Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.

The advent of advanced modern nanotechnology techniques offers new and exciting opportunities to develop novel nanotech-derived antimalarial nanodrugs with enhanced selective and targeting abilities that allow for lower effective drug dosages, longer drug persistence and reduced drug degradation within the body. Using a nanodrug approach also has the advantage of avoiding drug resistance problems that plague reconfigured versions of already-existing antimalarial drugs. In this study recombinant triosephosphate isomerase enzymes from Plasmodium falciparum (PfTIM) and Humans (hTIM) were recombinantly expressed, purified and characterised. PfTIM was shown to have optimal pH stability at pH 5.0-5.5 and thermal stability at 25°C with Km 4.34 mM and Vmax 0.876 μmol.ml⁻ₑmin⁻ₑ. For hTIM, these parameters were as follows: pH optima of 6.5-7.0; temperature optima of 30°C, with Km 2.27 mM and Vmax 0.714 μmol.ml⁻ₑmin⁻ₑ. Recombinant TIM enzymes were subjected to inhibition studies using polyvinylpyrrolidone (PVP) stabilised silver nanoparticles (AgNPs) of 4-12 nm in diameter. These studies showed that the AgNPs were able to selectively inhibit PfTIM over hTIM with an 8-fold greater decrease in enzymatic efficiency (Kcat/Km) observed for PfTIM, as compared to hTIM, for kinetics tests done using 0.06 μM of AgNPs. Complete inhibition of PfTIM under optimal conditions was achieved using 0.25 μM AgNPs after 45 minutes while hTIM maintained approximately 31% of its activity at this AgNP concentration. The above results indicate that selective enzymatic targeting of the important, key metabolic enzyme TIM, can be achieved using nanotechnology-derived nanodrugs. It was demonstrated that the key structural differences, between the two enzyme variants, were significant enough to create unique characteristics for each TIM variant, thereby allowing for selective enzyme targeting using AgNPs. If these AgNPs could be coupled with a nanotechnology-derived, targeted localization mechanism – possibly using apoferritin to deliver the AgNPs to infected erythrocytes (Burns and Pollock, 2008) – then such an approach could offer new opportunities for the development of viable antimalarial nanodrugs. For this to be achieved further research into several key areas will be required, including nanoparticle toxicity, drug localization and testing the lethality of the system on live parasite cultures.

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.

Malaria is a devastating disease which affects almost half of the world’s population. Since the description of the malaria genome sequence, various aspects of the parasite have been studied, including drug resistance mechanisms, epidemiology and surveillance systems. Alarmingly, very little is known about the basic biological processes such as the regulation of the expression of parasite genes. The parasite, Plasmodium falciparum, has developed highly specialized methods of regulating the transcription of genes, starting at the regulation of genes controlling basic cellular processes such as protein synthesis and erythrocyte invasion, followed by the transcriptional regulation of more specialized genes, such as those aiding in immune evasion and pathogenesis. The description of the P. falciparum transcriptome by Bozdech et al. in 2003 revealed a complex, just-in-time and tightly regulated transcription profile of P. falciparum genes. This suggests that the most probable Achilles heel for Plasmodium may be its unique mechanisms of regulating gene expression. Various cis- and trans-regulatory sequences have been identified in P. falciparum, along with possible DNA (and RNA) binding proteins. The first part of this research focussed on transcriptional regulatory mechanisms in which an in silico search identified cis-regulatory sequences in the 5’ untranslated region of the antigenically variant var gene family. Electrophoretic mobility shift assays (EMSA) were used to identify protein binding partners of these sequences, which could ultimately act as transcription factors in regulating the expression of this essential gene family. The second part of the research investigated the involvement of post-transcriptional regulatory mechanisms in the polyamine biosynthetic pathway of P. falciparum. Polyamines have been proven to be crucial for the