Malaria causes nearly 3 million deaths annually. The parasite species responsible for the most lethal forms of malaria is P. falciparum (Miller et.al., 2002). Its destructive effect is most evident in the developing African countries, which lack the infrastructure and financial support to effectively control the disease. The only viable means of control at this stage is the use of antimalarial chemotherapy and –prophylaxis, but these drugs are losing their effectivity due to parasite resistance. This underlines the need for new, safe, efficient and cheap drugs as a solution to the African malaria problem. Within the validated folate metabolic pathway of P. falciparum, the identification of three new genes has provided new options for drug inhibition (Lee et.al., 2001). One of these genes encodes the bifunctional dihydrofolate synthase-folylpolyglutamate synthase (DHFS-FPGS), which is unique to P. falciparum (Salcedo et.al., 2001). When compared to human folylpolyglutamate synthase (FPGS), the parasite enzyme is an attractive drug target for selective inhibition due to the additional DHFS activity and low sequence similarity. However, to assess the value of DHFS-FPGS as a drug target and rationally design new drugs against the enzyme, large amounts of enzyme are needed for activity studies and structural determination. The heterologous expression of malaria genes often result in low expression levels, due to its high A+T content and codon bias. To circumvent this problem, a modified P. falciparum dhfs-fpgs, adapted to E.coli codon preferences and with a lower A+T content was synthesised in this study for increased expression. A two-step overlap-extension PCR method was optimised for the synthesis of the 1586bp dhfs-fpgs from only 1 pmol each of partially overlapping oligonucleotides. The use of partially overlapping oligonucleotides, lower amounts of starting material and fewer PCR cycles cut the costs of gene synthesis and the optimisation increased PCR efficiency, when compared to other gene synthesis reports (Carpenter et al., 1999; Zhang et al., 2002). The correct sequences could be obtained from the sequencing of as little as three clones. The successfully constructed dhfs-fpgs gene was expressed in a variety of E. coli expression hosts and vector systems. In all the systems, expression levels of the synthetic gene were much higher than for the native P. falciparum gene. Functional complementation of a DHFS-FPGS deficient E. coli strain verified that the DHFS and FPGS activities were encoded by the synthetic gene, that complementation was achieved to a greater extent than for native P. falciparum dhfs-fpgs and that a synthetic tagless and C-terminal Histidine-tagged DHFS-FPGS had the highest levels of DHFS and FPGS activity. Preliminary purification studies for these two constructs were performed for optimised enzyme isolation, to be followed by activity assays. These optimisations will also serve as basis for future large-scale isolation strategies to obtain sufficient amounts of protein for the structural determination of the enzyme, which would be vital to drug target verification, drug development and –design, thus paving the way for a new generation of antifolate malaria therapy. / Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

The effect of infectious diseases on the population in the developing countries is of utmost concern. Malaria, tuberculosis (TB) and human immunodeficiency virus (HIV) are the three major infectious disease threats. They account for approximately half of the mortality caused by infectious diseases, which is almost half of the mortality in the developing countries. With no vaccine likely in the foreseeable future, drugs remain the best means of controlling infectious diseases. In the industrialized nations at the present time, some 50% of all prescribed drugs are derived or synthesized from natural products (animals, marine species, plants and micro-organisms). It has been estimated that plants are the most important source of medicine for more than 80% of the world’s population. As previous work on the leaves of Croton steenkampianus gave promising results and revealed that it still contained bioactive compounds that could be isolated, it was chosen for further work. The bioassay guided fractionation of the ethanol crude extract using silica and Sephadex column chromatography resulted in the isolation of six compounds: three flavoniods (quercetin, tamarixetin and eriodictyol), one new indane (1) (2,6-dimethyl-1-oxo-4 indanecarboxylic acid) and two new diterpenes (steenkrotin A (2) and steenkrotin B (3)) with novel skeletons. The structure