Browsing by Author "Egan, Timothy John"

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    A metallomic, proteomic and lipidomic investigation of the malaria parasites digestive vacuole and insights into the mediators of haemozoin formation
    (2019) Mohunlal, Roxanne; Egan, Timothy John
    Novel and unambiguous mechanistic details of the biochemical processes that enable the most virulent form of the malaria pathogen, Plasmodium falciparum, to thrive within the human host are desperately required in order to find innovative strategies to counteract emerging parasite resistance that will inevitably render current malarial therapies obsolete. This study focused on exploring the Plasmodium falciparum metallome and investigated the mediators of haemozoin formation, an ingenious parasite-specific process critical to host immune evasion as well as parasite survival and one that is surprisingly rather contentious. In order to obtain a comprehensive view of metal distribution, the trace metal content in parasites isolated at varying time-points over the 48 h intraerythrocytic cycle were measured by ICP-MS. The trace metals detected; namely, iron, magnesium, zinc, manganese and copper, were compared to control uninfected erythrocytes. With the malaria parasite being a haematophagous organism, iron was detected as the most abundant trace metal and found to exhibit a significant increase up to 32 h into the life cycle after which the measured iron content remained relatively constant and lower than the control. This was attributed to the maximum amount of haemoglobin having been ingested at this mature trophozoite stage and the active conversion of free haem (Fe(III)PPIX) into biocrystalline haemozoin. All other trace metals exhibited 2 to 4-fold increases in metal ion content as the parasite matured and were detected in amounts higher than those found in the erythrocyte control, demonstrating uptake of these ions from the external medium. These increases coincided with specific cellular events such as cell division and enhanced parasite metabolism. Qualitative proteomic analysis of parasite material identified several metalloproteins but most significant, was the discovery of magnesium and copper transporters. Together, these findings suggest that transition metal import is essential to promote important cellular events and parasite growth and are indicative of unique parasite-specific metal transport pathways. The mediators of crystal formation were investigated by interrogating the haemozoin proteome and lipidome. Haemozoin was obtained from parasitised erythrocytes following a fractionation approach which culminated in their release from purified digestive vacuoles (DVs) following multiple freeze-thaw cycles. Isolated crystals were extensively washed with aqueous sodium acetate buffer (0.5 M, pH 5.2), detergent(4% SDS) and organic solvents (acetone/methanol) prior to base dissolution (0.1 M NaOH). Mass spectrometry was used to investigate the biomolecules occluded by haemozoin by analysing extensively washed and dissolved crystals. Haem detoxification protein (HDP), a protein currently postulated to be potent in mediating haemozoin formation in vivo, was detected in high relative abundance in the dissolved haemozoin fraction by semi-quantitative label-free proteomics. Expression and purification of recombinant soluble HDP was optimised and both soluble and refolded protein were further investigated. Characterisation by circular dichroism and fluorescence spectroscopy revealed that soluble HDP differed in conformation to its refolded counterpart. In aqueous solution (pH 7.4), UV-vis spectrophotometric titrations showed soluble and refolded HDP to bind Fe(III)PPIX in a 1:1 ratio with a Kd of 1.2 ± 0.5 µM and 0.35 ± 0.04 µM respectively. Crucially, activity studies under biomimetic conditions (pH 5.2, 37°C) demonstrated that this protein was not competent to promote β-haematin (synthetic haemozoin) formation without the incorporation of detergent. Mass spectrometry-based lipidomics identified and quantified over 400 lipids in dissolved haemozoin. Neutral lipids were found to be the dominant class comprising 90% cholesterol, 2% cholesterol esters and 0.6% acylglycerols. The detected lipids were used to prepare a model lipid blend which was found to efficiently promote β-haematin formation in yields greater than 90% in under 10 minutes and at concentrations as low as 18 µM. Crystals synthesised using the model lipid blend were found to exhibit similar morphological traits to haemozoin naturally produced by the parasite. Live-cell imaging by spinning disk confocal microscopy revealed that neutral lipid bodies localised externally from the DV or in close contact with the DV membrane but were not found in the immediate vicinity of haemozoin. β-Haematin was found to occlude labelled protein and lipid when synthesised in their presence. Furthermore, occluded biomolecules were not readily displaced from the crystal surface through simple washing with aqueous buffer (pH 5.2) but were only released upon base dissolution of crystals. Pre-formed β-haematin and haemozoin incubated with labelled biomolecules resulted in no further occlusion which demonstrated that these crystals occlude material in a manner non-exchangeable with the DV lumen, thus providing a window into the molecules present at the time and site of crystal formation. Overall, this multidisciplinary omics approach has revealed that the malaria parasite has a unique metallome which may provide promising new drug targets. Significantly, this study has unequivocally demonstrated that haemozoin occludes proteins and lipids in detectable amounts in a non-exchangeable manner with the external milieu but, crucially, lipids occluded by haemozoin are present at the time and site of formation within the DV and are potent mediators of haemozoin formation in the malaria parasite.
