3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Inhibition of Schistosoma japonicum glutathione transferase by Cibacron Blue: insights from structural, functional and molecular modelling studies(2018) Hlabano, BabongiweSchistosomiasis is a leading neglected tropical disease, caused by blood flukes of the genus Schistosoma. Around 200 million people worldwide are affected, with the majority in Sub-Sahara Africa. Currently, only praziquantel is used for the treatment of schistosomiasis and its exclusive use has led to concerns of rise of praziquantel resistant Schistosomes. There is therefore a need for the development of new anti-schistosomal drugs. Schistosoma species lack the cytochrome P-450 detoxification mechanism, an important mechanism in human detoxification cycle, thus making Schistosoma glutathione S-transferase (GST) one of the main enzyme for detoxification of electrophilic and hydrophobic compounds. Schistosoma japonicum GST (SjGST) is an attractive drug/vaccine target against schistosomiasis. In this study, the mechanism of inhibition of SjGST by Cibacron Blue 3G-A (CB3GA) was investigated. Soluble SjGST was recombinantly expressed and purified successfully to homogeneity. SjGST maintained dimeric structure in the presence of CB3GA. IC50 value of CB3GA was determined to be 100 nM. Michaelis-Manten kinetic studies where performed in the presence and absence of CB3GA and showed that SjGST has high affinity for glutathione compared with CDNB. Lineweaver–Burk plots indicated that CB3GA is an uncompetitive and mixed inhibitor to the G-site and H-site respectively. Induced fit docking predicted that CB3GA binds to the L-site consistent with kinetic inhibition studies. MM-GBSA predicted free binding energy of SjGST and CB3GA was ΔGPred = -310 kJ/mol compared with experimental free energy of binding of ΔGExp = -49 kJ/mol. CB3GA is an efficient inhibitor of SjGST that binds to the dimer interface of SjGST altering catalytic activity of both the G-site and H-site. The unique characteristic of the L-site provides an opportunity for highly specific rational drug design.Item An investigation of plasmodium falciparum sortilin in trafficking of invasion proteins of the human malaria parasite(2018) Shunmugan, SerenaMalaria is arguably one of the most overwhelming infectious diseases throughout the world's existence. The most virulent parasite, Plasmodium falciparum, has a redundancy of invasion proteins, allowing it to switch between different receptors on the host red blood cell. These invasion proteins are stored in the apical organelles, the rhoptries and micronemes, but very little is known about how newly synthesized proteins are transported to these organelles. The hypothesis in this study was that a common protein is involved in trafficking invasion proteins from the trans-Golgi network and PfSORTILIN was investigated as a potential escorter protein. The CCys domain of PfMAEBL, a rhoptry protein, and the prodomain of PfAMA-l, a microneme protein, have been implicated in trafficking to the apical organelles. These domains and the VPS 10 domain of PfSORTILIN were cloned into expression vectors encoding a GST- or Histag. Recombinant proteins were expressed in E. coli and purified by affinity chromatography on glutathione- or Ni-particles. In vitro binding assays were performed, which showed that PfSORTILIN VPS 10 bound to PfMAEBL ccys but not to the PfAMA-1 prodomain, suggesting that PfSORTILIN is a rhoptry protein escorter and is not involved in microneme trafficking. To identify novel binding partners of PfSORTILIN VPS 10, the protein was biopanned against a P. falciparum phage display library. No binding partners were identified, most likely because the library is not schizont-stage specific, which is when PfSORTILIN and invasion proteins are predominantly expressed. The results from this study were integrated with other studies and a trafficking model for PfMAEBL was proposed. This study enhances our knowledge of trafficking pathways and suggests that PfSORTILIN may serve as a common rhoptry protein escorter.Item The total synthesis of 5-methoxy-3,4-dehydroxanthomegnin, dehydroherbarin lactone and 6-ferrocenyl-3,4-dihydroisochromene(2018) Sumani, Jimmy Yaphet EphetPyranonaphthoquinones, especially those with lactone functionality, are a class of naturally occurring compounds that exhibit a wide range of biological properties. For example, studies have shown that 10-hydroxy-5,7-dimethoxy-3-methyl-1H-naphtho-[2,3-c]-pyran-1,6,9-trione