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PubMed1999

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Animals↗
PubMed2000

Structure-function relationships in aminoquinolines: effect of amino and chloro groups on quinoline-hematin complex formation, inhibition of beta-hematin formation, and antiplasmodial activity.

Comparison of 19 aminoquinolines supports the hypothesis that chloroquine and related antimalarials act by complexing ferriprotoporphyrin IX (Fe(III)PPIX), inhibiting its conversion to beta-hematin (hemozoin) and hence its detoxification. The study suggests that a basic amino side chain is also essential for antiplasmodial activity. 2- And 4-aminoquinolines are unique in their strong affinity for Fe(III)PPIX, and attachment of side chains to the amino group has relatively little influence on the strength of complex formation. Association with Fe(III)PPIX is necessary, but not sufficient, for inhibiting beta-hematin formation. Presence of a 7-chloro group in the 4-aminoquinoline ring is a requirement for beta-hematin inhibitory activity, and this is also unaffected by side chains attached to the amino group. In turn, beta-hematin inhibitory activity is necessary, but not sufficient, for antiplasmodial activity as the presence of an aminoalkyl group attached to the 4-amino-7-chloroquinoline template is essential for strong activity. We thus propose that the 4-aminoquinoline nucleus of chloroquine and related antimalarials is responsible for complexing Fe(III)PPIX, the 7-chloro group is required for inhibition of beta-hematin formation, and the basic amino side chain is required for drug accumulation in the food vacuole of the parasite.

Aminoquinolines↗
PubMed1999

A novel endogenous antimalarial: Fe(II)-protoporphyrin IX alpha (heme) inhibits hematin polymerization to beta-hematin (malaria pigment) and kills malaria parasites.

The polymerization of hemoglobin-derived ferric-protoporphyrin IX [Fe(III)PPIX] to inert hemozoin (malaria pigment) is a crucial and unique process for intraerythrocytic plasmodia to prevent heme toxicity and thus a good target for new antimalarials. Quinoline drugs, i.e., chloroquine, and non-iron porphyrins have been shown to block polymerization by forming electronic pi-pi interactions with heme monomers. Here, we report the identification of ferrous-protoporphyrin IX [Fe(II)PPIX] as a novel endogenous anti-malarial. Fe(II)PPIX molecules, released from the proteolysis of hemoglobin, are first oxidized and then polymerized to hemozoin. We obtained Fe(II)PPIX on preparative scale by electrochemical reduction of Fe(III)PPIX, and the reaction was monitored by cyclic voltammetry. Polymerization assays at acidic pH were conducted with the resulting Fe(II)PPIX using a spectrophotometric microassay of heme polymerization adapted to anaerobic conditions and the products characterized by infrared spectroscopy. Fe(II)PPIX (a) did not polymerize and (b) produced a dose-dependent inhibition of Fe(III)PPIX polymerization (IC(50) = 0.4 molar equiv). Moreover, Fe(II)PPIX produced by chemical reduction with thiol-containing compounds gave similar results: a dose-dependent inhibition of heme polymerization was observed using either L-cysteine, N-acetylcysteine, or DL-homocysteine, but not with L-cystine. Cyclic voltammetry confirmed that the inhibition of heme polymerization was due to the Fe(II)PPIX molecules generated by the thiol-mediated reduction of Fe(III)PPIX. These results point to Fe(II)PPIX as a potential endogenous antimalarial and to Fe(III)PPIX reduction as a potential new pharmacological target.

Animals↗
PubMed1983

The inactivation of hemostatic factors by hematin.