of the compounds was determined using NMR, IR, UV, MS and X-ray crystallography. Ethanol crude extract, quercetin, steenkrotin A, steenkrotin B and the indane were tested against four strains of Plasmodium falciparum (D6, D10, Dd2 and W2). Quercetin showed good antiplasmodial activity against the D10 and Dd2 strains. The antiplasmodial activity of steenkrotin A and crude extract were moderate. The antimalarial activity of steenkrotin A in particular is promising, as it showed more activity against resistant strains. The indane, and steekrotin B were not active against the strains of P. falciparum used (IC50 > 10 μg/m). The IC50 of the compounds improved when they were combined with chloroquine. However, the IC50 of chloroquine was still the lowest. The compounds showed moderate bioactivity against Bacillus cereus and Escherichia coli. The three new compounds (1, 2 and 3) tested against Mycobacterium (H37Rv) were not active (IC50 > 10 μg/ml). The indane (1) showed anti-HIV activity at 50 μg/ml against reverse transcriptase. The antioxidant activity of the compounds tested ranged from weak to excellent (>280.00 μg/ml for compound 1 and 2 to 0.05 μg/ml for quercetin). The cytotoxicity of the compounds and extract were determined against Vero cells lines. Their IC50 values ranged from 34.0 to 305.9 ìg/ml, which is higher and better than that of chloroquine. The IC50 values obtained are: chloroquine (25.0), quercetin (33.6), steenkrotin A (35.0), ethanol extract (45.0), tamarixetin (53.8), indane (248.2) and steenkrotin B (305.9). / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

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 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

Le paludisme, maladie parasitaire et vectorielle, sévissant principalement dans les zones intertropicales où vit près de 40% de la population mondiale, reste un problème majeur en santé publique. Les cinq espèces de Plasmodium connues infectées le paludisme chez l'homme sont présentes au Cambodge, qui est reconnu comme l'épicentre de l'émergence de souches de P. falciparum multi-résistantes (chloroquine, sulfadoxine- pyriméthamine, méfloquine, artémisinine), pouvant entraver les progrès accomplis depuis plus d'une décennie. Le travail de thèse intitulé « Approche génomique et bio-informatique de l'émergence et de la diffusion des résistances chez Plasmodium au Cambodge » avait pour objectif de développer de nouveaux outils moléculaires et biologiques pour 1) une meilleure compréhension de l'impact des stratégies mises en place pour lutter contre le paludisme à P. falciparum sur les autres espèces de Plasmodium, 2) la mise en place d'outils biologique et moléculaire, permettant de mieux définir l'épidémiologie des parasites résistants, en particulier la résistance à la quinine et aux dérivés de l'artémisinine, 3) l'étude et la définition des sous-populations parasitaires circulant au Cambodge afin d'estimer les risques associés à la diffusion de la résistance à l'artémisinine. Cette thèse a été réalisée dans l'Unité d'Epidémiologie Moléculaire du Paludisme à l'Institut Pasteur du Cambodge (IPC) sous la codirection du Dr. Didier Ménard (Chef de laboratoire à l'IP) et du Pr. Emmanuel Cornillot (Professeur à l'Université Montpellier I). Le premier objectif visait à étudier l'impact de la pression médicamenteuse sur la dynamique d'évolution des populations parasitaires. Nous avons d'abord évalué le polymorphisme des gènes associés à la résistance à la pyriméthamine (gène dhfr, dihydrofolate reductase) chez Plasmodium malariae et Plasmodium ovale (article 1 et manuscrit en préparation1), et le polymorphisme du gène mdr-1 (multidrug resistance 1) associé à la résistance à la mefloquine chez P. vivax (article 2). De plus, en collaboration avec l'Institut Pasteur de Madagascar, nous avons étudié le lien pouvant exister entre le polymorphisme du gène candidat Plasmodium falciparum Na+/H+ exchanger (Pfnhe-1) et la résistance (clinique et in vitro) de P. falciparum à la quinine (articles 3 et 4).Le deuxième objectif s'est interessé au développement d'outils biologiques et moléculaires permettant d'évaluer la résistance des souches de P. falciparum aux dérivés de l'artémisinine. Les 3 articles présentés (articles 5, 6 et 7) decrivent la méthodologie d'approche originale utilisée associant la génomique, la biologie, la clinique et l'épidémiologie, qui a permis d'aboutir à la découverte d'un marqueur moléculaire (mutations au sein du gène Kelch 13) fiable pour identifier les souches résistantes aux dérivés de l'artémisinine.Le dernier objectif était consacré au développement de la technique PCR-LDR-FMA appliqué