parasite’s development and therefore, an RNA interference knock-down strategy was used to verify the importance of the polyamine biosynthetic enzymes S-Adenosylmethionine decarboxylase (AdoMetDC), Ornithine decarboxylase (ODC) and Spermidine synthase. It is clear that various mechanisms for gene regulation are used by the parasite and that this is critical for the survival of this organism. The results of this study suggest the potential presence of both double-stranded and single-stranded DNA regulatory proteins within P. falciparum nuclear extract. As controversial as RNA interference remains in P. falciparum, this technique was used as a plausible knock-down strategy of parasite specific genes and certain trends, regarding the visible decreases in gene transcript level after double-stranded RNA treatment, were observed. However, final conclusions as to the feasibility of using RNA interference in P. falciparum remain to be elucidated. This study therefore ultimately lends insight into the transcriptional and post-transcriptional levels of P. falciparum gene regulation. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

This thesis combines four published research papers and a book chapter investigating aspects of central metabolism in the human malaria parasite Plasmodium falciparum. The publications are preceded by a statement which explores features of the research not fully described in the published texts, incorporates a review of the development over time and the present state of relevant scientific knowledge, and discusses the place of the individual papers and book chapter within malaria research. An assessment of the impact of each publication on its field of study is also included. A general discussion of the combination of papers as representative of the progress of research into the metabolism of malaria parasites concludes the statement section. The first publication is a chapter from a book, which describes detailed methods for the in vitro cultivation of P. falciparum. Such methodology, both robust and reliable, is a prerequisite for any investigation of parasite metabolism. The following publications are all primary research papers. The second publication describes the isolation and characterisation of the gene encoding the glycolytic pathway enzyme enolase from P. falciparum. The inferred amino acid sequence included peptide insertions found only in the enolases of higher plants and other photosynthetic organisms. This raised implications concerning the deep evolutionary history of the malaria parasite and related species. The third is concerned with the elucidation of the molecular basis of resistance to the antimalarial drug sulfadoxine. Resistance was found to result from point mutations within the dihydropteroate synthetase domain of the bifunctional protein hydroxymethylpterin pyrophosphokinase-dihydroptero¬ate synthetase, an enzyme of the parasite folate pathway. Additionally, it was discovered that the presence of exogenous folate has an antagonistic effect on sulfadoxine in some parasites of a defined genotype. This highlighted the importance of folate salvage in parasite metabolism. Fourth is a paper representing the discovery of a novel metabolism in both P. falciparum and the related apicomplexan parasite Toxoplasma gondii. The use of parasite genes in rescuing an Escherichia coli tyrosine auxotroph resulted in a proof of function of the products of these genes as pterin-4a-carbinolaminedehydratases. Pterin recycling, hitherto undetected in apicomplexans, was therefore added to the known metabolic processes of these organisms. The final paper describes an investigation into the subcellular distribution of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) within P. falciparum erythrocytic stage parasites. The use of confocal laser scanning microscopy and immunofluorescent techniques showed that SHMTc, the sole enzymatically active parasite SHMT protein, was found in the cytoplasm but also showed a stage-specific localisation to both the mitochondrion and apicoplast organelles. The otherwise enigmatic, enzymatically inert, SHMTm paralogue revealed a possible function, when in complex, in allowing targeted localisation of SHMTc to the mitochondrion. The spatial distribution of SHMTm also suggested a possible role in the morphogenesis of elongating apicoplasts during schizogony.