Prolonged clotting times and reduced levels of clotting factors have been reported in hematin-treated patients. This effect persists for up to 5 hr after hematin infusion, associated with plasma levels ranging from 0.01 to 0.04 mg/ml. Therefore we performed in vitro studies to investigate the effects of hematin on fibrinogen, thrombin, factor VIII:C, and plasmin. Hematin in a final concentration of 0.01 mg/ml inhibited the clotting of bovine fibrinogen (1.3 to 2.6 mg/ml) by bovine thrombin (0.12 U/ml) and inhibited the hydrolysis of a synthetic substrate by human thrombin. However, if the hematin was first mixed with albumin (25 mg/ml), fourfold higher concentrations were required to prolong the thrombin clotting time. Hematin, 0.035 mg/ml, reduced VIII:C activity from 0.88 to 0.40 U/ml as measured by two-stage assay. Hematin (0.05 mg/ml) also inhibited the activation of VIII:C by thrombin (0.04 U/ml): baseline activity, 0.84 U/ml; thrombin-activated, 2.94 U/ml; with hematin added, 1.33 U/ml. Hematin also inhibited clot lysis. The inclusion of hematin (0.03 mg/ml) in the diluting buffer reduced the lysis of whole blood clots from 86% +/- 5 to 23% +/- 5 (p less than 0.001, mean +/- S.D. of four determinations) and decreased the lysis of 125I-fibrin clots induced by plasmin (0.02 CTA U/ml) from 100% to 27%. In concentrations as low as 0.09 microgram/ml, hematin inhibited the hydrolysis of a synthetic substrate by plasmin. Hematin was mixed with fibrinogen, albumin, or thrombin, and the mixtures applied to Sephadex G-200 columns. Adherence of the hematin to Sephadex was prevented by either prerinsing the column with albumin or using borate buffer at pH 9.2. Hematin co-eluted with each protein applied to the column and, in the case of fibrinogen, altered its electrophoretic mobility and markedly prolonged the thrombin clotting time of the eluted fibrinogen. We conclude that hematin binds to a variety of hemostatic proteins, inhibiting their biologic activity.

Animals↗
PubMed1999

Deferoxamine: stimulation of hematin polymerization and antagonism of its inhibition by chloroquine.

The iron chelator deferoxamine enhances the clearance of Plasmodium falciparum parasitemia and may be useful in drug combinations for the treatment of cerebral malaria. However, the deferoxamine-chloroquine drug combination is antagonistic, or at best additive, against P. falciparum in vitro. As chloroquine is thought to exert its antimalarial activity by interacting with hematin released from the proteolytic degradation of hemoglobin in the parasite food vacuole, we hypothesized that deferoxamine might interfere with the ability of chloroquine to inhibit hematin polymerization, since it was reported that deferoxamine interacts with hematin. Therefore, we assessed deferoxamine-hematin binding in more detail and investigated the effect of deferoxamine on hematin polymerization in the presence and absence of chloroquine. Isothermal titration calorimetry (ITC) experiments demonstrated an enthalpy-driven deferoxamine:hematin mu-oxo dimer binding with an association constant of 2.8 x 10(4) M(-1) at pH 6.5, a binding affinity 14-fold lower than that measured for chloroquine. At least two of the three hydroxamic acid functional groups of deferoxamine must be unionized for effective binding. We also discovered that deferoxamine antagonized chloroquine-mediated inhibition of hematin polymerization. Unexpectedly, deferoxamine increased the concentration of soluble forms of hematin and enhanced the rate of hematin polymerization. Deferoxamine also could initiate hematin polymerization. In contrast, chloroquine decreased the concentration of soluble forms of hematin and inhibited hematin polymerization. This work supports the postulate that initiation of hematin polymerization requires a higher concentration of soluble hematin monomer than does the elongation phase of polymerization and provides one possible explanation for the observed antagonism between deferoxamine and chloroquine against parasites in culture.

Animals↗
PubMed2000

Characterization of chloroquine-hematin mu-oxo dimer binding by isothermal titration calorimetry.

Numerous studies indicate that a key feature of chloroquine's (CQ) antimalarial activity is its interaction with hematin. We now characterize this CQ-hematin interaction in detail using isothermal titration calorimetry (ITC). Between pH 5.6 and 9.0, association constants (K(a) values) for enthalpy-driven CQ-hematin mu-oxo dimer binding fell in the narrow range of 2.3-4.4 x 10(5) M(-1). It is therefore probable that CQ-hematin mu-oxo dimer binding affinity does not diminish at the pH range (4.8-5.4) of the parasite food vacuole. The binding affinity was unaffected by high salt concentrations, suggesting that ionic interactions do not contribute significantly to this complexation. With increasing ionic strength, the entropic penalty of CQ-hematin mu-oxo dimer binding decreased accompanied by increased hematin mu-oxo dimer aggregation. A stoichiometry (n) of 1:4 in the pH range 6.5-9.0 indicates that CQ binds to two hematin mu-oxo dimers. At pH 5.6, a stoichiometry of 1:8 suggests that CQ binds to an aggregate of four hematin mu-oxo dimers. This work adds further evidence supporting the hypothesis that CQ impedes hematin monomer incorporation into hemozoin by producing a forward shift in the hematin monomer-hematin mu-oxo dimer equilibrium, contributing to a destructive accumulation of soluble forms of hematin in the parasite and leading to its death by hematin poisoning.