à la détection d'un panel de 24 SNPs permettant de caractériser par un « barcode » chaque isolat de P. falciparum. Cette technique couplée avec une analyse bio-informatique et statistique des données nous a permis d'étudier et de définir la structuration des populations parasitaires circulant au Cambodge afin d'estimer les zones à risque de diffusion de la résistance à l'artémisinine (manuscrit en préparation 2).A travers ce travail de thèse, nous nous sommes efforcés de montrer la puissance des techniques de biologie moléculaire disponibles couplées avec des approches génomique et bio-informatique pour améliorer notre compréhension de la dynamique d'évolution des populations parasitaires. Ce travail s'est essentiellement concentré sur les phénomènes liés à l'émergence et de la diffusion des parasites résistants aux antipaludiques, le but final de ce travail étant d'améliorer les stratégies de lutte mises en place pour atteindre l'ambitieux objectif d'élimination du paludisme. / Malaria, a protozoan vector-borne disease, is mainly prevalent in tropical areas, where nearly 40% of the world population is residing and remains one of the most concerns for public health worldwide. In Cambodia, the five Plasmodium species known to cause malaria in humans are present. The main feature of this country is that it is recognized as the epicenter of the emergence of multi-resistant P. falciparum parasites (to chloroquine, sulfadoxine-pyrimethamine, mefloquine, and artemisinin), a very significant menace to public health in the Mekong region that could impact the worldwide strategy to fight malaria. The thesis presented here, entitled “Genomics and Bioinformatics in the emergence and spread of resistant Plasmodium in Cambodia” aimed to develop new molecular and biological tools for:1) improving our understanding of the collateral impact of the strategies implemented to fight against falciparum malaria on the other Plasmodium species; 2) defining the molecular epidemiology of antimalarial resistant parasites, especially resistance to quinine and artemisinin derivatives;3) studying and defining the structure of P. falciparum parasite populations circulating in Cambodia to estimate areas at risk of spread of artemisinin resistance, using genomic approaches and bioinformatics. This thesis was carried out in the Malaria Molecular Epidemiology Unit at Pasteur Institute in Cambodia (IPC) under the co-direction of Dr. Didier Ménard (Head of the Unit, IP) and Pr. Emmanuel Cornillot (Professor, University of Montpellier I). The first objective of this work was to study the impact of drug used to treat falciparum malaria on the dynamics of other Plasmodium species. In a first step, we evaluated the polymorphism in gene associated to pyrimethamine resistance (dhfr gene, dihydrofolate reductase) in Plasmodium malariae and in Plasmodium ovale (article 1 and manuscript in preparation 1) and the polymorphism in mdr-1 gene (multidrug resistance 1 gene) associated to mefloquine resistance in P. vivax (article 2). Secondly, in collaboration with Pasteur Institute in Madagascar, we investigated the association between the polymorphism in Plasmodium falciparum Na + / H + exchanger gene (Pfnhe-1) and quinine resistance defined either by clinical or in vitro phenotypes (articles 3 and 4). The second objective was focused on the development of novel biological and molecular tools to assess the resistance of P. falciparum to artemisinin derivatives. The three papers presented (articles 5, 6 and 7) describe an original approach combining genomics, biological, clinical and epidemiological studies, which lead to the discovery of a molecular marker (mutations Kelch 13 gene) associated to artemisinin resistance.The third and final objective was devoted to the development of the PCR-LDR-FMA technology applied to the detection of a panel of 24 SNPs to characterize a "barcode" of P. falciparum isolates. This technic coupled with bioinformatics and statistical analysis allowed us to study and define the structure of the parasite populations circulating in Cambodia for estimating areas at risk of spread of artemisinin resistance (manuscript in preparation 2). Through this work, we have tried to show the usefulness of available molecular biology methods coupled with genomic and bioinformatics approaches to improve our understanding of the dynamics of the malaria parasite populations. This work has been mainly focused on the emergence and spread of antimalarial resistant parasites, keeping in mind that the ultimate goal of this work was to improve strategies implemented to achieve the ambitious goal of malaria elimination.

