Encapsulated metalloporphyrins have been widely studied for their use as efficient heterogeneous ... more Encapsulated metalloporphyrins have been widely studied for their use as efficient heterogeneous catalysts, inspired by the known catalytic activity of porphyrins in haemoproteins. The oxidation of organic substrates by haemoproteins is one of the well-known roles of these proteins, in which the haem (ferriprotoporphyrin IX = FePPIX) cofactor is the centre of reactivity. While these porphyrins are highly efficient catalysts in the protein environment, once removed, they quickly lose their reactivity. It is for this reason that they have garnered much interest in the field of heterogeneous catalysis of oxidation reactions. This review details current research in the field, focusing on the application of encapsulated haem, and other synthetic metalloporphyrins, applied to oxidation reactions.
Antimicrobial agents and chemotherapy, Jan 12, 2018
The rapid spread of strains of malaria parasites resistant to several drugs has threatened global... more The rapid spread of strains of malaria parasites resistant to several drugs has threatened global malaria control. Hence, the aim of this study was to predict the anti-malarial activity of chemical compounds possessing anti-hemozoin formation activity as a new means of anti-malarial drug discovery. After the initialanti-hemozoin formation high-throughput screening (HTS) of 9,600 compounds, a total of 224 hit compounds were identified as hemozoin inhibitors. These 224 compounds were tested forerythrocytic anti-malarial activity at 10 μM using the chloroquine-mefloquine sensitivestrain, 3D7A. Two independent experiments were conducted. The physicochemical properties of the active compounds were extracted from ChemSpider and SciFinder databases. We analyzed the extracted data using Bayesian model averaging (BMA). Our findings revealed that lower numbers of S atoms, lower values of log D pH 3, 4 and 5, and higher values of ACD log D pH 7.4 had a significant association with anti-malaria...
Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as hi... more Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as high-value partners for combination therapies. Here, we report the identification and characterization of hexahydroquinolines (HHQs) that show low nanomolar potency against both pathogenic and transmissible intra-erythrocytic forms of the malaria parasite Plasmodium falciparum. This activity translates into potent transmission-blocking potential, as shown by in vitro male gamete formation assays and reduced oocyst infection and prevalence in Anopheles mosquitoes. In vivo studies illustrated the ability of lead HHQs to suppress Plasmodium berghei blood-stage parasite proliferation. Resistance selection studies, confirmed by CRISPR-Cas9-based gene editing, identified the digestive vacuole membrane-spanning transporter PfMDR1 (P. falciparum multidrug resistance gene-1) as a determinant of parasite resistance to HHQs. Haemoglobin and haem fractionation assays suggest a mode of action that resu...
Current efforts to reduce the global burden of malaria are threatened by the rapid spread through... more Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically...
The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. ... more The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. New therapeutic alternatives targeting critical pathways within the parasite are therefore urgently needed. Haemozoin formation in Schistosoma presents one such target. We assessed in vitro antischistosomal activity of pyrido[1,2-a]benzimidazoles (PBIs), and investigated correlations with their ability to inhibit β-haematin formation. We further evaluated the in vivo efficacy of representative compounds in experimental mice and conducted pharmacokinetic analysis on the most potent. At 10 µM, 48/57 compounds resulted in >70% mortality of newly transformed schistosomula, while 37 of these maintained > 60% mortality of adult S. mansoni. No correlations were observed between β-haematin inhibitory and antischistosomal activities against both larval and adult parasites, suggesting possible presence of other target(s) or a mode of inhibition of crystal formation that is not adequately modelled by the assay. The most active compound in vivo showed 58.7% and 61.3% total and female worm burden reduction, respectively, and further pharmacokinetic analysis hinted at solubility-limited absorption and high hepatic clearance as possible contributors to the modest efficacy despite good in vitro activity. The PBIs evaluated in this report thus merit further optimization to improve their efficacy and to elucidate their possible mode of action.
In a previous study, target based screening was carried out for inhibitors of β-hematin (syntheti... more In a previous study, target based screening was carried out for inhibitors of β-hematin (synthetic hemozoin) formation, and a series of triarylimidazoles were identified as active against Plasmodium falciparum. Here, we report the subsequent synthesis and testing of derivatives with varying substituents on the three phenyl rings for this series. The results indicated that a 2-hydroxy-1,3-dimethoxy substitution pattern on ring A is required for submicromolar parasite activity. In addition, cell-fractionation studies revealed uncommonly large, dose-dependent increases of P. falciparum intracellular exchangeable (free) heme, correlating with decreased parasite survival for β-hematin inhibiting derivatives.
Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pr... more Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southe...
Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, s... more Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, suitable for cellular imaging have been synthesised by attaching the small extrinsic fluorophore, NBD. Interactions of these derivatives with ferriprotoporphyrin IX were evaluated to verify that insights generated by live-cell imaging were relevant to the parent molecules. These analogues are shown by confocal and super-resolution microscopy to accumulate selectively in Plasmodium falciparum. Localisation to the region corresponding to the digestive vacuole supports the putative primary role of these alkaloids as haemozoin inhibitors. Quantitative analysis revealed minimal accumulation within the nucleus, rejecting the disruption of DNA replication as a possible mode of action. While extensive localisation to phospholipid structures and associated organelles was observed, the analogues did not show evidence of association with neutral lipid bodies.
In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodi... more In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono-and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of β-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation, but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification. Intracellular neutral lipid particles or macromolecular lipid assemblies are present in nearly all organisms (1). Generally, these particles function as storage compartments and metabolic † Supported by NIH Grant 1R01AI083145
Kinetics of beta-haematin (synthetic malaria pigment) formation from haematin have been studied i... more Kinetics of beta-haematin (synthetic malaria pigment) formation from haematin have been studied in the presence of aqueous benzoic acid and derivatives of benzoic acid. Formation of the beta-haematin product is demonstrated by X-ray diffraction and IR spectroscopy. Reactions were followed by determining the fraction of unreacted haematin at various time points during the process via reaction of extracted aliquots with pyridine. The kinetics can be fitted to the Avrami equation, indicating that the process involves nucleation and growth. Reaction kinetics in stirred benzoic acid are similar to those previously observed in acetic acid, except that benzoic acid is far more active in promoting the reaction than acetic acid. The reaction reaches completion within 2 h in the presence of 0.050 M benzoic acid (pH 4.5, 60 degrees C). This compares with 1 h in the presence of 4.5 M acetic acid and 4 h in the presence of 2 M acetic acid. The reaction rate in benzoic acid is not affected if the stirring rate is decreased to zero, but very vigorous stirring appears to disrupt nucleation. The rate constant for beta-haematin formation in benzoic acid has a linear dependence on benzoic acid concentration and follows Arrhenius behaviour with temperature. There is a bell-shaped dependence on pH. This suggests that the haematin species in which one propionate group is protonated and the other is deprotonated is optimal for beta-haematin formation. When the reaction is conducted in para-substituted benzoic acid derivatives, the log of the rate constant increases linearly with the Hammett constant. These findings suggest that the role of the carboxylic acid may be to disrupt hydrogen bonding and pi-stacking in haematin, facilitating conversion to beta-haematin. The large activation energy for conversion of precipitated haematin to beta-haematin suggests that the reaction in vivo most likely involves direct nucleation from solution and probably does not occur in aqueous medium.
ABSTRACT (Chemical Equation Presented) The synthesis, cytotoxicity, and antimalarial activity of ... more ABSTRACT (Chemical Equation Presented) The synthesis, cytotoxicity, and antimalarial activity of resistance-reversing bifunctional dihydropyrimidone-chloroquinoline conjugates are reported herein. In vitro assay results indicate this class of compounds is highly active against both chloroquine-resistant and chloroquine-sensitive strains of P. falciparum.
The strength of inhibition of beta-hematin (synthetic hemozoin or malaria pigment) formation by t... more The strength of inhibition of beta-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, beta-hematin formation was brought about using 4.5M acetate, pH 4.5 at 60 degrees C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit beta-hematin formation when relatively short incubation times are used, it was found that beta-hematin eventually forms with longer incubation periods (<8h for chloroquine and >8h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC(50) for inhibition of beta-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 IC(50) 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 beta-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 beta-hematin inhibition on drug concentration.
Nucleation of malaria pigment (hemozoin) and β-hematin (synthetic hemozoin) can be promoted by li... more Nucleation of malaria pigment (hemozoin) and β-hematin (synthetic hemozoin) can be promoted by lipids. We determined, by grazing incidence X-ray diffraction and X-ray reflectivity, that 1-myristoyl-glycerol (MMG), mimicking a digestive vacuole lipid surface, ...