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    A pharmacokinetic and antimalarial efficacy evaluation of pyridodibemequines and their metabolites
    (2021) Redhi, Devasha; Wiesner, Lubbe; Egan, Timothy John
    The recurring challenge of the emergence of drug resistance necessitates the continual development of improved antimalarial treatments, which can target parasite strains that display reduced susceptibility towards current therapy. Consequently, a novel series of dual functioning pyridodibemequine (PDBQ) compounds were designed with the intention of reversing resistance in chloroquine-resistant (CQR) strains of Plasmodium falciparum. These hybrid molecules integrate a 4-amino-7-chloroquinoline antiplasmodial core with a modified dibenzylmethylamine side chain, which interacts with the CQR mutant P. falciparum chloroquine resistance transporter (PfCRT) to hinder the efflux of chloroquine (CQ) from its site of action, thereby reversing CQ resistance. The parent compounds of the PDBQ series, which differ in the ortho-, meta-, and para-orientation of the dibenzylmethylamine side chain, displayed favourable in vitro potency against chloroquine-sensitive (CQS) and CQR strains of P. falciparum; however, they were shown to be metabolically labile. Structure elucidation demonstrated that all formed PDBQ metabolites retained the 4-amino-7-chloroquinoline pharmacophore of the parent. Thus, the major metabolites, M1 and M2, generally conserved the in vitro antimalarial activity and selectivity of the parent compounds. Mechanistic studies revealed that the antiplasmodial activity of the PDBQ parent compounds and major metabolites primarily results from the inhibition of haemozoin formation, culminating in a toxic accumulation of ferriprotoporphyrin IX. The parents and major metabolites exhibited minimal toxicity against a mammalian cell line, and the metabolites are proposed to display reduced systemic toxicity, and human ether-a-go-go-related gene liability compared to the parent compounds. Furthermore, the major metabolites generally exhibited similar or improved in vitro solubility, permeability, lipophilicity, and metabolic stability compared to the parent compounds. Therefore, given their favourable in vitro characteristics, the major active metabolites of each parent PDBQ compound were further evaluated in this project to determine their potential as early preclinical antimalarial lead candidates. The proof of concept study presented herein investigated the in vivo pharmacokinetics (PK) of the PDBQ series of parents and major metabolites in a healthy murine model to allow the rational selection of candidates to be evaluated for their in vivo antimalarial efficacy and PK in a P. falciparum-infected murine model. For the PK studies in healthy and malaria-infected mice, analyte detection from whole blood was achieved using high-performance liquid chromatography coupled to tandem mass spectrometry. The bioanalytical methods were developed and partially validated based on a fit-for-purpose approach, which ensured that the generated PK concentration data was reliable and accurate. Lastly, given the significance of combination therapy in delaying the onset of drug resistance, fixed-dose ratio isobologram analyses were performed to probe the potential of the lead candidates to be used synergistically with an antimalarial partner drug possessing a distinct mechanism of action to haemozoin inhibition. Additionally, the ability of the lead candidates to reverse CQ resistance was evaluated in a CQR strain of P. falciparum. A comparative PK study was performed in a healthy murine model to determine whether the parent PDBQ compound should be used as a strategy to deliver the active metabolites or whether the active major metabolite should be directly administered. This was achieved by oral administration of the parent PDBQ compound and subsequent quantification of the parent compound and formation of the major metabolites. In addition, each pre-synthesised major metabolite was orally administered, at the equivalent parent dose, to characterise the PK profile of the individual active metabolite. These PK studies revealed that the overall oral exposure of the antimalarial pharmacophore was markedly greater after direct administration of the preformed metabolite compared to the cumulative oral exposure of the parent and formed metabolites after administration of the parent PDBQ compound. Furthermore, the metabolites attained higher maximal concentrations and maintained circulating concentrations which favourably exceeded their respective in vitro half-maximal inhibitory