Browsing by Author "Egan, Timothy J"
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- ItemRestrictedThe crystal structure of halofantrine–ferriprotoporphyrin IX and the mechanism of action of arylmethanol antimalarials.(Elsevier, 2008) de Villiers, Katherine A; Marques, Helder; Egan, Timothy JThe crystal structure of the complex formed between the antimalarial drug halofantrine and ferriprotoporphyrin IX (Fe(III)PPIX) has been determined by single crystal X-ray diffraction. The structure shows that halofantrine coordinates to the Fe(III) center through its alcohol functionality in addition to p-stacking of the phenanthrene ring over the porphyrin. The length of the Fe(III)–O bond is consistent with an alkoxide and not an alcohol coordinating group. The iron porphyrin is five coordinate and monomeric. Changes in the electronic spectrum of Fe(III)PPIX upon addition of halofantrine base in acetonitrile solution are almost identical to those observed upon addition of quinidine free base in the same solvent. This suggests homologous binding. Molecular mechanics modeling of Fe(III)PPIX complexes of quinidine, quinine, 9-epiquinine and 9-epiquinidine based on this homology suggests that the antimalarially active quinidine and quinine can readily adopt conformations that permit formation of an intramolecular salt bridge between the protonated quinuclidine tertiary amino group and unprotonated heme propionate group, while the inactive epimers 9-epiquinidine and 9-epiquinine have to adopt high energy conformations in order to accommodate such salt bridge formation. We propose that salt bridge formation may interrupt formation of the hemozoin precursor dimer formed during the heme detoxification pathway and so account for the strong activity of the two active isomers.
- ItemOpen AccessCrystallization of calcium oxalate on molecularly imprinted polymer surfaces(2004) Tewolde, Tewolde Siele; Egan, Timothy J; Rodgers, Allen LCalcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. As such, crystallization of this substance has been studied in several different model systems. However, molecular imprinting has not been previously used in this field. In the present study, template crystals of calcium oxalate mono- and di-hydrate (COM and COD respectively) were used to imprint a copolymer of 6-methacrylamidohexanoic acid and divinylbenzene.
- ItemRestrictedCrystallization of synthetic haemozoin (beta-haematin) nucleated at the surface of lipid particles(Royal Society of Chemistry, 2010) Hoang, Anh N; Ncokazi, Kanyile K; de Villiers, Katherine A; Wright, David W; Egan, Timothy JThe mechanism of formation of haemozoin, a detoxification by-product of several blood-feeding organisms including malaria parasites, has been a subject of debate; however, recent studies suggest that neutral lipids may serve as a catalyst. In this study, a model system consisting of an emulsion of neutral lipid particles was employed to investigate the formation of b-haematin, the synthetic counterpart of haemozoin, at the lipid–water interface. A solution of monoglyceride, either monostearoylglycerol (MSG) or monopalmitoylglycerol (MPG), dissolved in acetone and methanol was introduced to an aqueous surface. Fluorescence, confocal and transmission electron microscopic (TEM) imaging and dynamic light scattering analysis of samples obtained from beneath the surface confirmed the presence of homogeneous lipid particles existing in two major populations: one in the low micrometre size range and the other in the hundred nanometre range. The introduction of haem (Fe(III)PPIX) to this lipid particle system under biomimetic conditions (37 ◦C, pH 4.8) produced b-haematin with apparent first-order kinetics and an average half life of 0.5 min. TEM of monoglycerides (MSG or MPG) extruded through a 200 nm filter with haem produced b-haematin crystals aligned and parallel to the lipid–water interface. These TEM data, together with a model system replacing the lipid with an aqueous organic solvent interface using either methyl laurate or docosane demonstrated that the OH and C=O groups are apparently necessary for efficient nucleation. This suggests that b-haematin crystallizes via epitaxial nucleation at the lipid–water interface through interaction of Fe(III)PPIX with the polar head group. Once nucleated, the crystal grows parallel to the interface until growth is terminated by the curvature of the lipid particle. The hydrophobic nature of the mature crystal favours an interior transport resulting in crystals aligned parallel to the lipid–water interface and each other, strikingly similar to that seen in malaria parasites.