The discovery of new β-haematin inhibitors has become one focus for researches in response to the resistance of P. falciparum malaria parasites that emerged towards well-known antimalarials. While hundreds of new β-haematin inhibitors have been discovered using detergent mediated high-throughput screening methods, a crucial aspect is understanding exactly how these β-haematin inhibitors behave in the malaria parasite and inhibit the formation of haemozoin. What is known, is that well-known β-haematin inhibitors like chloroquine cause increased amounts of exchangeable haem in the parasite digestive vacuole and form a Fe(III)PPIX-inhibitor complex by accumulating at high concentrations which consequently inhibits parasite growth. Another important focus is on understanding the digestion of haemoglobin and its role in haemozoin formation. This research investigates the in vitro modelling of cellular haemozoin and inhibition by various β-haematin inhibitors across different scaffolds and the role of haemoglobin degradation in P. falciparum malaria parasites. The investigated β-haematin inhibitors resulted in micromolar IC50 (NF54) values and decreased parasite growth with increases in concentration. Using a pyridine-based parasite haem fractionation plate method, these β-haematin inhibitors were shown to target haemozoin formation by causing increased amounts of exchangeable haem that corresponded to decreasing amounts of haemozoin in chloroquine-sensitive parasites. The amounts of exchangeable haem were shown to be inversely proportional to the percentage of parasite growth in the presence of these β-haematin inhibitors. It was apparent that there was a tendency for parasite growth inhibition activity to decrease as the amount of exchangeable haem present in chloroquine sensitive parasites increased, although, the trend was not statistically significant. Moreover, it was observed that experimental cellular accumulation ratio values were low in comparison to chloroquine and amodiaquine. Based on the experimental cellular accumulation ratio values, it was deduced that the accumulation of these β-haematin inhibitors was not primarily due to pH trapping and more complex than previously proposed. Further investigations into the exchangeable haem amounts as a function of intracellular test compound amounts at the IC50 values of these β-haematin inhibitors highlighted that there was an apparent 1:1 relationship with the amount of intracellular exchangeable haem, indicative of complex formation. Transmission electron microscopy images were obtained for untreated parasites that showed intact parasites inside red blood cells with clearly visible haemozoin crystals dispersed throughout the parasite digestive vacuole, whilst, treated parasites showed less defined haemozoin crystals as a result of inhibition. Moreover, electron energy-loss spectroscopy revealed that untreated parasites exhibited a strong iron signal which was associated with haemozoin in the parasite digestive vacuole with a weaker signal attributed to the red blood cell cytoplasm. Similarly, a strong iron signal was shown in the digestive vacuole of treated parasites which was associated with less defined haemozoin crystals. A halo around these haemozoin crystals was observed and was suggested to be indicative of the build-up of exchangeable haem. Additionally, a strong bromine signal attributed to a bromine-containing β-haematin inhibitor, test compound 1, was also observed in the same region as the haemozoin crystals. Overlaid signal distribution maps for iron and bromine showed direct evidence of Fe(III)PPIX and test compound 1, suggesting complexation. High-quality Raman spectra were obtained for the Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetically prepared samples for the Fe(III)PPIX porphyrin dominated spectral region of 1700-500 cm-1 at an excitation wavelength of 532 nm. From the spectra, a putative Fe(III)PPIX-test compound 1 complex was identified and shown to be similar to the synthetically prepared counterpart, haematin-test compound 1 mixture. It was highlighted that a unique peak at 1080 cm-1 indicated π- π interactions between the