Antimalarials↗
PubMed2002

Hematin is one of the cytotoxic factors in pancreatitis-associated ascitic fluid that causes hepatocellular injury.

BACKGROUND: We recently demonstrated that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s), inducing apoptosis in hepatocytes, and that PAAF induces hepatic adenosine triphosphate depletion, hepatocellular acidosis, and accumulation of hepatic intracellular sodium. Because ascitic fluid and serum from patients with hemorrhagic pancreatitis contain a lot of hematin, we aimed to test the hypothesis that hematin can induce hepatocellular injury, and then we compared its cytotoxicity with that of PAAF. METHODS: In vivo effects of intraperitoneal injection of hematin into the liver of healthy rats were evaluated with in situ nick-end labeling, blood biochemical analysis, and nuclear magnetic resonance spectroscopy. In vitro cytotoxic and apoptosis-inducing activities of hematin on rat primary culture hepatocytes were investigated with a cellular proliferation assay kit and DNA fragmentation enzyme-linked immunosorbent assay, respectively. Furthermore, PAAF was fractionated with Sephacryl S-300 gel column chromatography, and cytotoxic activities of its fractions on a human hepatoma cell line (HuH-7) were compared with those of hematin. RESULTS: Intraperitoneal injection of hematin into healthy rats caused apoptosis in the hepatocytes and elevated serum glutamate oxaloacetic transaminase and lactate dehydrogenase levels. Intraperitoneal injection of hematin also caused a significant decrease in the hepatic beta-adenosine triphosphate/inorganic phosphate ratio, severe hepatic intracellular acidosis, and a significant increase of hepatic intracellular sodium (Na(+)) concentration, similar to the effects of PAAF. In vitro, hematin decreased hepatocyte viability and increased the DNA fragmentation of hepatocytes, similar to the effects of 10% PAAF. Albumin reversed the cytotoxic effects of hematin and PAAF on HuH-7 cells nearly completely and partially, respectively. Fractionation of PAAF and hematin by gel column chromatography revealed that the first peak of cytotoxic activity of PAAF corresponded to that of hematin and that the cytotoxic activity was reversed by albumin nearly completely. CONCLUSIONS: These results suggest that hematin is one of the cytotoxic factors in PAAF that causes hepatocellular injury and that cellular injuries caused by hematin may be involved in the development of multiple organ failure associated with severe acute pancreatitis.

Albumins↗
PubMed1984

Effect of hematin on endothelial cells and endothelial cell-platelet interactions.

Hematin, a therapeutic agent for acute porphyrias, induces a coagulopathy characterized by thrombocytopenia and prolonged clotting times. In vitro, it activates platelets and inhibits thrombin and plasmin. Hematin has also been shown to participate in the activation of microsomal cyclooxygenase. The apparent diversity of hematin's effects on multiple hemostatic components as well as its role in prostaglandin synthesis led the authors to investigate the effects of hematin on endothelial cells and endothelial cell-platelet interactions. Marked morphologic alterations of cultured bovine aortic endothelial cells ( BAECs ) were noted after exposure to as little as 2 micrograms/ml hematin in HEPES buffer or to 40 micrograms/ml of hematin in plasma (a level achieved during hematin therapy). These changes included cell retraction and surface vesiculation. Despite the apparent severity of the changes, there was no associated cell lysis or detachment. Furthermore, the cells resumed their cobblestone appearance after hematin had been removed. Platelet adhesion to hematin-treated monolayers was significantly increased, especially when low concentrations of hematin were included in the platelet suspensions. Prostacyclin production was not increased by BAECs exposed to hematin, presumably because of the inaccessibility of hematin to cyclooxygenase in intact cells. The endothelial changes observed in tissue culture are consistent with the clinical observations of phlebitis at the injection site and provide an additional explanation for the thrombocytopenia in patients receiving hematin therapy.