Encapsulated metalloporphyrins have been widely studied for their use as efficient heterogeneous ... more Encapsulated metalloporphyrins have been widely studied for their use as efficient heterogeneous catalysts, inspired by the known catalytic activity of porphyrins in haemoproteins. The oxidation of organic substrates by haemoproteins is one of the well-known roles of these proteins, in which the haem (ferriprotoporphyrin IX = FePPIX) cofactor is the centre of reactivity. While these porphyrins are highly efficient catalysts in the protein environment, once removed, they quickly lose their reactivity. It is for this reason that they have garnered much interest in the field of heterogeneous catalysis of oxidation reactions. This review details current research in the field, focusing on the application of encapsulated haem, and other synthetic metalloporphyrins, applied to oxidation reactions.
Antimicrobial agents and chemotherapy, Jan 12, 2018
The rapid spread of strains of malaria parasites resistant to several drugs has threatened global... more The rapid spread of strains of malaria parasites resistant to several drugs has threatened global malaria control. Hence, the aim of this study was to predict the anti-malarial activity of chemical compounds possessing anti-hemozoin formation activity as a new means of anti-malarial drug discovery. After the initialanti-hemozoin formation high-throughput screening (HTS) of 9,600 compounds, a total of 224 hit compounds were identified as hemozoin inhibitors. These 224 compounds were tested forerythrocytic anti-malarial activity at 10 μM using the chloroquine-mefloquine sensitivestrain, 3D7A. Two independent experiments were conducted. The physicochemical properties of the active compounds were extracted from ChemSpider and SciFinder databases. We analyzed the extracted data using Bayesian model averaging (BMA). Our findings revealed that lower numbers of S atoms, lower values of log D pH 3, 4 and 5, and higher values of ACD log D pH 7.4 had a significant association with anti-malaria...
Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as hi... more Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as high-value partners for combination therapies. Here, we report the identification and characterization of hexahydroquinolines (HHQs) that show low nanomolar potency against both pathogenic and transmissible intra-erythrocytic forms of the malaria parasite Plasmodium falciparum. This activity translates into potent transmission-blocking potential, as shown by in vitro male gamete formation assays and reduced oocyst infection and prevalence in Anopheles mosquitoes. In vivo studies illustrated the ability of lead HHQs to suppress Plasmodium berghei blood-stage parasite proliferation. Resistance selection studies, confirmed by CRISPR-Cas9-based gene editing, identified the digestive vacuole membrane-spanning transporter PfMDR1 (P. falciparum multidrug resistance gene-1) as a determinant of parasite resistance to HHQs. Haemoglobin and haem fractionation assays suggest a mode of action that resu...
Current efforts to reduce the global burden of malaria are threatened by the rapid spread through... more Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically...
The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. ... more The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. New therapeutic alternatives targeting critical pathways within the parasite are therefore urgently needed. Haemozoin formation in Schistosoma presents one such target. We assessed in vitro antischistosomal activity of pyrido[1,2-a]benzimidazoles (PBIs), and investigated correlations with their ability to inhibit β-haematin formation. We further evaluated the in vivo efficacy of representative compounds in experimental mice and conducted pharmacokinetic analysis on the most potent. At 10 µM, 48/57 compounds resulted in >70% mortality of newly transformed schistosomula, while 37 of these maintained > 60% mortality of adult S. mansoni. No correlations were observed between β-haematin inhibitory and antischistosomal activities against both larval and adult parasites, suggesting possible presence of other target(s) or a mode of inhibition of crystal formation that is not adequately modelled by the assay. The most active compound in vivo showed 58.7% and 61.3% total and female worm burden reduction, respectively, and further pharmacokinetic analysis hinted at solubility-limited absorption and high hepatic clearance as possible contributors to the modest efficacy despite good in vitro activity. The PBIs evaluated in this report thus merit further optimization to improve their efficacy and to elucidate their possible mode of action.
In a previous study, target based screening was carried out for inhibitors of β-hematin (syntheti... more In a previous study, target based screening was carried out for inhibitors of β-hematin (synthetic hemozoin) formation, and a series of triarylimidazoles were identified as active against Plasmodium falciparum. Here, we report the subsequent synthesis and testing of derivatives with varying substituents on the three phenyl rings for this series. The results indicated that a 2-hydroxy-1,3-dimethoxy substitution pattern on ring A is required for submicromolar parasite activity. In addition, cell-fractionation studies revealed uncommonly large, dose-dependent increases of P. falciparum intracellular exchangeable (free) heme, correlating with decreased parasite survival for β-hematin inhibiting derivatives.
Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pr... more Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southe...
Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, s... more Fluorescent derivatives of the archetypal antimalarial quinine and its diastereomer, quinidine, suitable for cellular imaging have been synthesised by attaching the small extrinsic fluorophore, NBD. Interactions of these derivatives with ferriprotoporphyrin IX were evaluated to verify that insights generated by live-cell imaging were relevant to the parent molecules. These analogues are shown by confocal and super-resolution microscopy to accumulate selectively in Plasmodium falciparum. Localisation to the region corresponding to the digestive vacuole supports the putative primary role of these alkaloids as haemozoin inhibitors. Quantitative analysis revealed minimal accumulation within the nucleus, rejecting the disruption of DNA replication as a possible mode of action. While extensive localisation to phospholipid structures and associated organelles was observed, the analogues did not show evidence of association with neutral lipid bodies.
In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodi... more In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono-and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of β-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation, but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification. Intracellular neutral lipid particles or macromolecular lipid assemblies are present in nearly all organisms (1). Generally, these particles function as storage compartments and metabolic † Supported by NIH Grant 1R01AI083145
Kinetics of beta-haematin (synthetic malaria pigment) formation from haematin have been studied i... more Kinetics of beta-haematin (synthetic malaria pigment) formation from haematin have been studied in the presence of aqueous benzoic acid and derivatives of benzoic acid. Formation of the beta-haematin product is demonstrated by X-ray diffraction and IR spectroscopy. Reactions were followed by determining the fraction of unreacted haematin at various time points during the process via reaction of extracted aliquots with pyridine. The kinetics can be fitted to the Avrami equation, indicating that the process involves nucleation and growth. Reaction kinetics in stirred benzoic acid are similar to those previously observed in acetic acid, except that benzoic acid is far more active in promoting the reaction than acetic acid. The reaction reaches completion within 2 h in the presence of 0.050 M benzoic acid (pH 4.5, 60 degrees C). This compares with 1 h in the presence of 4.5 M acetic acid and 4 h in the presence of 2 M acetic acid. The reaction rate in benzoic acid is not affected if the stirring rate is decreased to zero, but very vigorous stirring appears to disrupt nucleation. The rate constant for beta-haematin formation in benzoic acid has a linear dependence on benzoic acid concentration and follows Arrhenius behaviour with temperature. There is a bell-shaped dependence on pH. This suggests that the haematin species in which one propionate group is protonated and the other is deprotonated is optimal for beta-haematin formation. When the reaction is conducted in para-substituted benzoic acid derivatives, the log of the rate constant increases linearly with the Hammett constant. These findings suggest that the role of the carboxylic acid may be to disrupt hydrogen bonding and pi-stacking in haematin, facilitating conversion to beta-haematin. The large activation energy for conversion of precipitated haematin to beta-haematin suggests that the reaction in vivo most likely involves direct nucleation from solution and probably does not occur in aqueous medium.
ABSTRACT (Chemical Equation Presented) The synthesis, cytotoxicity, and antimalarial activity of ... more ABSTRACT (Chemical Equation Presented) The synthesis, cytotoxicity, and antimalarial activity of resistance-reversing bifunctional dihydropyrimidone-chloroquinoline conjugates are reported herein. In vitro assay results indicate this class of compounds is highly active against both chloroquine-resistant and chloroquine-sensitive strains of P. falciparum.
The strength of inhibition of beta-hematin (synthetic hemozoin or malaria pigment) formation by t... more The strength of inhibition of beta-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, beta-hematin formation was brought about using 4.5M acetate, pH 4.5 at 60 degrees C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit beta-hematin formation when relatively short incubation times are used, it was found that beta-hematin eventually forms with longer incubation periods (<8h for chloroquine and >8h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC(50) for inhibition of beta-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 IC(50) 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 beta-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 beta-hematin inhibition on drug concentration.
Nucleation of malaria pigment (hemozoin) and β-hematin (synthetic hemozoin) can be promoted by li... more Nucleation of malaria pigment (hemozoin) and β-hematin (synthetic hemozoin) can be promoted by lipids. We determined, by grazing incidence X-ray diffraction and X-ray reflectivity, that 1-myristoyl-glycerol (MMG), mimicking a digestive vacuole lipid surface, ...
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Papers by Timothy Egan