors involved in the regulation of parasite gene expression, and the possible dynamics thereof. This work focused on the use of high-content and super-resolution fluorescent microscopy for the study and graphical representation of the spatial organisation of various nuclear factors involved in transcriptional regulation in P. falciparum parasites. The first objective (chapter 2) establishes P. falciparum parasite sample preparation and fluorescent labeling techniques for microscopy. Immunofluorescent labeling of var gene associated transcription repressive and permissive histone modifications, H3K9me3 and H3K9ac, respectively, as well as serine 2- phosphorylated RNA polymerase II and the putative transcription and splicing factor PfMyb2, was optimised. DNA fluorescent in situ hybridisation was also optimised for labeling of var gene exons. In the second objective (chapter 3), the assays established in the previous chapter are used for high-content combinatorial labeling in thousands of nuclei, followed by analysis using a bespoke computational algorithm for the detection and classification of different labeling patterns. This approach revealed a high level of diversity in the nuclear distributions of each assayed target. Superresolution stochastic optical reconstruction microscopy was used to further study the sub-diffraction organisation of selected labeling patterns. The data presented in this dissertation reveal that the complex spatial organisation of certain nuclear factors is subject to greater diversity within the nucleus of P. falciparum parasites than previously thought. / Dissertation (MSc)--University of Pretoria, 2012. / gm2013 / Biochemistry / unrestricted

In spite of great effort aimed at eradication, the malaria epidemic still claims over 600,000 lives each year, and 50% of the world is at risk of contracting the disease. The most deadly form of malaria is caused by Plasmodium falciparum, which is spread from human to human via the female Anopheles mosquito. P. falciparum's lifecycle, which includes both sexual and asexual reproduction, facilitates rapid evolution in response to drug pressure, resulting in the emergence of resistant strains against every antimalarial medication that has been deployed. There is a great need for new antimalarial drugs. Chloroquine (CQ), an aminoquinoline drug deployed in the 1940s, was an inexpensive, effective and safe drug but now has been rendered ineffective throughout much of the tropical regions due to the emergence of CQ-resistant strains of P. falciparum. A new class of hybrid drugs, called Reversed-CQs, has been developed by linking a molecule with a CQ-like moiety to a molecule with a reversal agent (RA) moiety; an RA is a chemosensitizer that can reverse CQ-resistance. The prototype Reversed-CQ, PL01, was shown to be effective in vitro against sensitive and resistant P. falciparum cell cultures, with IC50 values of 2.9 and 5.3 nM, respectively, in comparison to IC50 values for CQ which were 6.9 and 102 nM, respectively. In the course of the Reversed-CQ research, PL74 was synthesized with a pyridine ring replacing the quinoline ring. It was expected that PL74 would display reversal agent activity but would not display antimalarial activity. However PL74 showed antimalarialactivity with IC50 values of 185 and 169 nM in vitro against CQ-sensitive and CQ-resistant strains, respectively. In the investigation of PL74 it has been found that this molecule has a pyridinium salt structure, novel to the Reversed-CQ compounds, and through a structure-activity relationship (SAR) study, it was shown to have activity that may indicate a mode of action different from the Reversed-CQ compounds. A study of the literature revealed that pyridinium salt compounds, with some similarity to PL74, were found to operate as choline analogs inhibiting the biosynthesis of phosphatidylcholine as their main antimalarial mode of action.

Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia [37]. According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of the human host, and human erythrocytes. When the parasite infects the erythrocyte, some induced cell host modifications are made in order to accommodate growth. During its intra-erythrocytic life cycle, the malaria parasite traffics numerous proteins to a set of unique destinations within its own plasma membrane including the digestive vacuole, the apicoplast, rhoptries, and micronemes. Vesicular transport is an essential process in eukaryotic cells. This coordinated process is responsible for moving thousands of proteins between compartments within the cell. Essential to the targeting and fusion of protein transport vesicles in eukaryotes are SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), a family of fusogenic proteins that are localized to distinct intracellular compartments [11]. Studies performed in our laboratory have identified 18 proteins putatively belonging to the PfSNARE family [2]. To date the exact role of PfSNAREs in the unique trafficking pathways of malaria is undetermined. Of particular interest to our study is PfVAMP8. In model eukaryotic organisms, VAMP8 containing vesicles deliver cargo to lysosomes and are involved in endocytosis. The food vacuole of the parasite is very similar to that of lysosomes and is essential to parasite survival. The study aims to identify the organelle(s) to which PfVAMP8 is localized and characterize membrane-association properties of this parasite’s R-SNARE protein. We believe that PfVAMP8 would localize to unique compartments in the parasite protein network flow. An in depth understanding of its mechanisms and localizations could be a key in developing novel anti-malarials. This study aims to identify the organelle(s) to which PfVAMP8 are localized, determine the trafficking determinants of this protein and determine this proteins’ expression and membrane association during the intra-erythrocytic stages of Plasmodium falciparum. Our immunofluorescence studies with known biological markers reveals that, PfVAMP8 passes through the endoplasmic reticulum, Golgi, and localizes to the food vacuole during trophozoite and schizont stage. Further characterization of the membrane association properties of the protein in this study reveals that PfVAMP8 is a soluble integral membrane protein with amphipathic characteristics.

A novel experimental approach for micro-absorption spectroscopy and high-pressure microscopy of single cells is developed and applied to the investigation of morphological, volume, and spectroscopic changes in healthy red blood cells (RBCs) and erythrocytes infected with the malaria parasite Plasmodium falciparum. Through real-time optical imaging of individual erythrocytes (size ~ 7[micrometer]) we determine the change in volume over the pressure range from 0.1 to 210 MPa. The lateral diameter of healthy RBCs decreases reversibly with pressure with an approximate slope of 0.015 [micrometer] / MPa. In infected cells, clear differences in the deformability and between the compression and decompression curves are observed. The results are discussed with respect to the elasticity of the phospholipid membrane and the spectrin molecular network. Employing micro-absorption spectroscopy with spatial resolution of 1.4 [micrometer] in the lateral and 3.6 [micrometer] in the axial direction the visible absorption spectrum of hemoglobin in a single red blood cell is measured under physiological conditions. The spectra of cells infected with the malaria parasite show changes in peak positions and relative intensities in the Soret and [alpha]- and [beta]- bands. These indicate hemoglobin degradation that can be correlated with the stages of the parasite multiplication cycle and can be used as a potential diagnostic marker. The research is further extended towards the understanding of pressure effects on the ligand binding kinetics to heme proteins. For a well characterized reaction at ambient pressure, CO binding to myoglobin in solution, we investigate the transient absorption following laser flash photolysis over eight decades in time at variable pressure and temperature. The data demonstrate that pressure significantly affects the amplitudes (not just the rates) of the component processes. The amplitude of the geminate process increases with pressure corresponding to a smaller escape fraction of ligands into the solvent and a smaller inner barrier.