Animals↗
PubMed1986

Hematin-derived anticoagulant. Generation in vitro and in vivo.

Prolongation of clotting times produced by hematin was investigated both in vitro and in vivo. Hematin-derived anticoagulant (HDA) was found to be due to a degradative product or derivative of hematin, and was generated in vitro in standing (aging) aqueous solutions of the parent compound. Generation of HDA in vitro was inhibited by antioxidants. The anticoagulant effect of HDA was inhibited by freshly prepared hematin, fresh Sn-protoporphyrin, imidazole, or the iron chelator desferrioxamine. Ferrioxamine did not inhibit HDA, and inhibition by imidazole was reversed with ferric citrate, suggesting a role for iron in the mechanism of HDA activity. HDA activity was dissociated from hematin in plasma by clotting with thrombin. HDA segregated into the clot fraction, whereas hematin remained largely in the serum fraction, suggesting that HDA may preferentially bind to fibrinogen. TLC and HPLC showed a single peak of HDA activity that was not associated with the parent compound. Evidence for HDA generation in vivo was found when rats were injected with fresh (no HDA) hematin. Prolongation of clotting times appeared after hematin appeared in the plasma, and anticoagulant activity persisted after a fall in plasma hematin concentration. Thus, there was a temporal dissociation between hematin and HDA, suggesting that a modification of hematin must occur in vivo before an anticoagulant effect is produced. Generation of HDA in vitro has implications for hematin preparation and administration. Generation of HDA in vivo suggests that similar modifications of endogenous heme or other porphyrins may occur to produce HDA under physiologic or pathophysiologic conditions.

Anticoagulants↗
PubMed1986

The interaction of the factor VIII/von Willebrand factor complex with hematin.

Intravenous infusion of hematin, used in the treatment of acute porphyria, induces a decline in the plasma factor VIII/von Willebrand factor complex (VIII/vWF) and thrombocytopenia. We investigated this problem by studying the interaction between hematin, purified VIII/vWF, and platelets in vitro. Hematin was labeled with either 59Fe or 3H and characterized by gel chromatography. Hematin self-aggregated, forming a complex with an average molecular weight of approximately 10,000 daltons. When incubated with VIII/vWF for 30 min at 37 degrees C and applied to Sepharose CL-4B, the hematin eluted with the VIII/vWF in the void volume. Hematin inhibited the dissociation of factor VIII antigen (VIII:Ag) from the von Willebrand antigen (vWF:Ag) in 0.25 M CaCl2, and reversed the aggregation of VIII:Ag induced by 0.1 M 6-aminocaproic acid. Both hematin and the hematin-VIII/vWF complex bound to washed normal platelets and to platelets from a patient with Bernard-Soulier syndrome. Thrombasthenic platelets were not aggregatable by hematin, and bound significantly less hematin-VIII/vWF than normal platelets suggesting that hematin-induced platelet activation was required for binding. Likewise, binding was inhibited by PGE1 which also prevented aggregation. We conclude that hematin forms complexes with VIII/vWF, alters the functional activity and dissociation of this compound, and participates in the binding of VIII/vWF to platelets.

Blood Platelets↗
PubMed1991

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes.

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.

Benzene Derivatives↗
PubMed1986

Cytosolic activation of hematin-dependent microsomal monooxygenase activity in the lung.