- ItemOpen AccessThe design and synthesis of new transition metal coordination complexes as potential anti-malarial agents(2011) Hager, Emma; Egan, Timothy JNew Rh(I), Rh(III), Ir(III) and Pd(II) coordination complexes bearing 1,10-phenanthroline (phen) or 2,2'-bipyridyl (bipy) ligands have been investigated for potential anti-malarial activity.
- ItemOpen AccessDevelopment of quinoline and non-quinoline based organometallic complexes and their ligands conjugated to polyamine scaffolds as pharmacological agents(2014) Stringer, Tameryn; Smith, Gregory S; Egan, Timothy JMalaria remains a major global health problem and to date, hundreds of thousands of people die as a result of this disease every year. Malaria has been able to adapt and rebound despite various efforts made to combat the disease. The decrease in efficacy of many front-line drugs against malaria, due to increased resistance, prompts investigation into obtaining effective compounds that are able to overcome this resistance. This study investigated the synthesis, characterisation and biological evaluation of new quinoline as well as non-quinoline based compounds. Selected Rh(I) metal complexes thereof were also studied. The compounds were screened for antiplasmodial activity, in addition, their activity against WHCO1 oesophageal cancer cells and the parasite, Trichomonas vaginalis (T vaginalis) was also evaluated.
- ItemOpen AccessDiscovery of benzamides and triarylimidazoles active against Plasmodium falciparum via haemozoin inhibition : high throughput screening, synthesis and structure-activity relationships(2015) Wicht, Kathryn Jean; Egan, Timothy J; Hunter, RogerNew antimalarials are desperately needed to overcome growing P. falciparum resistance to the current drugs. Successful quinoline-based drugs target haemozoin formation causing a cytotoxic accumulation of free haem (Fe(III)PPIX) in the parasite, a target which remains promising for future treatments. Much research has been undertaken on the quinoline antimalarials, which has led to several hypotheses of haemozoin inhibition and drug accumulation mechanisms, however, relatively few studies have been carried out for haemozoin antimalarials with alternate chemotypes. High throughput screening (HTS) can be used to identify novel scaffolds that inhibit β-haematin (βH - synthetic haemozoin) formation and which have favourable P. falciparum activities. In this project, HTS has been carried out on 43,520 small, organic, drug-like compounds as part of a larger screen of 144,330 Vanderbilt University Institute of Chemical Biology (VU) chemical library compounds and 530 were found to be good inhibitors of βH relative to the chloroquine (CQ) and amodiaquine (AQ) controls. A further 171 compounds were found to inhibit parasite growth, showing improved hit rates from previous HTS efforts. Two scaffolds (A=benzamides and B=triarylimidazoles) were selected for further analysis, whereupon analogues were synthesised.
- ItemRestrictedExperimental and time-dependent density functional theory characterization of the UV−Visible spectra of monomeric and μ‑Oxo dimeric ferriprotoporphyrin IX(American Chemical Society, 2012) Kuter, David; Venter, Gerhard A; Naidoo, Kevin J; Egan, Timothy JSpeciation of ferriprotoporphyrin IX, Fe(III)PPIX, in aqueous solution is complex. Despite the use of its characteristic spectroscopic features for identification, the theoretical basis of the unique UV−visible absorbance spectrum of μ- [Fe(III)PPIX]2O has not been explored. To investigate this and to establish a structural and spectroscopic model for Fe(III)PPIX species, density functional theory (DFT) calculations were undertaken for H2O−Fe(III)PPIX and μ- [Fe(III)PPIX]2O. The models agreed with related Fe(III)porphyrin crystal structures and reproduced vibrational spectra well. The UV−visible absorbance spectra of H2O−Fe(III)PPIX and μ-[Fe(III)PPIX]2O were calculated using time-dependent DFT and reproduced major features of the experimental spectra of both. Transitions contributing to calculated excitations have been identified. The features of the electronic spectrum calculated for μ-[Fe(III)PPIX]2O were attributed to delocalization of electron density between the two porphyrin rings of the dimer, the weaker ligand field of the axial ligand, and antiferromagnetic coupling of the Fe(III) centers. Room temperature magnetic circular dichroism (MCD) spectra have been recorded and are shown to be useful in distinguishing between these two Fe(III)PPIX species. Bands underlying major spectroscopic features were identified through simultaneous deconvolution of UV−visible and MCD spectra. Computed UV−visible spectra were compared to deconvoluted spectra. Interpretation of the prominent bands of H2O−Fe(III)PPIX largely conforms to previous literature. Owing to the weak paramagnetism of μ-[Fe(III)PPIX]2O at room temperature and the larger number of underlying excitations, interpretation of its experimental UV−visible spectrum was necessarily tentative. Nonetheless, comparison with the calculated spectra of antiferromagnetically coupled and paramagnetic forms of the μ-oxo dimer of Fe(III)porphine suggested that the composition of the Soret band involves a mixture of π→π* and π→dπ charge transfer transitions. The Q-band and charge transfer bands appear to amalgamate into a mixed low energy envelope consisting of excitations with heavily admixed π→π* and charge transfer transitions.