pyrrole-imidazole ring and thus confirming that the formation of this putative Fe(III)PPIX-inhibitor complex occurs. The confocal Raman true mapping technique proved to be efficient and reliable for imaging the signal distribution of haemozoin at the Raman peak of 754 cm-1 and 1080 cm-1 for the Fe(III)PPIX-test compound 1 complex which co-localized in the digestive vacuole of chloroquine sensitive parasites. Moreover, oxy- and deoxy-haemoglobin was observed to be localized to the red blood cell, where, deoxy-haemoglobin was located on the outer parts of the parasite. Principle component analysis, based on the Raman peak positions, exhibited significant differences in the spectra for Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetic samples where clusters were observed to separate mainly along principle component 1. These data proved that the spectra of the Fe(III)PPIX-test compound 1 complex was the same as its synthetically prepared counterpart but different from the remaining Fe(III)PPIX species. In comparison to the Fe(III)PPIX-test compound 1 complex, the cluster separations were observed to be significant, where, no significant separation was observed for the Fe(III)PPIX-test compound 1 complex and the haematin-test compound 1 mixture. Based on this, it was evident that a Fe(III)PPIX-test compound 1 complex existed in the digestive vacuole of treated chloroquine sensitive parasites. To fully understand the inhibition of haemozoin, the development of a haem pathway model is necessary, but, requires certain prerequisites. Bioinformatics data from PAXdb and ExPASy revealed that chloroquine resistance (Dd2) parasites, containing 1337 previously identified proteins with an average abundance-weighted molecular weight of 40,483 ± 77 g/mol. With this, the protein mass per cell for red blood cells, chloroquine-sensitive and - resistant parasites were consistent across three protein quantification methods was measured and revealed that chloroquine resistant parasites had a significantly higher protein mass per cell than chloroquine sensitive parasites and in turn a higher total number of protein molecules per cell. Aspartic proteases are 4-fold higher in concentration than cysteine proteases with histo-aspartic protease having the highest concentration in chloroquine resistant parasites. Along with these data, a time point quantification for chloroquine sensitive parasites throughout the blood-stage showed that the amount of haemoglobin decreased in a sigmoidal manner and corresponded to a linear increase in the amount of haemozoin and relatively constant exchangeable haem amount. This was consistent with Giemsa smears that showed that for early time points, large initial decreases in the amount of haemoglobin were observed between the early trophozoite to late trophozoite stage.

Malariakontrolle ist von Resistenzen gegen Malariamedikamente wie Chloroquin (CQ) und Artemisininderivaten (ART) bedroht. Hier untersuchten wir das Ausmaß dieser Resistenzen in Fünf Feldstudien in Nigeria, Ruanda und Südwestindien unter Beurteilung der Prävalenzen Arzneimittelresistenz-assoziierter Mutation der Plasmodium-Parasiten (P. falciparum: K13, dhps, dhfr, mdr1 und P. vivax: mdr1) z.T. in Korrelation mit klinischen Patientendaten und ex-vivo Überlebensraten (ÜLR) unter Zugabe von ART. K13 wurde in 360 zwischen 2010-2018 gesammelte ruandischen P. falciparum Isolaten genotypisiert. Erstmals fanden wir dort niedrige Frequenzen der mit ART-Resistenz assoziierten K13-Mutation. Jedoch lassen Mutation mit niedrigen ÜLR, sowie ein Isolat mit hohen ÜLR aber ohne K13-Mutation eines Patienten der die Infektion unter Therapie nicht eliminieren konnte, Fragen offen. Ca.100 indische P. falciparum und P. vivax Isolaten aus 2015 wurden auf Mutationen in P. falciparum Markern für die Resistenz gegen Sulfadoxin-Pyrimethamin (SP) (d.h. pfdhps/pfdhfr), Artesunat (AS) (d.h. K13) und Lumefantrin (d.h. pfmdr1) sowie P. vivax Marker für CQ-Resistenz (pvmdr1) untersucht. Der Großteil der Isolate zeigt Mutationen die SP-Resistenz hervorrufen, daher könnte die Effizienz der AS+SP-Therapie begrenzen sein. Außerdem eignet sich Lumefantrin nicht als