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.

Malaria is a major health problem, mainly in developing countries, and causes an estimated 1 million deaths per year. Plasmodium falciparum is the major type of human malaria parasite, and causes the most infections and deaths. Malaria drugs, like any other drugs, suffer from possible side effects and the potential for emergence of resistance. Chloroquine, which was a very effective drug, has been used since about 1945, but its use is severely limited by resistance, even though it has mild side effects, and is otherwise very efficacious. Research has shown that there are chloroquine reversal agents, molecules that can reinstate antimalarial activity of chloroquine and chloroquine-like drugs; many such reversal agents are composed of two aromatic groups linked to a hydrogen bond acceptor several bonds away. By linking a chloroquine-like molecule to a reversal agent-like molecule, it was hoped that a hybrid molecule could be made with both antimalarial and reversal agent properties. In the Peyton Lab, such hybrid "Reversed Chloroquine" molecules have been synthesized and shown to have better antimalarial activity than chloroquine against the P. falciparum chloroquine-sensitive strain D6, as well as the P. falciparum chloroquine-resistant strains Dd2 and 7G8. The work reported in this manuscript involves simplifying the reversal agent head group of the Reversed Chloroquine molecules, to a single aromatic ring instead of the two rings groups described by others; this modification retained, or even enhanced, the antimalarial activity of the parent Reversed Chloroquine molecules. Of note was compound PL154, which had IC50 values of 0.3 nM and 0.5 nM against chloroquine-sensitive D6 and chloroquine-resistant Dd2. Compound PL106 was made to increase water solubility (a requirement for bioavailability) of the simplified Reversed Chloroquine molecules. Molecular modifications inherent to PL106 were not very detrimental to the antimalarial activity, and PL106 was found to be orally available in mice infected with P. yoelli, with an ED50 value of about 5.5 mg/kg/d. Varying the linker length between the quinoline ring and the protonatable nitrogen, or between the head group and the protonatable nitrogen, did not have adverse effects on the antimalarial activities of the simplified Reversed Chloroquine molecules, in accord with the trends observed for the original design of Reversed Chloroquine molecules as found from previous studies in the Peyton Lab. The simplified Reversed Chloroquine molecules even tolerated aliphatic head groups (rather than the original design which specified aromatic rings), showing that major modifications could be made on the Reversed Chloroquine molecules without major loss in activity. A bisquinoline compound, PL192, was made that contained secondary nitrogens at position 4 of the quinoline ring (PL192 is a modification of piperaquine, a known antimalarial drug that contains tertiary nitrogens at position 4 of the quinoline ring); this compound was more potent than piperaquine which had an IC50 value of 0.7 nM against CQS D6 and an IC50 of 1.5 nM against CQR Dd2, PL192 had IC50 values of 0.63 nM against chloroquine sensitive D6 and 0.02 nM against chloroquine resistant Dd2. Finally, the mechanism of action of these simplified "Reversed Chloroquines" was evaluated; it was found that the simplified "Reversed Chloroquines" behaved like chloroquine in inhibiting β-hematin formation and in heme binding. However, the simplified "Reversed Chloroquines" were found to inhibit chloroquine transport for chloroquine resistant P. falciparum chloroquine resistance transporter expressed in Xenopus oocytes to a lesser extant than the classical reversal agent verapamil. From these studies it was noted that the simplified "Reversed Chloroquines" may not behave as well as classical reversal agents would in restoring chloroquine efficacy, but they are very potent, and so could be a major step in developing drug candidates against malaria.