Malaria parasites are responsible for an increase in the morbidity and mortality in several tropical regions in Southern Africa. In this thesis, research was undertaken on Plasmodium, which is responsible for more than 95% of these cases. Although pharmacological prophylaxis is available, a worldwide resistance against existing drugs have been encountered. A study on chloroquineresistance in North-Eastern Transvaal in 1988, indicated that 11% of the strains in this area were resistant against chloroquine. Only 78% of these parasites were sensitive to the new drug, Mefloquine, which could serve as a substitute in chloroquine-resistant infections. Furthermore, no strain was found to be resistant or insensitive to both anti-malarial drugs. Malaria parasites can be obtained for research purposes from long term in vitro cultures. The initiation of cultures of some wild isolates of P.falciparum appears to be problematic at certain levels above sea-level. The gas composition of the medium was identified as a probable cause due to its dependence on the partial pressures of gasses at different heights above sealevel. The toxic effect of a too high oxygen concentration on the parasite, caused by the lower atmospheric pressure of the Highveld area, was prevented after the concentration of dissolved carbon dioxide in the medium was increased through equilibration with the special gas mixture. Possible shortcomings in the long-term culture method for malaria parasites that could retard optimum growth, were also investigated. Local parasite isolates could be supported on medium enriched with bovine serum although the growth rate was lower than when human serum was used. By increasing the frequency of medium replacement with progressing parasitemia, less stress were placed on the system since parasites was exposed for shorter periods and to lower concentrations of byproducts such as lactic acid. In addition, the ATP-concentration in infected cultures decreased by nearly 50 % over a growth period of 4 days. The stress in the host cell was reflected by the decrease in the total adenyl-nucleotide pool and the increase in AMP-concentration. An increase in the intracellular IMP-concentration indicated that the purine salvage pathway was inhibited which may explain the decrease in the ATP-concentration. It therefore appears that the regular replacement of medium and replenishment of erythrocytes only partially contribute to the successful establishment of malaria cultures. More research is necessary to identify the factors that are responsible for the inhibition of the purine salvage pathway. The stress placed on the human body by the parasite is complicated by the thrombocytopenia observed in some infections. Increases in the concentrations of thrombocyte-associated immunoglobulin G and M which are a characteristic for this condition, can be determined by a modified microo-method developed in our laboratory. By monitoring the parasite-infected patient over a period of time with the micro-method, the increase in thrombocytes and decrease in thrombocyte-associated antibodies were correlated with the recuperation of the patient. This method does not destroy the thrombocytes, thereby allowing displacement studies to be undertaken with purified parasite antigens of synthetic peptides. An investigation of parasite-infected erythrocytes by means of light microscopy, transmission and scanning electronmicroscopy, indicated that the local isolate could be composed of a mixture of strains, of which some have the ability to induce knobs on the erythrocyte. Furthermore, the investigation illustrated that fast fixation with gluteraldehyde is superior to slow fixation when transmission electronmicroscopy is performed. However, no difference could be observed when scanning electron microscopy was performed on infected erythrocytes that had been fixed by either of these methods. AFRIKAANS : Malaria parasiete is verantwoordelik vir 'n toenemende morbiditeit sowel as mortaliteit in verskeie tropiese streke in Suider-Afrika waarvan meer as 95 % van die gevalle deur Plasmodium falciparum veroorsaak word. Navorsing in die tesis is gevolglik op hierdie parasiet toegespits. Ten spyte daarvan dat far'makologiese profilakse toegepas word, kan malaria nog steeds opgedoen word as gevolg van n wereldwye weerstandigheid teen bestaande geneesmiddels. 