has a plethora of biological activities which includes, among others, cytotoxicity against McCoy cell lines and significant antibacterial property against Helicobacter pylori. This PhD project describes the total synthesis of 7,9-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,5,10-trione from simple starting materials in 14 steps in an overall percentage yield of 5%. The synthesis was achieved by subjecting the commercially available, 2,4-dimethoxybenzaldehyde, to a number of synthetic transformation protocols we have developed in our laboratory group. The key steps in the synthesis included the PIFA mediated methoxylation of the naphthalene nucleus that was constructed from the Stobbe condensation reaction and the palladium assisted Wacker oxidation reaction of the naphthoic acid intermediates. We also successfully synthesized 10-hydroxy-5,7-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,6,9-trione starting from 2,4,5-trimethoxybenzaldehyde in 15 steps in an overall percentage yield of 1%. This was achieved by employing the protocol we established during the synthesis of 7,9-dimethoxy-3-methyl-1H-benzo[g]isochromene-1,5,10trione. In the course of achieving the total syntheses of 7,9-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,5,10-trione and 10-hydroxy-5,7-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,6,9-trione a number of unexpected products were isolated and characterized. The second part of the project was aimed at developing a methodology for coupling a ferrocene moiety to 3,4-dihydroisochromene and subjecting the resultant ferrocenyl 3,4dihydroisochromene to oxidative demethylation protocols. Ferrocene coupled hybrid compounds are currently receiving a lot of attention because of their application in medicine. Abstract iii For example, ferroquine is active against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. The synthesis of 6-ferrocenyl-(±)-trans-3,4-dihydro-5,8-dimethoxy-1,3-dimethyl-6-vinyl-1Hisochromene was achieved starting from 2,5-dihydroxyacetophenone in 10 steps in an overall percentage yield of 10%. The key steps in this methodology involved the base mediated ring closing reaction which delivered (±)-trans-3,4-dihydro-5,8-dimethoxy-1,3-dimethyl-6-[(E)-1propenyl]-1H-isochromene and the McMurray cross-coupling metathesis reaction which afforded 6-ferrocenyl-(±)-trans-3,4-dihydro-5,8-dimethoxy-1,3-dimethyl-6-vinyl-1Hisochromene. The oxidative demethylation of 6-ferrocenyl-(±)-trans-3,4-dihydro-5,8-dimethoxy-1,3dimethyl-6-vinyl-1H-isochromene to complete the total synthesis of 6-ferrocenyl-(±)-trans3,4-dihydro-1,3-dimethyl-6-vinyl-1H-isochromene-5,8-dione was unsuccessful and an alternative protocol was developed which was aimed at coupling of ferrocene moiety to (±)trans-6-bromo-3,4-dihydro-1,3-dimethyl-1H-isochromene-5,8-dione at the last step. This methodology did not yield the target molecule, 6-ferrocenyl-(±)-trans-3,4-dihydro-1,3dimethyl-6-vinyl-1H-isochromene-5,8-dione. However, a number of potentially biologically important serendipitously obtained compounds were isolated. The last part of this PhD project involved in vitro biological screening of 3 selected compounds synthesized in this project against the Gambian FCR-3 strain of Plasmodium falciparum. All the 3 compounds namely, (±)-trans-15-bromo-5,13-dimethoxy-2,4,10,12tetramethyl-4,7,9,10,12,13-hexahydro-1H-7,13-methanopyrano[4',3':6,7]oxocino[2,3-f]isochromen-8(2H)-one, 3-(chloromethyl)-3,4-dihydro-5,7,10-trimethoxybenzo[g]isochromene1,6,9-trione and bis[(±)-trans-3,4-dihydro-8-methoxy-1,3-dimethyl-1H-isochromen-5yloxy]methane showed promising activity. (±)-Trans-15-bromo-5,13-dimethoxy-2,4,10,12tetramethyl-4,7,9,10,12,13-hexahydro-1H-7,13-methanopyrano[4',3':6,7]oxocino[2,3-f]isochromen-8(2H)-one showed the best activity with an IC50 value of 0.0018 µM. This value was better than the activity shown by the reference compounds, quinine and dihydroartemisinin, which showed activity of 2.81 µM and 0.0061 µM, respectively, under the same assay conditions.Item The characterization of the phosphatidyl-inositol-3-kinase in plasmodium falciparum and the effect of selective inhibitors of this enzyme on the parasite(2004-05-04) Mtombeni, NokuhleMalaria is the most prevalent parasitic disease in the world and the emergence of drug resistant strains of Plasmodium falciparum has made the search for new antimalarial drugs important. Protein kinases play an important role in cellular function and the phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in