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A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine in Epidemiology and Biostatistics Johannesburg, 2002. / To systematically evaluate the efficacy of double dose of sulfadoxine-pyremithamine (SP/SP) treatment in pregnancy in malaria endemic areas. Methods - The relevant articles were retrieved by a computerized search of Medline, Cochrane Review, Pub Med and Google with the following key words, sulfadoxine-pyrimethamine, intermittent, pregnancy, Quasi- experimental studies and Randomised Control Trials. Three reviewers identified only 2 papers meeting the inclusion criteria set for the study. Systematic quantitative review was performed. / IT2018

PhD, Faculty of Science, University of the Witwatersrand, 1999

Background Promotion of job opportunities and tourism in African countries has led to an increase in expatriates in malaria endemic areas. A paucity of data exist on characteristics and numbers of expatriates and long-term travellers being evacuated from sub-Saharan Africa for suspected malaria infections diagnosed while still in Africa. Methods A retrospective flight record review of a South African fixed-wing air-ambulance provider from June 2006 through July 2009 was performed. Adult expatriates and long-term travellers with suspected malaria being evacuated from sub-Saharan African countries to Johannesburg, South Africa were included. Results Suspected malaria was the single most common diagnosis for dispatching airambulances with 81 (11.9%) of the 679 flights. Accuracy of the initial diagnosis, based on confirmation of malaria at the receiving facility was 78.4% for blood smears, 92.3% for rapid detection tests and 42.8% for clinical signs alone. P. falciparum (alone, or in combination with other Plasmodium species) was the most frequently isolated species at both the referring (100%) and receiving (88.2%) facilities in cases where the species was documented. The suspected malaria patients were predominantly male 69 (84.1%), with a mean age of 42.1 ±12.8 years, and were in sub-Saharan Africa for occupational reasons 65 (79.3%). Angola, the Democratic Republic of Congo and Mozambique were the countries of origin in 48 (58.5%) of the suspected malaria flights. Compliance on appropriate malaria chemoprophylaxis was documented in two (2.4%) suspected malaria patients. Intubation as a marker of severity was required for 15 (18.3%) patients, and one (1.2%) patient died inflight. No statistically significant difference (p=0.50) was shown for intubation requirements when comparing patients who had utilised malaria chemoprophylaxis with the patients who had not utilised chemoprophylaxis. Conclusions Patients presented in advanced stages of severe/complicated malaria with concurrent poor chemoprophylaxis utilisation and compliance. Appropriate chemoprophylaxis did not decrease the severity of presentation (based on intubation requirements) and did not guarantee complete malaria protection.