Additions of micromolar concentrations of hematin to washed rat pulmonary microsomal preparations resulted in marked (5-7-fold) increases in the NADPH-dependent generation of phenolic metabolites of benzo[a]pyrene (BaP). 9-Hydroxy-BaP was identified as the major reaction product. Additions of pulmonary cytosolic fractions to microsomes produced no measurable effect but cytosol and hematin added together elicited 25-30-fold increases in total phenolic products. Cytosolic fractions from other tissues, including rat kidneys and perfused rat livers, were also highly effective in enhancing the hematin-mediated increases in monooxygenase activity. However, cytosol from human placental tissues was only minimally effective when either pulmonary or placental microsomes were utilized as enzyme source. Superoxide dismutase and catalase (alone or in combination) had no measurable effect on hematin-mediated increases. Horseradish peroxidase effectively inhibited the hematin-dependent reactions but hematin-independent reactions were inhibited with equal effectiveness. Carbon monoxide profoundly inhibited all hematin-mediated increases in metabolite formation. The activating cytosolic component was non-dialyzable, inactivated by trypsin and heat, and eluted in the void volume from Sephadex G-150 columns. This suggested that the cytosolic factor(s) responsible for the increased hematin-dependent oxidation was a protein(s) with a high molecular weight or perhaps an aggregate or oligomer of proteinaceous material. HPLC profiles indicated a major effect on the generation of phenolics; quinones were also increased but only minimal increases in diols were observed. Results were consistent with the hypothesis that hematin-mediated increases in pulmonary monooxygenase activity result from an increased association of a small pool of pulmonary P-450-apoprotein(s) with the hematin prosthetic group to result in increased levels of an unidentified holocytochrome(s) with a relatively high substrate turnover number. The current data suggest a quaternary interaction among P-450 apoprotein(s), heme prosthetic group, reaction products (particularly 3-hydroxy-BaP) and a cytosolic protein(s). We postulate that the mechanism of action of the cytosolic factor is to facilitate the interaction of hematin with the apocytochrome.

Animals↗
PubMed1997

Investigations of B- and beta-hematin.

The preparation from ferriprotoporphyrin IX (FP) in aqueous acid medium of the related pigments beta- and B-hematin [see G. Blauer and M. Akkawi, Biochem. Mol. Biol. Int. 35, 231 (1995)] is presented under different conditions. Both pigments are characterized by infrared spectra which differ in the range of 1600-1700 cm-1 in their strong bands with absorption peaks measured at 1648 +/- 2 cm-1 for B-hematin and at 1663 +/- 1 cm-1 for beta-hematin. The pH dependence of B-hematin formation at 37 degrees C and at different concentrations of acetic acid and FP exhibits a maximum yield near pH 4. The formation of beta-hematin at 70 degrees C shows high yield in 6 M acetic acid or in the presence of 0.028 M trichloroacetate at pH 4.6. The dependence of the yield of the pigments on the time and temperature of incubation, concentration of FP, and the presence of different electrolytes was investigated. Both B- and beta-hematin are either insoluble or very slightly soluble in different solvents at room temperature, and appear to dissociate into regular FP in strongly alkaline aqueous medium. In the presence of different quinoline-based drugs, the formation of both B- and beta-hematin at pH 4-5 is inhibited. Under certain conditions, the effect of added carboxylic acids on pigment formation is suggested to be due, at least in part, to the prevention of initial hydrogen bonding among FP carboxyl groups. For both B- and beta-hematin, branched and cyclic macromolecular structures are proposed involving linkages between an FP iron and a side-chain carboxylate group of another FP, in addition to hydrogen bonds between FP carboxyl groups. B- and beta-hematin are assumed to differ in molecular weight and the extent of bond formation. Possible mechanisms for beta-hematin production from B-hematin and certain relations between the synthetic pigments and the malaria pigment are suggested.

Amodiaquine↗
PubMed2001

On the mechanism of hemozoin production in malaria parasites: activated erythrocyte membranes promote beta-hematin synthesis.

The ferriprotoporphyrin IX (FP) molecules released by intraerythrocytic malaria parasites during hemoglobin digestion are converted to beta-hematin and are stored in the parasites' food vacuoles. It has been demonstrated in cell-free medium that the incorporation of FP into beta-hematin under physiological conditions requires a catalyst from parasite lysates or pre-formed beta-hematin. In the present studies, lysates of Plasmodium falciparum-infected erythrocytes were suspended in 1 M NaOH and were washed with phosphate buffer, pH 7.6. When the cell extracts were incubated with hematin in 0.5 M sodium acetate buffer, pH 5, for 20 hr at 37 degrees C, a large quantity of beta-hematin was formed. To determine whether parasite components were necessary for the beta-hematin formation, normal erythrocyte ghosts were similarly treated with 1 M NaOH and then incubated with hematin. In repeated experiments it was found that, on the average, 70% of the hematin was converted to beta-hematin. Membranes treated with HCl or CH(3)COOH also promoted the formation of beta-hematin, while untreated membranes were ineffective. The possibility that metabolic activities in the food vacuoles of malaria parasites may activate membrane fragments, from hemoglobin vesicles, to promote beta-hematin formation is discussed in this paper.