diverse cellular processes such as glucose transport, cell survival and proliferation. A homology based approach identified an open reading frame (ORF) coding for the catalytic region of part of the 6.4 Kb ORF of PFE0765w gene sequence found at plasmoDB. The ORF consisted of 1 758 base pairs which coded for a 586 amino acid protein with a molecular weight of 68.5 KDa. The PfPI3K ORF was amplified from P.falciparum DNA, subcloned into an expression vector and the sequence verified. Analysis of the expressed protein obtained by Western blotting and probing with anti-His monoclonal antibody showed a protein of 68.5 KDa as well as some smaller products.Item An investigation of p-glycoprotein in plasmodium falciparum and the isolation of haemozoin(1992) De Almeida, Maria, RosarioChloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptical parasites, and this is thought to be the basis of their resistance ( Fitch, 1970 ). Martin et. al ( 1987 ), recently demonstrated that in the presence of verapamil, a calcium channel blocker, chloroquine-resistant P falciparum becomes chloroquine-sensitive, with an increase in the chloroquine accumulation.The mechanism of such reversal has yet to be elucidatedItem Investigating a BIR-containing plasmodium falciparum protein and identifying its binding partners(2018) Liebenberg, DaleThe majority of the worldwide malaria deaths are caused by the Plasmodium falciparum parasite and unfortunately parasite strains are emerging that are resistant to not only artemisinin, but also the partner drugs used in current antimalarial combination therapy. The intraerythrocytic P. falciparum life stage is characterised by exponential, asexual proliferation that could cause the premature death of the human host before the sexual gametocytes have had enough time to develop and be taken up by the mosquito vector to continue its lifecycle. It is hypothesised that P. falciparum maintains its population at a level low enough to allow for the transmission of these gametocytes by using a form of regulated cell death (RCD). The molecular members of this cell death pathway are currently unclear, but a putative P. falciparum inhibitor of apoptosis protein (PfIAP; PF3D7_0519600) has previously been identified. Metazoan IAP proteins play anti-apoptotic roles in cells by interacting and inhibiting pro-apoptotic caspases, but also perform other functions. Analysis of the PfIAP protein using bioinformatic tools revealed that it contains one conserved baculoviral IAP repeat (BIR) domain and that this P. falciparum BIR domain is structurally similar to the BIR domains of various human IAP proteins. mRNA extracted from asexual P. falciparum parasites was used to construct a biotin-tagged phage display library, which was used in biopanning experiments with two regions of the PfIAP protein, expressed as recombinant GST-tagged proteins. Four binding partners were identified for the N-terminal BIR domain of the protein, while two proteins were identified as interacting partners for the C-terminal region of PfIAP. Of these, a double C2-like domain-containing protein (PfDOC2) and the high molecular weight rhoptry protein 3 (PfRhopH3) were expressed as recombinant His-tagged proteins and verified as PfIAP binding partners by in vitro binding assays. Transgenic P. falciparum parasites were generated expressing a GFP-tagged PfIAP BIR domain, which localised to the cytoplasm under both normal and high temperature conditions which mimic febrile malaria, a physiological trigger of RCD. Knockout experiments of the pfiap gene using the CRISPR-Cas9 genome editing tool suggested that this gene could be essential for the survival of asexual P. falciparum parasites. This study offers the first details of a putative P. falciparum inhibitor of apoptosis protein and suggests that it could have non-apoptotic roles in the parasite, given the diverse functions of the binding partners that comprise the PfIAP protein-protein network.Item The effect of 7-chloroquinoline derivatives on the life cycle of the malaria parasite(2017) Kathrada, FatimaWith the widespread resistance to current antimalarials and the great burden of malaria, the aim of this study was to determine the antimalarial activity of novel derivatives against the Plasmodium falciparum NF54 strain alone and in combination with quinine using the pLDH assay. The derivatives were designed such that they maintained the core chloroquinoline structure with known antimalarial activity with the addition of