- ItemOpen AccessHaematin-Quinoline interactions and structure-activity relationships in the antimalarial chloroquine and related compounds(2002) Kaschula, Catherine Hart; Egan, Timothy J; Hunter, RogerThe nature of the ferriprotoporphyrin IX (Fe(III)PPIX) antimalarial drug target and its interactions with aminoquinolines was investigated spectrophotometrically. The antiquity of malaria, which is caused by protozoan parasites of the genus Plasmodium, is demonstrated by the host specificity of over 100 parasite species found in reptiles, birds and mammals. The four species of plasmodia that infect man are P. vivax, P. malariae, P. ovale and P. falciparum; of which P. falciparum is the most deadly (Bruce-Chwatt 1981 ).
- ItemRestrictedHaemozoin formation(Elsevier, 2008) Egan, Timothy JFormation of malaria pigment or haemozoin is the major route of haem detoxification in the malaria parasite Plasmodium falciparum as well as several other species of haematophagous organisms, including other Plasmodium species, helminth worms such as Schistosoma mansoni and blood-sucking insects such as Rhodnius prolixus. Recent advances in our understanding of the formation of haemozoin, both from new observations that it is formed within lipid bodies in P. falciparum and S. mansoni and biomimetic studies on the formation of its synthetic counterpart -haematin are reviewed. The review also covers methods available for screening compounds for their ability to inhibit -haematin formation.
- ItemOpen AccessIncrease on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro(Public Library of Science, 2010) Stiebler, Renata; Hoang, Anh N; Egan, Timothy J; Wright, David W; Oliveira, Marcus FBACKGROUND: Hemozoin (Hz) is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH) (the synthetic counterpart of Hz) formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro . METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO) and a series of polyethyleneglycols (PEGs). We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000) increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300) caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels.
- ItemOpen AccessInsights into the mechanism of action of quinoline antimalarials against Plasmodium falciparum revealed by novel fluorescent analogues and chemical proteomics(2016) Woodland, John Geoffrey; Egan, Timothy J; Hunter, RogerFor centuries, quinoline-based drugs have formed the cornerstone of antimalarial treatment. Despite recent challenges posed by resistance, interest in these molecules persists. It is thus surprising that crucial details of their mechanism of action against the most virulent malaria parasite, Plasmodium falciparum, remain unresolved. This thesis develops new tools to generate deeper insights into the modes of action of the two major classes of the quinoline antimalarials against P. falciparum. These are the quinoline methanols, represented by the diastereomeric Cinchona alkaloids quinine and quinidine, and the 4-aminoquinolines, represented by chloroquine. Mechanistic studies of these antimalarials have typically focused on the inhibition of haemozoin biocrystallisation within the acidic digestive vacuole of P. falciparum. In order to conduct a comprehensive survey of the subcellular localisation of these antimalarials across the entire infected erythrocyte, a suite of novel fluorescent derivatives was designed and synthesised. Key physicochemical properties of these antimalarials were retained in order to preserve the interactions of these drugs with their putative target, ferriprotoporphyrin IX or Fe(III)PPIX. Versatile derivatisation of the alkaloids was enabled by a regioselective radical-mediated thiolene click reduction. 7-Nitrobenz-2-oxa-1,3-diazole (NBD) was identified as a suitable reporter fluorophore and was attached to the quinoline core by nucleophilic aromatic substitution. The length of the spacer chain between the quinoline and the fluorophore was varied by preparing NBD-labelled amino acids and their corresponding succinimidyl esters. A novel NBD-labelled chloroquine derivative was prepared by using its N-dealkylated analogue as a key intermediate. A single quinine derivative with an alternative fluorophore, bimane, was also prepared.
