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A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Master of Science June, 30, 2015 / The world suffers under a serious threat of malaria with about 584 000 deaths reported each year and most of these fatalities being children under five years of age. Malaria is caused by the protozoan parasite of the genus Plasmodium. Five different malaria species infect humans and cause disease: P. vivax, P. malariae, P. ovale, P. knowlesi and the cause of most malaria deaths, P. falciparum. The main reason for this disturbing situation is the emergence of drug resistance which reduces the effectiveness of most antimalarials. Hence, there is an urgent need for new drugs that will possibly be effective against both wild type and mutant strains of Plasmodium species. Pyrimethamine, a dihydrofolate reductase (DHFR) inhibitor, has been used most widely as an antimalarial antifolate drug for the treatment of malaria. However, rapid development of parasite resistance to this drug occurred because of its rigidity. Parasitic resistance to antimalarial antifolates arises from single mutations at various amino acid residues surrounding the PfDHFR active site. In this project, we aimed to design and synthesise a novel series of flexible pyrimidine analogues of a dihydrotriazine hit compound prepared in a previous study. These compounds were designed to target folate metabolism in the malaria parasite. The initial series of compounds prepared in this project were synthesised over 5 steps in an overall yield of 10%. The flexible pyrimidine analogues were screened for antimalarial activity in an in vitro P. falciparum screen on the Gambian FCR-3 strain (chloroquine and cycloguanil resistant strain) with dihydroartemisinin, methotrexate and quinine as controls. 5-(3-(3,5-Dichlorophenoxy)propyl)-6-phenylpyrimidine-2,4-diamine displayed the best antimalarial activity (IC50 = 0.09 μM) of the compounds in this series. Surprisingly; this was the only compound prepared in this series that proved to be as effective as our original hit dihydrotriazine (IC50 ~50 nM). In the second generation of compounds prepared in this study, we used a multicomponent coupling approach to synthesise three flexible pyrimidines bearing a non-aromatic side chain at the 6-position of the pyrimidine ring. For comparison, two analogues bearing a phenyl group at the 6-position of the pyrimidine ring were also prepared. Once again; only one compound of this series [5-((4-chlorophenethylamino)methyl)-6-cyclopropylpyrimidine-2,4-diamine, (IC50 = 0.03 μM)] showed activity comparable with our original hit compound. iv Finally, ten substituted pyrimidines bearing a flexible side chain at the 6-position of the pyrimidine ring, were prepared. These compounds are structurally similar to P65, [6-methyl-5-(3-(2,4,5-trichlorophenoxy)propoxy)pyrimidine-2,4-diamine] an analogue of a potent antifolate, WR99210, found to have good oral bioavailability in rats. Once again, the antimalarial activity of the compounds prepared was assessed in an in vitro P. falciparum screen on the Gambian FCR-3 strain. The most promising compound of this series was 6-(3-(3,4-dichlorophenoxy)propoxy)pyrimidine-2,4-diamine, which exhibited antimalarial activity in the low micromolar range (IC50 = 4.46 μM).

Malaria continues to be an enormous health-threat in the developing world and the emergence of drug resistance has further compounded the problem. The parasite-specific enzyme, 1-deoxY-D-xylulose-S-phosphate reductoisomerase (DXR), has recently been validated as a promising antimalarial drug target. The present study comprises a combination of synthetic, physical organic, computer modelling and bioassay techniques directed towards the development of novel DXR inhibitors. A range of 2-heteroarylamino-2-oxoethyl- and 2- heteroarylamino-2-oxopropyl phosphonate esters and their corresponding phosphonic acid salts have been synthesised as analogues of the highly active DXR inhibitor, fosmidomycin. Treatment of the heteroarylamino precursors with chloroacetyl chloride or chloropropionyl chloride afforded chloroamide intermediates, Arbuzov reactions of which led to the corresponding diethyl phosphonate esters. Hydrolysis of the esters has been effected using bromotrimethylsilane. Twenty-four new compounds have been prepared and fully characterised using elemental (HRMS or combustion) and spectroscopic (1- and 2-D NMR and IR) analysis. A 31p NMR kinetic study has been carried out on the two-step silylation reaction involved in the hydrolysis of the phosphonate esters and has provided activation parameters for the reaction. The kinetic analysis was refined using a computational method to give an improved fit with the experimental data. Saturation transfer difference (STD) NMR analysis, computer-simulated docking and enzyme inhibition assays have been used to evaluate the enzyme-binding and -inhibition potential of the synthesised ligands. Minimal to moderate inhibitory activity has been observed and several structure-activity relationships have been identified. In silica exploration of the DXR active site has revealed an additional binding pocket and information on the topology of the active site has led to the de novo design of a new series of potential ligands. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.

Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.

The purpose of this study was to describe the characteristics and findings of antimalarial drug efficacy studies conducted in Ethiopia and to use the findings to formulate recommendations for antimalarial drug efficacy monitoring and use of evidence to inform antimalarial treatment policy for the Ethiopian setting. This study reviewed 44 antimalarial efficacy studies conducted in Ethiopia from 1974 to 2011. The analysis of results indicated that chloroquine as the first-line antimalarial drug for the treatment of malaria due to Plasmodium falciparum had a 22% therapeutic failure in 1985. Chloroquine was replaced with sulfadoxine-pyrimethamine in 1998, more than 12 years later, when its therapeutic failure had reached 65%. Sulfadoxinepyrimethamine at the time of its introduction had a treatment failure of 7.7%; it was replaced after seven years in 2004 by artemether-lumefantrine; by then its treatment failure had reached 36%. The WHO recommends the replacement of a first-line antimalarial drug when more than 10% of treatment failure is reported. The replacement drug should have a therapeutic efficacy of more than 95%; while the change itself should be completed within two years. The prolonged delay to replace failing antimalarial drugs in Ethiopia seems to have been influenced mainly by the lack of systematic antimalarial drug efficacy data collection and pragmatic use of the data and evidence gathered.Almost eight years after its introduction, isolated studies show that the efficacy of artemether-lumefantrine has decreased from 99% in 2003 to around 96.3% in 2008. Though this decrease is not statistically significant (chi-square 1.5; P=0.22) and has not reached the threshold of 10%, it is plausible that its efficacy may drop further. This is mainly due to regulatory provisions in the country that allow marketing of oral artemisinin mono-therapies that are not recommended for malaria treatment, use of less effective antimalarial combination drugs in the neighboring countries and widespread drug quality problems. The situation calls for and this study recommends the establishment of stringent drug efficacy monitoring and early warning system and alignment of the antimalarial drug regulatory practices with recommendations of the WHO. / Health Studies / D. Litt. et Phil. (Health Studies)

Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

The purpose of this study was to describe the characteristics and findings of antimalarial drug efficacy studies conducted in Ethiopia and to use the findings to formulate recommendations for antimalarial drug efficacy monitoring and use of evidence to inform antimalarial treatment policy for the Ethiopian setting. This study reviewed 44 antimalarial efficacy studies conducted in Ethiopia from 1974 to 2011. The analysis of results indicated that chloroquine as the first-line antimalarial drug for the treatment of malaria due to Plasmodium falciparum had a 22% therapeutic failure in 1985. Chloroquine was replaced with sulfadoxine-pyrimethamine in 1998, more than 12 years later, when its therapeutic failure had reached 65%. Sulfadoxinepyrimethamine at the time of its introduction had a treatment failure of 7.7%; it was replaced after seven years in 2004 by artemether-lumefantrine; by then its treatment failure had reached 36%. The WHO recommends the replacement of a first-line antimalarial drug when more than 10% of treatment failure is reported. The replacement drug should have a therapeutic efficacy of more than 95%; while the change itself should be completed within two years. The prolonged delay to replace failing antimalarial drugs in Ethiopia seems to have been influenced mainly by the lack of systematic antimalarial drug efficacy data collection and pragmatic use of the data and evidence gathered.Almost eight years after its introduction, isolated studies show that the efficacy of artemether-lumefantrine has decreased from 99% in 2003 to around 96.3% in 2008. Though this decrease is not statistically significant (chi-square 1.5; P=0.22) and has not reached the threshold of 10%, it is plausible that its efficacy may drop further. This is mainly due to regulatory provisions in the country that allow marketing of oral artemisinin mono-therapies that are not recommended for malaria treatment, use of less effective antimalarial combination drugs in the neighboring countries and widespread drug quality problems. The situation calls for and this study recommends the establishment of stringent drug efficacy monitoring and early warning system and alignment of the antimalarial drug regulatory practices with recommendations of the WHO. / Health Studies / D. Litt. et Phil. (Health Studies)