further scaffolds with a different mechanism of action in an attempt to produce compounds with good antimalarial activity with a lower probability of developing resistance. The twenty-seven 7-chloroquinolin-4-yl piperazine-1-yl acetamide (CQPA) derivatives and the nine 4-((7-chloroquinolin-4-yl) piperazine-1-yl) pyrrolidin-2-yl) methanone (CQPPM) derivatives all possessed antimalarial activity with 4 and 8 derivatives from each class respectively showed inhibitory activity below 10 µM. The most active derivative from each set of derivatives, CQPA-26 and CQPPM-9 displayed good antimalarial activity with IC50 values of 1.29 µM and 1.42 µM, respectively compared to the standard antimalarial, quinine (IC50: 0.18 µM). The two most active derivatives displayed drug-like properties with favourable ionisation properties to confirm its ability to accumulate in the parasites’ digestive vacuole. In combination with quinine, these derivatives displayed synergistic and additive interactions, respectively. Morphological studies were carried out over a period of 48 hours to identify which parasite stage was most sensitive to the most active derivative from each class of derivatives. Both derivatives proved to show similar schizonticidal activity as the standard quinine with a lag in progression from the trophozoite stage. The inability to induce haemolysis indicated that the derivatives acted against the parasite and not the host red blood cell (<1% lysis), conferring a good safety profile. The low toxic nature of the derivatives was further demonstrated with the minimal toxicity towards human embryonic kidney epithelial cells and no effect on Artemia fransiscana brine shrimp viability (<5% lethality). Although the cyclopropyl and methyl substituted derivatives displayed good activity towards the intra-erythrocytic parasite, all the derivatives lacked larvicidal activity towards Anopheles arabiensis (mortality range: 0 - 5%). The derivatives showed no effect on the morphological structure of the Artemia fransiscana brine shrimp and the An. arabiensis larvae. This study highlights the good antimalarial activity of these derivatives coupled with a favourable safety profile as antimalarial agents and not larvicides. The introduction of less bulky side chains proved to show a greater antimalarial activity against Plasmodium falciparum.Item Characterisation of Southern African strains of the malarial parasite plasmodium falciparum(1993) Freese, Janet AnneThis thesis describes the characterisation of southern African isolates of Plasmodium falciparum according to isoenzyme type, antigen variants and drug sensitivity. Nineteen southern African isolates and a Gambian reference isolate were cultured in vitro in gassed tissue culture flasks. Polyacrylamide gel electrophoresis with use of 5 enzymes revealed little variation amongst the isolates and the frequencies of enzyme forms were similar to those of isolates from other parts of the world. Antigenic diversity was demonstrated using a panel of 9 monoclonal antibodies in the indirect fluorescent antibody test. The antigenic composition of 70% of isolates was markedly different to any obtained in other geographical areas. Both characterisation techniques revealed a mixture of parasite types in some isolates. However I the characteristics of most of these heterogeneous isolates were not stable with time in culture. Most isolates proved to be resistant to chloroquine in a 48-hour growth inhibition test. A wide range in Pyrimethamine susceptibilities was detected although most isolates exhibited a low level of resistance to this drug. In the radioisotope uptake assay, the halofantrine IC50 values obtained were comparable with those of West African isolates but were higher than the TC50s of south-East Asian isolates. All of the isolates resistant to chloroquine in the 48-hour test were resistant in the radioisotope assay. However, 1 isolate shown to be sensitive to chloroquine in the first test was found to be resistant in the 3Hhypoxanthine incorporation method. This was assumed to be the result of selection of resistant clones. Southern African isolates were shown to be fully susceptible to mefloquine, but had reduced susceptibility to quinine and sulphadoxine/ pyrimethaInine. Most isolates were sensitive to amodiaquine. A field study of 31 KwaZulu isolates of P. falciparum obtained in 1987 and 1988 showed 29 to be resistant to chloroquine in the 24-hour microtechnique, with the majority being highly resistant. Chloroquine