- ItemOpen AccessInteractions of quinoline antimalarials with haematin and their effect on ß-haematin formation(2005) Ncokazi, Kanyile K; Egan, Timothy JThermodynamic compensation in the interaction of quinoline antimalarials with haematin in 40% (v/v) aqueous OMSO has been compared with that in pure aqueous solution. The data indicate that the degree of desolvation and loss of conformational freedom is identical in both systems. The nature of interactions between quinoline drugs and haematin was investigated spectroscopically in organic and mixed solvents. Free energies of association of chloroquine, quinine and quinidine with haematin are largely insensitive to the increasing concentration of NaCI0. This demonstrates that electrostatic interactions play a minor role in the overall stability of these complexes under these conditions. Increasing concentration of OM SO weakens association of chloroquine, amodiaquine, quinine, quinidine and 9-epiquinine with haematin. These effects suggest that the interactions are hydrophobic. Furthermore, it has been demonstrated that free energy of association with haematin weakens as a function of decreasing solvent polarity in organic solvents. However, free energies of association are weaker in mixed aqueous solvents than in pure organic solvents. This indicates that dispersion and electrostatic interactions are relatively stronger in a non-aqueous environment. A new assay has been developed for measuring inhibition of ,ß-haematin formation using 5% (v/v) aqueous pyridine solution. This pyridine solution forms a low complex with haematin in aqueous solution but not with ,ß-haematin. Formation of ß-haematin is brought about in 4.5 M acetate, pH 4.5 at 60°C. The assay is rapid (60 min incubation) and requires no centrifugation or expensive radioactive material. This assay is compatible with high throughput screening and analysis can be done by visual inspection of ,ß- haematin inhibitors. The IC⁵⁰ values obtained were compared with those reported in other assays for 13 compounds investigated. There was generally a good correlation between the pyridine assay and other assays. Used qualitatively, the method was also employed for screening 48 compounds for ,ß-haematin inhibition. All of these compounds produced results in agreement with expectation from previous investigations.
- ItemOpen AccessInterplay between Plasmodium falciparum haemozoin and l-arginine: implication for nitric oxide production(BioMed Central, 2018-12-06) Corbett, Yolanda; D’Alessandro, Sarah; Parapini, Silvia; Scaccabarozzi, Diletta; Kalantari, Parisa; Zava, Stefania; Giavarini, Flavio; Caruso, Donatella; Colombo, Irma; Egan, Timothy J; Basilico, NicolettaAbstract Background Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), l-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between l-arginine and haemozoin. Methods Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of l-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 μM of haemozoin, followed by a decrease with doses up to 100 μM, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose–response manner. Similar results were seen when l-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC–MS/MS a complete depletion of l-arginine was observed in this haemozoin-treated, conditioned medium. Conclusions It is proposed that haemozoin interacts with l-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.