alternatives Medikament auf Grund der beobachteten Dominanz des pfmdr1-Haplotyps „NFD“. Die Abwesenheit der pvmdr1-Mutation Y976F und erfolgreiche Behandlungen zeigen, die Wirksamkeit von CQ gegen vivax Malaria im Studiengebiet. Auch Isolate von nigerianischen Schwangeren mit asymptomatischer P. falciparum Infektion zeigten hohe Prävalenzen von pfdhfr/pfdhps Vier- und Fünffachmutanten darum ist die Wirksamkeit der präventiver Therapie Schwangerer mit SP in Nigeria ernsthaft gefährdet. Die Daten spiegeln die Häufigkeit der Resistenzen gegen Malariamittel in diesen Gebieten wieder mit großen Unterschieden zwischen Regionen und Medikamenten. / The spread of resistance to antimalarial drugs such as chloroquine (CQ) and artemisinins (ART) is a great threat to malaria control. Here, we investigated the extent of such resistance in Nigeria, Rwanda and south-western India. We assessed the prevalence of mutations in few Plasmodium parasites’ markers of resistance, namely P. falciparum genes K13 (ART), pfdhps/pfdhfr (sulfadoxine-pyrimethamine, SP) and pfmdr1 (lumefantrine) as well as P. vivax gene pvmdr1 (CQ) in 5 field studies conducted in 2010-2018, and partially correlated the results to patients’ clinical outcome. Few isolates from Rwanda, were also evaluated for their parasite ex vivo survival rates (SR) upon exposure to ART. We tracked ART resistance in Rwanda by genotyping K13 in 360 P. falciparum isolates from 2010-2018. We showed for the first time that K13 mutations associated with ART resistance are present here, thus in Africa, at a low frequency. However, mutations occurred in patients who recovered and/or had low SR. Of note, one patient with high SR but no K13 mutation was still parasitemic after ART treatment. Moreover, we assessed the presence of mutations in K13, pfdhps/pfdhfr, pfmdr1 and pvmdr1 in ca 100 P. falciparum and 100 P. vivax isolates from south-western India. Most of P. falciparum isolates carried pfdhfr/pfdhps mutations conferring SP resistance, menacing the efficacy of SP-ART treatment. Also, the high prevalence of pfmdr1 haplotype “NFD” advised against the introduction of lumefantrine. The low rates of P. vivax pvmdr1 Y976F and patients’ successful parasite clearance, indicated that CQ remains effective in the area. Finally, a high rate of pfdhfr/pfdhps quadruple and quintuple mutant was observed in Nigerian pregnant women with asymptomatic P. falciparum infection, hence the effectiveness of preventive treatment with SP in pregnancy might be threatened. The data reflected the abundance of antimalarials resistance in these areas with important differences between regions and drugs.

Malaria is a severe public health concern in certain regions, causing 445,000 deaths and over 200 million cases in 2016 (Ashley et al., 2018). The vast majority of these cases and deaths are located in warm climates where the Anopheles mosquito is present, especially in sub-Saharan Africa and southeast Asia. Current efforts aim to prevent the disease through vaccination, which has proven to be challenging. Plasmodium falciparum, the pathogen responsible for malaria, is transmitted between humans via the female vector Anopheles mosquito. This parasite has a complex life cycle that is not fully understood, making it difficult to treat the infection and even more difficult to inoculate a population through vaccination. P. falciparum is also capable of polymorphism, changing structure once a host antibody has identified a pathogenic antigen. For this reason, it is very technically challenging to develop a vaccine that is able to confer a high enough immune response through sufficient host antibody production. This thesis will begin with a review on the prevalence and severity of malaria and an overview of the principles of immunology and vaccine development. We will then discuss the parasitic life cycle and how it results in the pathogenesis of the disease. Current antimalarial treatments and resistance to those treatments will be analyzed. This thesis will conclude with an in-depth analysis of the current prophylactic vaccines against P. falciparum, focusing on their mechanism, efficacy, and probability of success.