resistance was confirmed in this area in a field study carried out in 1989 but in 1990 all 4 isolates tested were sensitive to the drug. In 1989, 12 out of 13 KwaZulu isolates were resistant to amodiaquine and most isolates showed reduced susceptibility to quinine and sulphadoxine/ pyrimethamine.Item Blood group polymorphisms in Southern Africa and innate resistance to plasmodium falciparum(1992) Field, Stephen PaulThe observation by Haldane in 1949 that the distribution of malaria and certain thalassaemias were similar and that the former disease must be a selective force tor the continued existence of the latter by preservation of the heterozygotes. This theory which later became known as lithe malaria hypothesis" has been applied to other inherited conditions such as G6PD deficiency, membranopathies, certain blood group polymorphisms, other heamoglobinopathies such as sickle cell disease, blood group polymorphisms and more recently HLA phenotypes. It has been shown that the Duffy blood group antigens are the receptors for. Plasmodium vivax and since these antigens are lacking in most black Africans this species of malaria is virtually absent in Africa. It has also been shown that the glycophorins are at least in part the receptors for Pfalciparum. Several variants of the glycophorins exist and the biochemistry and, where known, the molecular mechanisms by which these arise is reviewed. Experimental work is carried out to establish the growth characteristics of Pfalciparum in an in vitro culture system using cells with glycophorin variants on their membranes. Three such variants were compared to normal cells and two (S~s-U-and Dantu) were found to be partially resistant to invasion by Pfalciparum merozoites whereas the third (Henshaw) was found to be no different to controls.Item Trafficking of plasmodium falciparum invasion proteins to the parasite micronemes(2017) Churchyard, AlisjeMalaria continues to be a global health problem. Despite a marked reduction in mortality over the last 15 years, these hard-fought gains are threatened by growing resistance of the Plasmodium falciparum malaria parasite to artemisinin, the frontline drug used in treatment of the disease. Clinical symptoms of malaria are caused by the intra-erythrocytic phase of the parasite life cycle. Entry into the erythrocyte is accomplished by several specialised invasion proteins, which are stored in unique apical secretory organelles known as micronemes and rhoptries. Very little is known about the trafficking signals and transport mechanisms of invasion proteins to these organelles. Three micronemal proteins, Apical Membrane Antigen-1 (PfAMA-1), Subtilisin-like protease 2 (PfSUB2) and Erythrocyte Binding Antigen 181 (PfEBA181) were investigated with the aim of identifying domains responsible for targeting the micronemes. Selected domains were amplified and mini-genes were created by overlap extension PCR. A pARLmCherry plasmid containing a Pfama-1 stage-specific promoter that is only active during the schizont stage of parasite development when micronemes are formed, was used to create mCherry-tagged constructs. P. falciparum parasites were transfected by electroporation of the plasmid constructs. Transgenic parasites were selected by drug pressure and the expression of red fluorescent mCherry-tagged chimaeric proteins was visualised in live parasites. Co-localisation studies were performed with a microneme marker to assess if the transgenic mini-proteins reached their destination. Interestingly, all three proteins required different domains to target the micronemes: PfEBA181 required an extended region of a conserved cysteine-rich domain, PfAMA-1 required the prodomain, and PfSUB2 required the transmembrane domain. Since no common targeting signal was identified, the possibility of a protein escorter was explored. The PfAMA-1 prodomain was expressed as a recombinant histidine-tagged protein and immobilised onto Nickel-coated beads, which were exposed to a P. falciparum phage display library for four rounds of biopanning. Two novel binding partners were identified: a putative Chaperone Binding Protein and a putative Formin 2. The identification of the molecular trafficking determinants of three invasion proteins, as well as a potential protein escorter for microneme targeting, represent novel findings that extend our knowledge of a fundamental biological process in the malaria parasite. This pathway may be exploited for drug development and new malaria treatment strategies.