- ItemOpen AccessInvestigating the structure-function relationships of Plasmodium Haem Detoxification Protein and Phosphatidylinositol 4-kinase(2022) Wambua, Lynn; Chibale, Kelly; Egan, Timothy J; Arendse, Lauren BThe human malaria parasite Plasmodium falciparum relies on proteins and protein-mediated processes to survive, replicate in the host and evade the host's immune response. This study focuses on two Plasmodium proteins: (i) Haem Detoxification Protein (HDP) which has been reported to catalyse haemozoin formation in Plasmodium and (ii) Phosphatidylinositol-4- Kinase β (PI4Kβ) which plays an essential role in multiple stages of the parasite lifecycle and is a clinically validated drug target for malaria chemotherapy. Previous efforts to characterise HDP have been limited to experiments using the refolded form of recombinant protein and the role of HDP in haemozoin formation is still unclear. The aim of this research project was to optimise expression and purification of soluble Plasmodium falciparum HDP and characterise the protein's role in haemozoin formation Efforts to optimise soluble HDP expression were effective albeit exhaustive efforts to purify HDP from the soluble fraction were unsuccessful. Purification of HDP under denaturing conditions was achieved using previously reported methods. Refolded HDP was evaluated for β-hematin formation activity, but the results indicated β-hematin formation was mediated by sodium dodecyl sulfate used in the assay rather than by HDP. HDP was co-crystalised in the presence of haem but crystals diffracted poorly. Evaluation of the predicted HDP structure based on homology modelling showed that the four histidine residues predicted to facilitate haemozoin formation are not aligned in a way that would facilitate haemozoin crystal growth. This, coupled with the data from the biochemical assays, suggests HDP is unlikely to be involved in haemozoin formation. The second aim of this project was to use Plasmodium vivax PI4Kβ inhibition assays coupled with site-directed mutagenesis and mass spectrometry to support target-based malaria drug discovery programs focused on the development of both ATP-competitive and covalent Plasmodium PI4Kβ inhibitors. Two residues of interest in Plasmodium vivax PI4Kβ unique to Plasmodium, F832 and C1327, were mutated to alanine. F832 is thought to form key Pi-Pi interactions with inhibitors and C1327, found on the periphery of the catalytic site, is a potential target for covalent inhibitors. Site-directed mutagenesis was used to introduce mutations at F832 and C1327 in wild-type Plasmodium vivax PI4Kβ and wild-type PI4Kβ and the two mutants were expressed and purified. Kinetic characterisation revealed the three enzymes had similar kinetic parameters. Inhibition data indicated the F832 mutation to alanine had a minimal effect on inhibition of PI4Kβ. A time-dependent inhibition assay was established to evaluate targeted covalent inhibitors (TCIs) using wild-type PI4Kβ and the PI4Kβ C1327A mutant. A distinct decrease in wild-type PI4Kβ IC50 was observed with increasing enzyme-inhibitor pre-incubation time for inhibitors containing chloroacetamide and acrylamide warheads. In contrast, PI4Kβ C1327A IC50 values were independent of enzyme-inhibitor pre-incubation time supporting C1327- mediated covalent inhibition. Mass spectrometry was used to confirm covalent modification of the targeted cysteine residue and to assess the rate of covalent bond formation. These assays provide valuable insights, which can be used to guide the optimisation of PI4Kβ inhibitors and the choice of warheads for TCIs.
- ItemOpen AccessKinetics of β-haematin formation in benzoic acid(2005) Tshivhase, Mmboneni Gifty; Egan, Timothy JHaemozoin is a cyclic dimer of ferriprotoporphyrin (IX) that occurs in malariaparasites and some other blood-eating organisms. It is chemically identical to β-haematin. In this work, the kinetics of β-haematin formation were investigated in 0.05 M benzoic acid acid solution using a newly developed pyridine-based assay.
- ItemRestrictedThe mechanism of beta-hematin formation in acetate solution. Parallels between hemozoin formation and biomineralization processes(American Chemical Society, 2001) Egan, Timothy J; Mavuso, Winile W; Ncokazi, Kanyile KFormation of â-hematin in acidic acetate solution has been investigated using quantitative infrared spectroscopy, X-ray diffraction, and scanning and transmission electron microscopy. The process occurs via rapid precipitation of amorphous (or possibly nanocrystalline) hematin, followed by slow conversion to crystalline â-hematin. Definitive evidence that the reaction occurs during incubation in acetate medium, rather than during the drying stage, is provided by X-ray diffraction and infrared spectroscopy of the wet material. The reaction follows a sigmoidal function indicative of a process of nucleation and growth and was modeled using the Avrami equation. Reaction rates and the dimensionality of growth (as indicated by the value of the Avrami constant) are strongly influenced by stirring rate. The reaction follows Arrhenius behavior, and there is a strong dependence of both the rate constant and the Avrami constant on acetate concentration. Acetate may act as a phase transfer catalyst, solubilizing hematin and facilitating its redeposition as â-hematin. The pH dependence of the process indicates that only the monoprotonated species of hematin is active in forming â-hematin. The formation of â-hematin closely parallels many mineralization processes, and this suggests that hemozoin formation may be a unique biomineralization process. Inferences are drawn with respect to the formation of hemozoin in vivo.