Animals↗
PubMed2005

A colorimetric high-throughput beta-hematin inhibition screening assay for use in the search for antimalarial compounds.

Antimalarial drugs such as chloroquine are believed to act by inhibiting hemozoin formation in the food vacuole of the malaria parasite. We have developed a new assay for measuring and detecting inhibition of synthetic hemozoin (beta-hematin) formation. Aqueous pyridine (5% v/v, pH 7.5) forms a low-spin complex with hematin but not with beta-hematin. Its absorbance obeys Beer's law, making it useful for quantitating hematin concentration in hematin/beta-hematin mixtures, allowing compounds to be investigated for inhibition of beta-hematin formation. The assay is rapid (60 min incubation) and requires no centrifugation. The beta-hematin inhibition data show good agreement with alternative assay methods reported by four laboratories. The assay was adapted for high-throughput colorimetric screening, allowing visual identification of beta-hematin inhibitors. In this mode, the assay successfully detected all 18 beta-hematin inhibitors in a set of 47 compounds tested, with no false positive results. The quantitative in vitro antimalarial activities of a set of 13 aminoquinolines and quinoline methanols were found to correlate significantly with beta-hematin inhibition values determined using the assay.

Animals↗
PubMed2003

Antimalarial 9-anilinoacridine compounds directed at hematin.

Antimalarial 9-anilinoacridines are potent inhibitors of parasite DNA topoisomerase II both in vitro and in situ. 3,6-diamino substitution on the acridine ring greatly improves parasiticidal activity against Plasmodium falciparum by targeting DNA topoisomerase II. A series of 9-anilinoacridines were investigated for their abilities to inhibit beta-hematin formation, to form drug-hematin complexes, and to enhance hematin-induced lysis of red blood cells. Inhibition of beta-hematin formation was minimal with 3,6-diamino analogs of 9-anilinoacridine and greatest with analogs with a 3,6-diCl substitution together with an electron-donating group in the 1'-anilino position. On the other hand, the presence of a 1'-N(CH3)2 group in the anilino ring produced compounds that strongly inhibited beta-hematin formation but which did not appear to be sensitive to the nature of the substitutions in the acridine nucleus. The derivatives bound hematin, and Job's plots of UV-visible absorbance changes in drug-hematin complexes at various molar ratios indicated a stoichiometric ratio of 1:2. The drugs enhanced hematin-induced red blood cell lysis at low concentrations (<4 microM). These studies open up the novel possibility of development of 9-anilinoacridine antimalarials that target not only DNA topoisomerase II but also beta-hematin formation, which should help delay the rapid onset of resistance to drugs acting at only a single site.

Amsacrine↗
PubMed1983

Hematin: unique effects of hemostasis.

Hematin is clinically useful in the treatment of acute intermittent porphyria. Recently, hematin-induced coagulopathy has been reported, and a patient we treated bled during hematin therapy. On 3 separate occasions, infusions of hematin (4 mg/kg) induced thrombocytopenia, prolongation of the prothrombin time, partial thromboplastin time. Reptilase time, and apparent decreases in fibrinogen and increases in fibrin(ogen) degradation products (FDP). However, fibrinogen assayed by heat precipitation was unchanged, the protamine paracoagulation test was negative, there was no red blood cell fragmentation, and plasminogen and antithrombin III remained normal, excluding the presence of disseminated intravascular coagulation. Furthermore, premedication with heparin, 5000 U i.v., failed to prevent the lengthening of the Reptilase time and exacerbated the thrombocytopenia. In vitro studies revealed that hematin, 0.1 mg/ml, aggregated platelets and induced the release of 14C-serotonin and adenosine triphosphate (ATP). Hematin also aggregated washed or gel-filtered platelets but had no effect on formalin-fixed platelets. Aggregation was inhibited by aspirin (0.12 mg/ml), adenosine triphosphate, and apyrase, suggesting that hematin aggregated platelets by inducing adenosine diphosphate (ADP) release. Hematin (0.07 mg/ml) progressively inactivated thrombin and 0.1 mg/ml prolonged the Reptilase time. Thus, hematin is unique in that it both induces platelet aggregation and inhibits coagulation.

Adult↗