The strides made in malaria eradication efforts have been aided by a combination of vector control and chemoprevention. However, Plasmodium resistance to first-line artemisinin-based combination therapies (ACTs), and mosquito resistance to insecticides threatens the progress made. Innovative vector control measures, vaccines and antimalarial drugs with novel modes of action are key to disease eradication. High-throughput phenotypic screening of chemical libraries tested directly against all the stages of the Plasmodium lifecycle have been the mainstay of antimalarial drug discovery efforts and have identified compounds that are effective in parasite clearance. Unfortunately, these screens are handicapped in that they are unable to specify the actual compound targets in the Plasmodium parasites. As a result, many candidate hits have had to be re-screened in specific assays to determine putative mechanisms of antiplasmodial action. Predictably, this has elevated target-specific screens as the next frontier in drug discovery. This shift has been aided by a number of factors, including the cost effectiveness of these screens and the fact that target-specific screens do not always require specialized access to parasites. When combined with knowledge of the target’s structure, where known, target-specific screens have the potential to give lead compounds with impeccable potency and selectivity. This approach has already been successfully put to use, for example, in the identification of P. falciparum p-type ATPase 4 (PfATP4) and P. falciparum phosphatidylinositol 4-kinase (PfPI(4)K) inhibitors. The new challenge now is the identification of quality targets. Here, computational biology ‘omics’ tools have proved to be an invaluable resource. Two of the more commonly used of these tools are genomics and metabolomics. In-vitro evolution assays followed by whole genome sequencing analysis is a popular genomics approach and helps unveil novel target genes. Plasmodium parasites are exposed to sublethal doses of a compound until an upward shift in the half-maximal inhibitory concentration (IC50), indicative of resistant parasites, is observed in the culture. Sequenced genomes of the resistant parasite clones are compared to those of the drug-naive parent to reveal genetic changes, which include both single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). While these genomic changes may point to genes encoding actual drug targets, they often reveal mediators of drug resistance or tolerance. Follow-up assays like SNP validation through gene editing are necessary to distinguish between actual targets, resistance mechanisms and random background mutations. Expectedly, genetic changes in uncharacterized Plasmodium genes are the bottle-necks in the identification of novel druggable targets. Even so, this genomics method has uncovered or reconfirmed novel antimalarial drug targets, including the proteasome, aminophospholipid-transporting P-type ATPase (PfAT-Pase2) and cGMP-dependent protein kinase (PfPKG). Metabolomic profiling and transcriptomics narrows down a compound’s mode of action. Here, parasites are treated with a compound of interest and the metabolites extracted and analyzed using liquid chromatography-mass spectrometry (LC-MS). The metabolomics fingerprint or metaprint is then compared to that of untreated parasites. While this method rarely provides the exact drug target, it narrows down the compound’s mode of action, which is valuable for target validation and characterization. The issue of non-specific or non-viable phenotype metabolite signals is easily filtered out by treating parasites with various drug concentrations and/or over a period of time. Other areas that limit the effectiveness of this tool and need to be addressed include the analysis of compounds that do not act through metabolic pathway disruption and potential host contamination. Nonetheless, metabolomics are a key player in drug discovery and have successfully been used in the study of pantothenamides (MMV689258) where the observed CoA analog buildup helped identify their mechanism of action in sequestering coenzyme A to block acetyl-CoA anabolism. Presented herein is a culmination of my graduate research in antimalarial drug discovery. Three independent projects are presented, and they all have either been published or are currently under reviewership. Chapter 1 is an introduction to malaria, a disease that has and continues to claim hundreds of thousands of lives, especially in my home continent of Africa. In chapter 2, I detail the experimental procedures used to generate the data presented in chapters 3-5. Chapter 3 is a detailed susceptibility profiling and metabolomic fingerprinting of Plasmodium falciparum asexual blood stages (ABS) to clinical and experimental antimalarials. This work, published in Cell Chemical Biology (2020), presents to the malaria research community a medium-throughput assay that can be utilized to identify new antimalarial lead compounds and novel assayable targets. Chapter 4 presents a detailed analysis of a novel ATP-binding cassette (ABC) transporter that confers pleiotropic antimalarial drug resistance in P. falciparum and that was first identified through in vitro evolution assays. This work is currently under review in Cell Chemical Biology. Chapter 5 presents work on an promising new preclinical compound, MMV688533, that provides single-dose cure and that was discovered using an innovative orthology-based screen by the Sanofi drug discovery team. In this chapter, I also present in detail the assays performed to better understand this compound’s mode of antiplasmodial action and the potential drivers of parasite resistance. This work has been accepted, pending minor textual revisions, in Science Translational Medicine. Finally in chapter 6, I summarize chapters 3-5 and share future follow-up work needed to strengthen and contextualize some of the experimental findings presented here.

Malaria is a vector borne disease that has been plaguing mankind since before recorded history. The disease is carried by three subspecies of mosquitoes Anopheles gambiae, Anopheles arabiensis and Anopheles funestu. These mosquitoes carry one of four type of Plasmodium specifically: P. falciparum, P. vivax, P. malariae or P. ovale. The disease is a killer; the World Health Organization (WHO) estimates that about 40% of the world's total populations live in areas where malaria is an endemic disease and as global warming occurs, endemic malaria will spread to more areas. The malaria parasite kills a child every 30 seconds. In Africa alone, as many as one million children die annually from malaria before they reach the age of 5. The World Health Organization has an estimate of 100-200 million victims annually. Malaria has many mathematical models and this paper will examine several different models in order to achieve a greater understanding of this disease.

Malaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for the design of rational intervention strategies. To achieve this goal, we performed an isobaric tag-based approach for a system-wide quantitative analysis of protein expression and site-specific phosphorylation events of the Plasmodium asexual developmental cycle in the red blood cells. This study identified 2,767 proteins, 1,337 phosphoproteins, and 6,293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Because the links between Plasmodium protein kinases as key cell cycle regulators to cellular events are largely unknown, it is of importance to define their cognate physiological substrates. To test the hypothesis that genetic screening would be a useful approach for discovery of candidate substrates of a protein kinase, we used the orphan kinase PfPK7 as a model. Our comparison of the phosphoproteome profiles between the wild-type 3D7 and PfPK7- parasites identified 146 proteins with 239 phosphorylation sites exhibiting decreased phosphorylation in the absence of PfPK7 at the developmental stages where nuclear division and merozoite formation occur. Further analysis of the decreased phosphorylated events revealed three motifs that are enriched among phosphorylated sites in proteins that are down regulated. In vitro kinase assays were done to validate the potential substrates of PfPK7 and to elucidate the signaling events that are regulated by PfPK7. In parallel to our experimental analysis, we used a computational approach for substrate prediction from our phosphoproteome dataset. This analysis identified 43 distinct phosphorylation motifs and a range of proline-directed potential MAPK/CDK substrates. To identify substrates/ interactors of Plasmodium CDK-like kinases, we also used HA-tagged CDK-like kinases, PfPK6 and Pfmrk lines. Co-immunoprecipitation of the HA-tagged PfPK6 and Pfmrk baits, followed by mass spectrometric analyses, identified the components of the protein interaction complexes of these kinases. Our analyses of HA-PfPK6 and HA-Pfmrk immunoprecipitates identified 15 and 21 proteins in the interaction complex, respectively. The ability of recombinant PfPK6 and Pfmrk to interact and/or utilize any of the proteins identified in the interaction complex as substrates was verified through in vitro kinase assays and pull-down analysis. This study is the most comprehensive definition of the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, and offered an insight into the dynamics of phosphorylation during the asexual cycle progression [1]. In summary, this study has 1) defined the constitutive and regulated expression of the Plasmodium proteome during its asexual life cycle, 2) demonstrated that fluctuation and reversible phosphorylation is important for the regulation of P. falciparum*s unique cell cycle, 3) provided the foundation for quantitative phosphoproteomic analysis of kinase negative mutants to understand their function, 4) provided a major step towards defining kinase-substrate pairs operative within parasite*s signaling networks, and 5) generated a preliminary interactome for PfPK6.

The complementarity of ancient DNA lends itself to integration with paleopathological inquiries of disease, particularly in scenarios where there is limited or conflicting historical, skeletal, and archaeological information in a given spatio-temporal context. This thesis expands on molecular approaches applied to the detection of “invisible” pathogens associated with non-catastrophic morbidity and mortality that are embedded in a unique biosocial context of the disease experience. Presented in ‘sandwich-thesis’ format, I explore the historical narrative surrounding malaria in Imperial period Italy (1st-4th c. A.D.) using a molecular approach that is integrated with an ecosocial framework, as well as addressing the methodological challenges of identifying pathogens in contexts without a priori knowledge or incongruous evidentiary sources. My research presents the first partial mitochondrial genome for P. falciparum recovered from two adults (prioritized from a subset of 58 individuals) in disparate ecological and social localities in Imperial period Italy. This provides a timestamp for this ancient protozoan parasite, with an emphasis on a multi-faceted approach to frame the human-parasite-vector-environment interactions in the studied localities. Additionally, I successfully applied an in-solution hybridization capture technique designed to detect over 1,000 human pathogens in archaeological samples, both of known and unknown pathogen constituents. This technique qualitatively and quantitatively assesses the likelihood of low abundance pathogenic targets that are present, in order to prioritize candidates to further pursue with downstream analyses, as well as beginning to explore the synergistic landscape of pathogen-pathogen interactions. In combination, the research outlined in this thesis emphasizes the molecular and biosocial experience of disease as interconnected elements in dynamic epidemiological environments of the past. / Dissertation / Doctor of Philosophy (PhD)