- ItemOpen AccessPhysicochemical, biological and β-haematin inhibiting activity of pyrido-dibemequines, pyrido[1,2-α]benzimidazoles and their derivatives(2018) Okombo, Ojwang' John; Egan, Timothy J; Chibale, KellyThere is an urgent need for new antimalarials following the emergence of Plasmodium falciparum strains with reduced sensitivity to the currently used artemisinin combination therapies. Classical aminoquinoline-based drugs inhibit the formation of haemozoin (HZ) thereby causing parasite death from the cellular accumulation of toxic 'free' haem. Coincidentally, this immutable pathway also exists in Schistosoma mansoni, and presents a vulnerable target for drug design in these haematophagus organisms. Therefore, it would be of interest to explore novel scaffolds that can inhibit HZ formation as well as exploit the merits of established drugs via structural modifications that would harness their pharmacological and pharmacokinetic advantages while circumventing their therapeutic shortcomings. This project investigated the physicochemical, biological and mechanistic profiles of pyrido-dibemequine (pDBQ) and pyrido[1,2-α]benzimidazole (PBI) derivatives whose structural motifs were informed by previously synthesised prototype molecules. Specifically, the aqueous solubility, membrane permeability, lipophilicity, metabolic stability and potential for cardiotoxicity of seven pDBQs, their metabolites and ten PBIs were tested through computational and experimental methods. In addition, their antiplasmodial and antischistosomal activities were determined and correlated with their respective physicochemical properties. As regards mechanistic evaluation, their ability to inhibit formation of abiotic HZ, β-haematin (βH), was assessed and intracellular inhibition of HZ formation probed. The pDBQs constitute reversed chloroquines with a 4-aminoquinoline nucleus hybridised to a dibenzylmethylamine side group that serves as a chemosensitising moiety. The pDBQ derivatives showed moderate to high solubility (52 - 197 μM) and permeability (LogPₐₚₚ: -4.6 - -3.6) at pH 6.5. Their lipophilicity, indexed by cLogP, ranged between 3.7 and 5.6 while the mean LogD at both cytosolic (7.4) and vacuolar (5.0) pH was 3.15 and 0.93, respectively. The compounds also showed low-nanomolar range antiplasmodial activity against both chloroquine (CQ)-sensitive (CQS) and resistant (CQR) strains (IC₅₀ range CQS: 14.4 - 126.6 nM, CQRᴰᵈ²: 44.5 - 162 nM and CQR⁷ᴳ⁸: 69.6 - 307.1 nM), with no discernible cross-resistance with CQ and the antiplasmodial activity directly correlated with lipophilicity. Mechanistically, all the pDBQs inhibited βH formation (IC₅₀: 13 - 25 μM) and haem-pyridine fractionation profiles revealed they produced a CQ-like dose-dependent increase in toxic 'free' haem with corresponding decrease in HZ levels. Predicted human-Ether-a-Go-Go-Related Gene (hERG) channel inhibition pIC₅₀ ranged between 6.2 and 6.6, and correlated strongly with the cLogP and molecular weight. The derivatives were also highly susceptibility to microsomal metabolism, with N-dealkylation identified as the main biotransformation route. The pDBQ metabolites exhibited solubility and membrane permeability profiles similar to the parent compounds at pH 6.5, albeit with reduced lipophilicity (cLogP range: 2.3 - 3.5). Their βH inhibition activity (IC₅₀: 15 - 24 μM) was also comparable to the parent compounds as were the haem-pyridine fractionation profiles. However, they showed greater antiplasmodial activity, with 4/7 derivatives exhibiting IC₅₀ < 80 nM against PƒDd2 (CQR strain). The metabolites had reduced hERG channel inhibition potential (pIC₅₀: 5.0 - 5.7) and significantly improved metabolic stability upon incubation with mouse and human liver microsomes. The PBIs comprise molecules with structural likeness to CQ, including a planar heterocyclic