concentration at 24 h post-oral administration compared to the parent compounds at the equivalent oral dose. These findings substantiated the direct administration of the preformed PDBQ major metabolite over the parent compound. From the series of PDBQ metabolite derivatives, compounds 43M1 and 47M1 displayed the highest maximal concentrations of 8 ± 1 and 9.4 ± 0.5 μM, respectively and the greatest oral exposures of 62 ± 3 and 93 ± 9 µM.h, respectively, after single 20 mg/kg oral administrations of either 43M1 or 47M1. Given their encouraging PK profiles, 43M1 and 47M1 were selected to progress to the subsequent phase of the study which evaluated their in vivo antimalarial efficacy and pharmacokinetic/pharmacodynamic (PK/PD) relationship in a P. falciparum-infected humanised murine model. 43M1 and 47M1 were efficacious against asexual intraerythrocytic P. falciparum infection in humanised mice, where both compounds displayed a 98% reduction in parasitaemia after 4 consecutive daily oral administrations of 20 mg/kg of either 43M1 or 47M1 compared to the untreated control. The PK/PD analysis revealed dose-dependent reductions in parasitaemia; and the doses required to produce 90% of the maximal parasiticidal response (ED90) were 12 and 7.7 mg/kg for 43M1 and 47M1, respectively. Additionally, the oral exposures required to achieve the effect at the ED90 were 6.2 and 18.6 µM.h for 43M1 and 47M1, respectively. 43M1 or 47M1 demonstrated overall in vitro antimalarial synergy with dihydroartemisinin or atovaquone and additivity with methylene blue in CQS and CQR strains of P. falciparum. 43M1 or 47M1 with mefloquine or lumefantrine displayed in vitro antimalarial synergy in a CQS strain of P. falciparum; however, in a CQR strain, antagonism and additivity were displayed with mefloquine and lumefantrine, respectively. Additionally, 43M1 and 47M1 were unable to potentiate the in vitro antiplasmodial activity of CQ in a CQR strain of P. falciparum which suggested that, unlike the parent PDBQ compound, the metabolite did not possess the ability to reverse CQ resistance. The promising in vivo antimalarial efficacy of 43M1 and 47M1 against P. falciparum and their prospective in vitro antimalarial synergy with dihydroartemisinin and atovaquone underlines the potential of 43M1 and 47M1 for further development as preclinical antimalarial candidates.
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    Encapsulation of iron(III) protoporphyrin IX and tetraphenylporphyrin in metal-organic frameworks for application as heterogeneous oxidation catalysts
    (2018) Dare, Nicola A; Egan, Timothy John; Bourne, Susan
    Two MOFs, [H2N(CH3)2][Zn3(TATB2(HCOO)]·HN(CH3)2·DMF·6H2O (1) and ZnHKUST-1 (2) (TATB = 4,4′,4″-s-triazine-2,4,6-triyl-tribenzoate) were investigated as potential hosts to encapsulate Fe(III) protoporphyrin IX (ferrihaem = Fe(III)PPIX) and Fe(III) tetraphenylporphyrin (Fe(III)TPP). Methyl orange (MO) adsorption was used as an initial model for substrate uptake in MOFs 1 and 2. MOF 1 showed good adsorption of MO (10.3 ± 0.8 mg.g-1 ) which could undergo in situ protonation upon exposure to aqueous HCl vapour. By contrast MO uptake by 2 was much lower (2 ± 1 mg.g-1 ) and PXRD indicated structural instability on exposure to water was the likely cause. Two methods for Fe(III)PPIX incorporation into 1 were investigated: soaking and encapsulation. Encapsulation was verified by SEM-EDS and showed comparable concentrations of Fe(III)PPIX on exposed interior surfaces and on the original surface of fractured crystals. SEM EDS results were consistent with ICP-OES data on bulk material (1.2 ± 0.1 mass % Fe). PXRD data showed that the framework in 1 was unchanged after encapsulation of Fe(III)PPIX. MO adsorption (6 ± 1 mg.g1 ) by Fe(III)PPIX-1 confirmed there is space for substrate diffusion into the framework, while the UV-visible spectrum of solubilized crystals confirmed that Fe(III)PPIX retained its integrity. A solid-state UV-visible spectrum of Fe(III)PPIX-1 indicated that Fe(III)PPIX was not in a µ-oxo dimeric form. Although single-crystal XRD data did not allow for full refinement of the encapsulated Fe(III)PPIX molecule owing to disorder of the metalloporphyrin, the Fe atom and pyrrole N atoms were located, enabling rigid-body modelling of the porphine core. For comparison, Fe(III)PPIX was further encapsulated in 2, forming Fe(III)PPIX-2. Reaction ABSTRACT of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) with H2O2, catalysed by Fe(III)PPIX-1 and -2 showed that Fe(III)PPIX-1 is significantly more efficient than Fe(III)PPIX-2 and is superior to solid Fe(III)PPIX-Cl