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.

Plasmodium falciparum, the organism responsible for the most prevalent and most virulent cases of malaria in humans, poses a major burden to the developing world. The parasite is increasingly developing resistance to traditional therapies, such as chloroquine, so the need to determine novel drug targets is more prevalent than ever. One such method involves targeting proteins unique to the malarial proteome that do not have homologues in humans. An especially promising group of targets are protein kinases, which are involved in many different biochemical pathways within the cell. Eukaryotic cell cycle progression is moderated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). These kinases depend on the binding of a cognate regulatory unit (cyclin) in order to enter its activated state. Once activated, these cyclins then mediate phosphorylation events that are crucial to cell cycle advancement . Cyclin Dependent Kinases (CDKs) are common to most eukaryotes and are responsible for regulating the cell cycle of growth and proliferation. Proteins have been previously identified in Plasmodium that have sequence homology to traditional CDK and have a potential function to be classified as "CDK-like" kinases. Three kinases that fit this description are Plasmodium falciparum Kinase 5, 6, and mrk, or MO15- Related Kinase. These kinases are expected to have roles in both malarial growth and regulation of the cell cycle. Bacterial constructs were generated to express and purify recombinant forms of these kinases and potential substrates. Once the potential interactors were isolated, in vitro protein kinase assays were used to validate the interactions to the kinases as substrates. In summary, the study has identified substrates that are directly phosphorylated by PfPK6, and demonstrated that the identified proteins are not directly phosphorylated by PfPK5 and Pfmrk.

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.

An estimated 500 million cases of malaria occur each year. The increasing prevalence of drug resistant strains of Plasmodium in most malaria endemic areas has significantly reduced the efficacy of current antimalarial drugs for prophylaxis and treatment of this disease. Therefore, discovery of new, inexpensive, and effective drugs are urgently needed to combat this disease. Marine biodiversity is an enormous source of novel chemical entities and has been barely investigated for antimalarial drug discovery. In an effort to discover novel therapeutics for malaria, we studied the antimalarial activities of a unique marine-derived peak fraction library provided by Harbor Branch Oceanographic Institute (HBOI). Within this unique library, we have screened 2,830 marine natural product (MNP) peak fractions through a medium throughput screening effort utilizing the SYBR Green-I fluorescence based assay, and have identified 253 fractions that exhibit antimalarial activity. From those inhibiting fractions we have identified twenty species of marine organisms that inhibit Plasmodium falciparum growth, from which thirty-five fractions were selected for further study. Among those thirty-five, eighty-three percent were also found to inhibit the chloroquine resistant strain of P. falciparum, Dd2. The most potent inhibitors were then screened for their cytotoxic properties using the MTT cell viability assay. Among the samples that exhibited potent inhibition of P. falciparum growth were fractions derived from a sponge of the genus Spongosorites sp.. This genus of sponge has been reported to contain the nortopsentin and topsentin class of bis-indole imidazole alkaloids. Nortopsentin A inhibited the parasite growth at the trophozoite stage with an IC50 value of 1.6 µM. This is the first report of antimalarial activity for this class of compound.

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