moiety and a basic amine side group. PBI analogues showed low to moderate solubility (<5 - 80 μM) and were moderately lipophilic (mean LogD7.4: 3.04). Although most of the derivatives were stable in liver microsomes, their predicted hERG channel inhibition potential was higher (pIC₅₀: 6.11 - 7.50), presumably due to their high molecular weights. All but one derivative had submicromolar activity against CQS and CQR strains, with analogues bearing halo-substituents on the left of the PBI core showing the best antiplasmodial activity (mean IC₅₀: CQS = 26.7 nM and CQR = 30.0 nM), highest selectivity (188 - 341) as well as complete cures in P. berghei-infected mice. The PBIs also inhibited βH formation (IC₅₀: 6.8 - 120 μM) but did not all display intracellular inhibition of HZ formation. All derivatives were active against juvenile (mean IC₅₀: 1.97 μM) and adult (mean IC₅₀: 4.38 μM) schistosomes, with the 3, 4-dichloro-substituted analogue exhibiting 48% reduction of worm burden in vivo. In summary, the pDBQs evaluated in this project constitute potent antiplasmodial inhibitors of HZ formation but whose activity is compromised by metabolic and hERG liability while their metabolites seem to possess improved biological and physicochemical features. The observed activity of the PBIs against P. falciparum and S. mansoni complements the already-established broad antimicrobial potency of this chemotype.
- ItemRestrictedQuinoline antimalarials decrease the rate of beta-hematin formation(Elsevier, 2005) Egan, Timothy J; Ncokazi, Kanyile KThe strength of inhibition of b-hematin (synthetic hemozoin or malaria pigment) formation by the quinoline antimalarial drugs chloroquine, amodiaquine, quinidine and quinine has been investigated as a function of incubation time. In the assay used, b-hematin formation was brought about using 4.5 M acetate, pH 4.5 at 60 C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit b-hematin formation when relatively short incubation times are used, it was found that b-hematin eventually forms with longer incubation periods (8 h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC50 for inhibition of b-hematin formation increases markedly with increasing incubation times in the case of the 4-aminoquinolines chloroquine and amodiaquine. By contrast, in the presence of the quinoline methanols quinine and quinidine the IC50 values increase much more slowly. This results in a partial reversal of the order of inhibition strengths at longer incubation times. Scanning electron microscopy indicates that b-hematin crystals formed in the presence of chloroquine are more uniform in both size and shape than those formed in the absence of the drug, with the external morphology of these crystallites being markedly altered. The findings suggest that these drugs act by decreasing the rate of hemozoin formation, rather than irreversibly blocking its formation. This model can also explain the observation of a sigmoidal dependence of b-hematin inhibition on drug concentration.
- ItemOpen AccessThe role of haem in the mechanism of action of antimalarials in Plasmodium falciparum(2016) Combrinck, Jill Michelle; Egan, Timothy J; Smith, PeterThe malaria parasite detoxifies host red blood cell derived haem by conversion into the inert biocrystal haemozoin. Inhibiting this critical pathway is proposed to be the mechanism of action of chloroquine and related antimalarials and several studies have linked inhibition of the formation of synthetic haemozoin, β-haematin, to parasite survival. However, haemozoin inhibition with a dose related increase in "free" haem correlated to decreased survival has not been demonstrated in the parasite. This project investigated the role of haem in the mechanism of action of several clinically relevant and novel antimalarials in the malaria parasite, Plasmodium falciparum.