due to the faster initial rate of reaction as well as the greater conversion of ABTS to ABTS●+ . Both frameworks 1 and 2 were also investigated as potential hosts to encapsulate Fe(III) tetraphenylporphyrin (Fe(III)TPP). Attempts to encapsulate Fe(III)TPP into 1 were unsuccessful, but Fe(III)TPP was successfully encapsulated into 2, forming Fe(III)TPP-2. The framework was characterised by PXRD and SEM-EDS confirmed uniform distribution of Fe(III)TPP through the framework. The loading of Fe(III)TPP determined using ICP-OES (0.604 ± 0.008 Fe mass %) agreed well with SEM-EDS data. Single crystals of Fe(III)TPP-2 were obtained and structure determination showed that the Fe(III) porphyrin was positionally disordered over three positions. The instability of Fe(III)TPP-2 in the presence of H2O resulted in it being an inappropriate choice as an oxidation catalyst. The kinetics of ABTS oxidation by H2O2 catalysed by Fe(III)PPIX-1 were further investigated. The peroxidatic activity of this heterogeneous system conforms to a rate law identical to that observed in solution with no discernible influence of particle size, suggesting that the MOF system closely mimics the solution state. The proposed rate law indicates a reaction mechanism with two possible pathways, as suggested for the same reaction in solution. The major pathway describes the coordination of H2O2 to the Fe(III) centre and subsequent formation of a high valent intermediate, while the minor pathway describes the same process preceded by ABTS coordination to the Fe(III) centre forming a six-coordinate complex. The further application of Fe(III)PPIX-1 as an oxidation catalyst was probed by investigating the catalytic oxidation of hydroquinone, thymol, benzyl alcohol and phenyl ethanol by tert-butyl-hydroperoxide ( tBuOOH). Reactions were successful and showed t1/2 values that increase with increasing substrate molecular volume.
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    In vitro modelling of cellular haemozoin and inhibition by β-haematin inhibitors and their derivatives
    (University of Cape Town, 2020) Openshaw, Roxanne; Egan, Timothy John
    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.
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    Interactions of ferriprotoporphyrin IX with neutral lipids and detergents: insights into their role in β-haematin formation
    (2015) Omar, Aneesa; Egan, Timothy John
    The malaria parasite ingests between 80-100% of the host red blood cell's iron in the form of haemoglobin, which is catabolised to amino acids and ferriprotoporphyrin (Fe(III)PPIX), that it subsequently detoxifies to form the bio crystal, haemozoin. Neutral lipids have been implicated as the biological molecules responsible for the nucleation of haemozoin in vivo, though their exact role is unclear. This thesis has investigated the interaction between these lipids and Fe(III)PPIX. By exploiting the fluorescence quenching ability of Fe(III)PPIX, Stern-Volmer plots were generated to estimate the concentration of Fe(III)PPIX partitioned into Nile red stained neutral lipid droplets (NLBDs) over a pH range. The pH dependence was found to correlate with the charge of the Fe(III)PPIX molecule and the largest amount of Fe(III)PPIX partitioning was observed under the acidic pH conditions of the parasite digestive vacuole (pH 4.80), where haemozoin is formed. From the fluorescence maximum of Nile red, the relative polarity inside the lipid droplets was shown to lie between that of acetone and octanol with a Dimroth-Reichart ET(30) parameter of 45 kcal/mol. The lipophilicity of Fe(III)PPIX was validated by measuring the octanol-water partition coefficient, log DOW, as1.8 for the ionised form at pH 7.5 and a log P of 2.8 for the neutral form. 4-aminoquinoline drugs, chloroquine and amodiaquine, decreased the partitioning of Fe(III)PPIX into NLBDs at low pH owing to the consistently charged nature of the drug-Fe(III)PPIX complex. In contrast, the quinoline methanols, quinidine and quinine, strongly increased the observed lipophilicityof Fe(III)PPIX in a pH dependent manner. Quinidine was found to effect the largest increase eon Fe(III)PPIX partitioning into NLBDs which was proposed to be due to a previously established inherent increased lipophilicity of the quinidine-Fe(III)PPIX complex.
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    Synthesis and investigation of benzimidazole and carbazole ß-haematin inhibiting scaffolds with antimalarial activity
    (2018) L'abbate, Fabrizio P; Egan, Timothy John; Hunter, Roger
    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.