Chloroquine was one of the main malarial treatments until the late 1960s when resistance began to emerge. This antimalarial targets haemozoin formation which causes a cytotoxic accumulation of free haem in the malaria parasite leading to parasite death. This is still one of the most promising pathways for treatment of the most prevalent species of malaria parasite, Plasmodium falciparum to date but, owing to growing resistance to chloroquine and other current antimalarial drugs, there is a dire need for new drugs. One strategy is to investigate non-chloroquine haemozoin inhibitors. High-throughput screening (HTS) was previously used to investigate novel β-haematin (synthetic haemozoin) inhibitors with promising P. falciparum growth inhibition activities. Of the 144 330 compounds screened, two hit compounds were selected for investigation in this project with two different scaffolds, namely benzimidazole and carbazole indole. In order to preselect benzimidazole derivatives for synthesis, Discovery Studio and Pipeline Pilot where used in tandem to enumerate 325 728 in silico compounds. These were filtered according to predicted β-haematin inhibition activities, followed by predicted malaria parasite growth activities using previously developed models based on Bayesian statistics. The predicted active compounds were further subjected to an in silico aqueous solubility model and separated according to predicted solubility values however, only 68 out of the 35 124 active compounds showed moderate solubility whilst the rest were poorly soluble. From this data, eighteen compounds were chosen for synthesis with varying functional groups. Using the same Bayesian models, biological activities for seven fragment compounds derived from the benzimidazole hit compound were predicted. Six out of seven were predicted to be β-haematin inhibitors while five out of seven were predicted active against the malaria parasite growth inhibition model. Similar Bayesian predictions were carried out on the seven proposed carbazole indole compounds with three compounds predicted to be β-haematin inhibitors while six compounds were predicted to be active against the malaria parasite growth inhibition model. The eighteen benzimidazole compounds were synthesized using a two-step synthesis, via a condensation reaction using polyphosphoric acid (PPA), 4-aminobenzoic acid and o-phenylenediamine to form the primary amine benzimidazole intermediate after which ani acylation reaction with the appropriate acid chloride furnished the desired compounds. β-haematin inhibition analysis revealed a 78% hit rate compared to the Bayesian predictions which resulted in a 24-fold enrichment compared to random screening. SAR analysis revealed an activity trend related to the position of substituents on the ring system as follows: para < ortho < meta. The type of ring system was also investigated, with a trend of phenyl < furan < pyrrole < thiophene < pyridyl found. The fragment compounds were either purchased or synthesized via standard acylation conditions using acid chlorides or acetic anhydride with primary amines as before. β-haematin inhibition analysis showed all these compounds to be inactive at the 100 µM cut-off but these compounds were still carried through to the next stage of testing in spite of these results. Molecular docking was carried out on all eighteen benzimidazole compounds in Materials Studio using the (001) and (011) β-haematin crystal faces for adsorption, together with a modified CVFF force-field. This showed a correlation between adsorption energies of the (011) β-haematin crystal face with the experimental β-haematin inhibition values. This indicated that the (011) β-haematin crystal face was the most important for β-haematin inhibition. Analysis of the benzimidazole compounds and their π-π and hydrogen bonding interactions was performed. The number of π-π interactions were found to be important for β-haematin inhibition activity. Both sets of benzimidazole compounds were tested against the NF54 chloroquine sensitive malaria parasite using growth inhibition assays with a 50% hit rate shown for the benzimidazole compounds and a 71% hit rate for the fragment study leading to a 26-fold and 36-fold enrichments compared to random screening. SAR analysis of the benzimidazole compounds revealed a trend for activity in relation to substituent position