- ItemOpen AccessThe role of lipid in malaria pigment (Haemozoin) formation under biomimetic conditions(2013) Ambele, Melvin Anyasi; Egan, Timothy JPrevious studies have proposed lipids and/or proteins to be involved in the biomineralization process of haemozoin formation in the malaria parasite which is a target for many antimalarial drugs. This study therefore investigated the biomolecules involved and established the role of lipids in mediating Hz formation under biomimetic conditions. Hz crystals were isolated from mature trophozoites obtained from saponin-lysed iRBCs. Trophozoites were triturated to release digestive vacuoles and subjected to freeze/thaw cycles to release Hz. Supernatant from SDS-washed Hz crystals and solution of dissolved crystals in NaOH were used for SDS PAGE and LC-MS/MS protein identification. None of the proteins HRPII, HDP, or those belonging to the proposed protein complex previously suggested to be involved in Hz formation in vivo, were identified in the proteome associated with Hz crystals. In addition, no 'stand out' proteins with expected Hz-mediating activity in the malaria parasite were identified. Contrastingly, putative lipoproteins and uncharacterized proteins with roles in lipid binding and transport were identified for the first time in the proteome associated with Hz crystals. Lipids consisting of MSG, MPG, DPG, DLG and DOG (NLB) previously reported to be associated with Hz crystals were easily identified to be associated with Hz crystals. Lipid was also found to be in vastly greater abundance than proteins in the Hz crystal. The absence, therefore, of all proteins previously suggested to be involved in Hz formation from the proteome associated with the Hz crystal, coupled with the identification of lipoproteins and the easily detected abundance of lipids with Hz-mediating activity, strongly suggest that lipids are the biomolecules responsible for the biomineralization of Hz formation in vivo. A solution mixture of NLB and haematin both prepared in acetone/methanol deposited on citrate buffer (50 mM, pH 4.8, 37oC), was allowed to incubate at 37oC for different lengths of time to investigate β-haematin formation. The product formed was confirmed by FT-IR to be β-haematin. The reaction was very fast giving yields of about 90% at 2.15 and 1.07 lipid-tohaem mol ratios and was completed in less than 10 min. The reaction only slowed when there were two haem molecules to one fatty acid chain, but still gave yields of about 90% in under 30 min. On the other hand, this slower kinetics allowed for the effects of relevant biological ions on the process to be investigated. Relevant biological ions at concentrations which they exist in RBC and/or blood are unlikely to greatly affect the lipid mediated process of Hz formation in vivo as only a 2.3 fold decrease in kinetics was observed in the presence of these ions. β-Haematin formed over time during the reaction showed an increase in fluorescence measured using confocal laser microscopy. This qualitatively corresponds to that of bulk kinetic measurements and also demonstrates the formation of well-ordered crystals in situ in association with the lipid component of the emulsion. However, the β-haematin crystals formed seemed to have a distinct external morphology different from Hz itself and thus this was investigated further. Solutions of NLB and haematin in acetone/methanol were mixed by means of vortexing, ultrasonication or micromixing before being used for β-haematin formation reactions. The β-haematin crystals formed at the emulsion layer as well as lipid droplets were characterized by TEM, TEM-ED and ESI/EELS and were compared to Hz. β-Haematin crystals formed from the vortexed mixture appeared to be smaller in size with external morphology and hkl indices different from Hz while ultrasonication or micromixing produced very long β-haematin crystals that are identical to Hz both in overall crystal habit and form. An observed difference in texture between the β-haematin crystals and Hz was likely due to the presence of proteins and lipids on the Hz crystal as shown in the proteomic and lipid studies of this work. The external morphology of the β-haematin formed was not affected by the NLB-to-haematin ratio at any given time nor by the kinetics of the crystal growth process. β-Haematin crystals formed at the emulsion layer were always seen to make contact with non-hollow lipid droplets with continuous hydrophobic interiors via their {100} faces. The lipid droplets in contact with crystals were found to control the length of the crystal. Growth stopped when the angle of contact between the lipid and crystal reached approximately 38o. The invariable observation of β-haematin crystals with their {100} face in contact with lipid droplets that control their sizes strongly suggests that lipids act as the nucleator, providing a favourable microenvironment suitable for the interaction with the propionic acid groups at the {100} surface of the crystal via the glycerol –OH group of the lipid. The proposal that lipids are the sole biomolecules responsible for Hz formation in vivo is strongly supported by the following findings: Firstly, all the proteins previously suggested to be involved in Hz formation were absent from the proteome associated with Hz. Interestingly, it was lipids that were found to be in relative abundance over these proteins in the Hz crystal. Secondly, these lipids were highly efficient in mediating β-haematin formation under biomimetic conditions producing crystals that were identical in overall habit and form to that of Hz itself. Finally, the lipid droplets invariably observed in contact with the crystals, controls their size and probably nucleate crystal growth via their {100} faces.