'n Ondersoek van die klorokien-weerstandigheidstatus in die Noord-Oostelike Transvaal het in 1988 getoon dat 11 % van die stamme in die area weerstandig is teenoor klorokien. Slegs 78% van die parasiete was sensitief vir die nuwe middel, Meflokien, wat as moontlike plaasvervanger in klorokien-weerstandige parasietinfeksies gebruik kan word. Geen parasietstam was egter weerstandig of onsensitief teen beide middels nie. Langtermyn kultuurkweking van malaria parasiete is essensieël vir die verkryging van uitgangsmateriaal vir navorsing op die parasiet. Dit blyk egter dat inisiasie van kulture van wilde stamme van P. falciparum problematies by sekere hoogtes bo seespieël is. Die gassamestelling van die medium is as 'n moontlike oorsaak gëidentifiseer aangesien dit afhanklik is van die parsiële drukke van gasse by verskillende hoogtes bo seespieël. Die toksiese effek van te hoë suurstofkonsentrasies op die parasiet a.g.v. die laer atmosferiese druk op die Hoëveldstreek, is oorkom deur die konsentrasie van opgeloste koolsuurgas in die medium te verhoog deur vooraf 'n spesiale gasmengsel daardeur te borrel. Moontlike tekortkominge in die langtermyn kultuur metode vir malaria parasiete wat optimale groei kan belemmer, is ook ondersoek. Plaaslike parasietstamme kon op medium wat verryk is met beesserum onderhou word, alhoewel die groeitempo laer is as wanneer mensserum gebruik word. 'n Toenemende tempo van mediumvervanging soos die parasitemia in kulture toeneem, het getoon dat minder stres op die sisteem geplaas word deurdat die parasiete vir 'n korter periode en aan laer konsentrasies van byprodukte soos melksuur blootgestel word. Die ATP-konsentrasies in gëinfekteerde kulture daal egter met ongeveer die helfte oor 'n groeiperiode van 4 dae. Tesame hiermee, is daar ook 'n daling in die totale adeniel-nukleotied poel van die gëinfekteerde rooibloedsel en 'n styging in die AMP-konsentrasie wat die stres in die gasheersel weerspieël. 'n Verhoging in die intrasellulêre IMP-konsentrasie dui op 'n moontlike inhibisie van die purienherwinningspadweg wat die verlaagde ATP-konsentrasie mag verklaar. Uit die studie blyk dit dus of meer gereelde vervanging van medium en toevoeging van rooibloedselle slegs 'n gedeeltelike bydrae maak tot die suksesvolle vestiging van malariakulture. Die identifikasie van die faktore wat lei tot die inhibisie van die purienherwinningspadweg, verg nog verdere ondersoeke. Die stres wat die parasiet in die menslike liggaam veroorsaak, word ook weerspieël deur onderandere die trombositopenie wat in sommige infeksies waargeneem word. Verhogings in die konsentrasie van trombosiet-geassosieërde immunoglobuliene G en M wat kenmerkend is van die toestand, kan gemeet word deur 'n gemodifiseerde mikrometode wat in ons laboratoriums ontwikkel is. Deur die parasiet-gëinfekteerde pasiët oor 'n paar dae met die mikrometode te monitor, kon die verhoging in trombosiete en afname in trombosiet-geassosieërde antiliggame met herstel van die pasiënt gekorreleer word. Die metode veroorsaak geen skade aan die trombosiete nie sodat antigeenverplasingstudies ook na die tyd gedoen kan word. In 'n ondersoek van die parasiet met behulp van ligmikroskopie, deurstraal- sowel as skandeer-elektronmikroskopie, is gevind dat die plaaslike isolaat moontlik uit 'n mengsel van stamme bestaan, waarvan sommige die vermoë het om knoppe op die rooibloedsel te induseer. Verder het die ondersoek getoon dat indien deurstraal-elektronmikroskopie gebruik word, 'n vinnige fikseringsmetode met gluteraldehied beter is as 'n stadige fikseringsmetode. In vergelyking hiermee kon geen verskil waargeneem word met skandeer-elektronmikroskopie van geparasiteerde rooibloedselle wat met enige van hierdie metodes gefikseer is nie. Copyright / Dissertation (MSc)--University of Pretoria, 1992. / Biochemistry / unrestricted

The multistage life cycle of malaria-causing P. falciparum is complex, making prevention and treatment difficult. As a result of resistance to many antimalarial drugs, novel compounds with unexplored targets are constantly sought after for the purpose of treating the symptoms of malaria. Here, novel compounds were screened for antiplasmodial activity against the symptom-causing asexual intraerythrocytic malaria-causing parasites. Unfortunately, many novel compounds in the drug discovery pipeline and drugs in clinical use possess underlying pharmacological issues that makes administration challenging. These include low aqueous solubility and short half-life which negatively impact bioavailability resulting in toxicity. This, in turn, increases patient non-compliance and the emergence of drug-resistant strains. Nanoparticles (NP) have the ability to mask drugs from the external environment while increasing circulation time and often alleviate many issues at once. Furthermore, the selected drugs do not need to be modified. Drug conjugation NPs with a targeting ligand and stimuli-responsive linkers have been extensively researched in many diseases, however, none have been reported for malaria clinically. Here, the first acid-labile targeted NP (tNP) that exploits the biology of infected erythrocytes and the specialised food vacuole (FV) of P. falciparum is interrogated for ability to decrease toxicity while retaining