of para ≈ ortho < meta and a ring system trend of phenyl < pyridyl < thiophene < furan < pyrrole. The benzimidazole compounds were further tested against the chloroquine resistant Dd2 P. falciparum strain which showed that disubstituted compounds were more active against this strain. Cellular haem fractionation studies revealed an increase in free haem and decrease in haemozoin confirming that haemozoin inhibition is the mode of action for the benzimidazole compounds. QSAR analysis of these compounds revealed a correlation between the -Log(P. falciparum IC50) which is also known as pLog(P. falciparum IC50) and 1/βhaematin IC50, number of hydrogen bond donors and molecular depth with 1/β-haematin IC50 the most dominant term. iv The first four carbazole indole compounds were synthesized using a two-step synthesis via deprotonation of carbazole and reaction with epichlorohydrin or 1,3-dibromopropane to furnish the epoxide or alkylbromine intermediates. These intermediates underwent a further SN2 reaction using deprotonated indole to furnish four final compounds. Synthesis of another three derivatives required benzyl protection of 7-hydroxyindole alcohol first, followed by reaction with the epoxide intermediates via an SN2 mechanism to furnish the final three compounds. Analysis using the turbidimetric solubility assay revealed the best aqueous solubility range of this series of compounds to be 10-20 µM (moderately soluble). β-haematin inhibition studies were carried out on this series of compounds with a 100% hit rate found when compared to the Bayesian model data which lead to 30-fold enrichment when compared to random screening. SAR analysis showed an increase in the number of hydroxyl groups led to an increase in β-haematin inhibition activity. Docking studies were performed on these seven compounds and showed that hydrogen bonding played a role in anchoring the molecules in the binding pocket on the crystal surface with increased adsorption energies seen with an increase in the number of hydroxyl groups. Malaria parasite growth inhibition studies showed no compounds to be active against the NF54 and Dd2 strains at the 2 µM cut-off. Cellular haem fractionation studies on the carbazole indole compounds showed that this series of compounds acts via a mechanism that results in inhibition of haemoglobin uptake into the food vacuole and not via haemozoin inhibition.

Malaria, a parasitic disease, was commonly associated with third world countries, with the highest mortality in nations in Sub-Saharan Africa and Asia. But, travel increases the risk of spread to more temperate regions, such as Western Europe and the United States where Malaria has been successfully eradicated. In the past 40 years, with a better understanding of the mosquito vector and the parasite itself, advancements in treatment and containment have been made. Understanding the parasite as well as its pathogenesis is vital in formulating effective treatments. Following the incidences of Plasmodium falciparum, knowlesi, vivax, malaria, ovale, and less commonly cynomolgi and simium over time as well as region helps to better illuminate the methods of Malarial transmission, interplay with environmental factors, and methods of treatment. While each species of parasite is similar in terms of mode of infection, they differ slightly when considering incubation periods and diagnostic and treatment techniques. Many drugs have been developed to treat Malaria and include Chloroquine, Primaquine, and derivatives of Artemisinin. While the discovery of these drugs was a significant breakthrough that dramatically reduced incidence and deaths caused by Malaria, improper administration of treatment has led to a recent increase in strains of the parasite which have developed drug resistance to Artemisinin Combination Therapies (ACT’s). Of these species, P. falciparum and P. vivax, the most common causes of malaria, are also so far the only species to have developed drug resistance. The goal of this thesis is to explore popular interventions, both drug and public health based, and how research focus has now shifted to better understanding the mechanism of parasitic drug resistance, specifically linked to mutations found in the Kelch protein in P. Falciparum. The recent findings of Kelch mutations pave the way towards addressing the growing problem of anti-Malarial resistance.