antimalarial activity. This dissertation describes the effect of tNPs on the efficacy and toxicity of selected compounds. In vitro haemolysis and cytotoxicity assays revealed that the tNPs are biocompatible to erythrocytes and HepG2 cells. The data also shows that tNPs decrease the toxicity of drugs and the chosen novel compound against human cells. A decrease in antiplasmodial activity was observed in vitro for the tNPs when compared to the novel compound and drugs on their own. However, this was due to the biogenesis of the FV and a shortened window of release. Nonetheless, the NP backbone was not active against P. falciparum intraerythrocytic parasites whereas tNPs were, showing activity due to released drug. The targeting ligand was also not specific for antiplasmodial activity. Although a significant loss in activity is observed, the results presented here suggests that these novel acid-labile tNPs serve as an attractive starting point for targeted treatment of malaria with an improved patient tolerance. Furthermore, novel compounds with issues can be selected without having to be modified or completely discarded. Therefore, increasing the chances of finding a variety of compounds that can be used to treat malaria while keeping patients safe. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2020. / NRF / Biochemistry / MSc (Biochemistry) / Unrestricted

Malaria is considered the most prevailing human parasitic disease. Despite various chemotherapeutic interventions being available, the parasite responsible for the most lethal form of malaria, Plasmodium falciparum, is continuously developing resistance towards drugs targeted against it. This, therefore, necessitates the need for validation of new antimalarial development. Polyamine biosynthetic enzymes, particularly S-adenosylmethionine-L-decarboxylase (PfAdoMetDC), has been identified as a suitable drug target for protozoan parasitic diseases due to its essential role in cell proliferation. Furthermore, in Plasmodium polyamine biosynthesis, PfAdoMetDC is organised into a unique bifunctional complex with ornithine decarboxylase (PfAdoMetDC/ODC) covalently linked by a hinge region, distinguishing this enzyme as unique a drug target. However, inhibitors targeting this pathway have not been successful in clinical assessment, creating the need for further research in identifying novel inhibitors. This study focused on the structural and functional characterisation of protein-specific properties of the AdoMetDC domain in P. falciparum parasites, as well as identifying novel inhibitors targeting this enzyme as a potential antimalarial therapeutic intervention. In order to develop novel inhibitors specifically targeting PfAdoMetDC through a structure-based drug discovery approach, the three-dimensional structure is required. However, due to a lack of structural and functional characterisation, determination of the crystal structure has been challenging. Heterologous expression of monofunctional PfAdoMetDC was achieved from a wild-type construct of the PfAdoMetDC domain including the covalently linked hinge region. In chapter 2, deletion of a large non-homologous, low-complexity parasite-specific insert (A3) in monofunctional PfAdoMetDC resulted in an increased yield, purity and sample homogeneity, whilst maintaining protein functionality and structural integrity. However, truncation of the proposed non-essential hinge region resulted in low-level expression of insoluble protein aggregates and a complete loss of protein activity, indicating that the hinge region is essential for monofunctional PfAdoMetDC. However, in the absence of the three-dimensional PfAdoMetDC crystal structure, novel derivatives of a well-known AdoMetDC inhibitor, MDL73811, were tested for their activity against heterologous PfAdoMetDC, as well as their potency against P. falciparum parasites, in chapter 3. The compound Genz-644131 was identified as a lead inhibitor of PfAdoMetDC, however, the poor membrane permeability of the compound resulted in low in vitro activity. Drug permeability of Genz-644131 into P. falciparum infected erythrocytes and its potency was significantly improved by its encapsulation into a novel immunoliposome based drug delivery system. The results presented here provide essential information for development of a unique strategy in obtaining suffiecient levels of fully active recombinant PfAdoMetDC of sufficient purity for crystallisation studies and subsequent structure-based drug design efforts. The combination of Genz-644131 with the novel drug delivery system, which markedly improved its potency against PfAdoMetDC may proof to be a viable antimalarial chemotherapeutic strategy for future investigations. / Thesis (PhD)--University of Pretoria, 2014. / tm2015 / Biochemistry / PhD / Unrestricted