In vitro beta-hematin formation assays with plasma of mice infected with Plasmodium yoelii and other parasite preparations: comparative inhibition with quinoline and endoperoxide antimalarials.

Formation of beta-hematin in vitro could be catalyzed in the presence of various preparations related to the malaria parasite viz., the cell free homogenate of Plasmodium yoelii, lipid extract of the parasite homogenate, purified malarial hemozoin and synthetic beta-hematin. Plasma from mice infected with P. yoelii also catalyzed in vitro beta-hematin formation with highly significant efficiency. The plasma based beta-hematin formation assay was highly sensitive, as the background absorbance was almost negligible due to absence of any preformed hemozoin. The plasma beta-hematin synthesizing activity was recovered in the lipid extract. The quinoline and endoperoxide antimalarials act by inhibiting hemozoin biosynthesis in the malaria parasite. Therefore, the in vitro beta-hematin formation assay is useful for the screening and identification of blood schizontocidal antimalarials acting through interruption of heme detoxification in the parasite. Quinoline and endoperoxide antimalarials showed about three fold greater inhibition of beta-hematin synthesizing activity in the plasma-based assays as compared to that of P. yoelii homogenate-based assays. The specificity of the inhibition was similar in both preparations. The plasma-based assay therefore provides a better alternative than the parasite homogenate-based assay for in vitro screening and identification of novel inhibitors of hemozoin biosynthesis as potential blood schizontocidal antimalarials.     

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.     

Standardization of the physicochemical parameters to assess in vitro the beta-hematin inhibitory activity of antimalarial drugs

Intraerythrocytic plasmodia form hemozoin as a detoxification product of hemoglobin-derived heme. An identical substance, beta-hematin (BH), can be obtained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inhibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al., Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH and salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degrees C, pH = 2.7), several salts including chloride and phosphate inhibited the process. Aminoquinoline drugs formulated as salts (chloroquine-phosphate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at higher pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta-hematin inhibitory activity). By replacing hematin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by salts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and chloroquine-sulfate at pH 5.1. Primaquine was not inhibitory. The final product, characterized by solubility in DMSO, consists of pure BH by FT-IR spectroscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h at 37 degrees C) is designed to screen for those molecules forming pi-pi interactions with hematin and thus inhibiting beta-hematin formation.     

Spectrophotometric determination of de novo hemozoin/beta-hematin formation in an in vitro assay

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.     

Metalloporphyrins inhibit beta-hematin (hemozoin) formation

Metal-substituted protoporphyrin IXs (Cr(III)PPIX (1), Co(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6), and Sn(IV)PPIX (7)) act as inhibitors to beta-hematin (hemozoin) formation, a critical detoxification biopolymer of malarial parasites. The central metal ion plays a significant role in the efficacy of the metalloprotoporphyrins to inhibit beta-hematin formation. The efficacy of these compounds correlates well with the water exchange rate for the octahedral aqua complexes of the porphyrin's central metal ion. Under these in vitro reaction conditions, metalloporphyrins 5, 6 and 7 are as much as six times more efficacious than the free ligand protoporphyrin IX in preventing beta-hematin formation and four times as efficacious as chloroquine, while metalloporphyrins 3 and 4 are three to four times more effective at preventing beta-hematin formation than the free protoporphyrin IX base. In contrast, the relatively exchange inert metalloporphyrins 1 and 2 are only as efficacious as the free ligand and only two-thirds as effective as chloroquine. Aggregation studies of the heme:MPPIX using UV-Vis and fluorescence spectroscopies are indicative of the formation of pi-pi hetero-metalloporphyrin assemblies. Thus, hemozoin inhibition is likely prevented by the formation of heme:MPPIX complexes through pi-stacking interactions. The ramifications of such hetero-metalloporphyrin assemblies, in the context of the emerging structural picture of hemozoin, are discussed.     

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.     

Quinoline antimalarials decrease the rate of beta-hematin formation

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.     

A physiochemical mechanism of hemozoin (beta-hematin) synthesis by malaria parasite.

Malaria parasite homogenate, the lipid extracts, and an unsaturated fatty acid, linoleic acid, which have been shown to promote beta-hematin formation in vitro, were used to investigate the mechanism of hemozoin biosynthesis, a distinct metabolic function of the malaria parasite. In vitro beta-hematin formation promoted by Plasmodium yoelii homogenate, the lipid extracts, and linoleic acid were blocked by ascorbic acid, reduced glutathione, sodium dithionite, beta-mercaptoethanol, dithiothreitol, and superoxide dismutase. Oxidized glutathione did not show any effect. Preoxidized preparations of the lipids extracts or the P. yoelii homogenate failed to catalyze beta-hematin formation. Depletion of oxygen in the reaction mixtures also inhibited the lipid-catalyzed beta-hematin formation. Under the reaction conditions similar to those used for the in vitro beta-hematin formation assay, the antioxidants and reducing agents mentioned above, except the DTT and beta-mercaptoethanol, did not cause degradation of heme. beta-Hematin formation was also inhibited by p-aminophenol, a free radical chain reaction breaker. Hemozoin biosynthesis within the digestive vacuoles of the malaria parasite may be a lipid-catalyzed physiochemical reaction. An oxidative mechanism may be proposed for lipid-mediated beta-hematin formation, which may be mediated by generation of some free radical intermediates of heme.     

The mechanism of beta-hematin formation in acetate solution. Parallels between hemozoin formation and biomineralization processes

Formation of beta-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 beta-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 beta-hematin. The pH dependence of the process indicates that only the monoprotonated species of hematin is active in forming beta-hematin. The formation of beta-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.     

Synthesis, antimalarial activity, structure-activity relationship analysis of thieno-[3,2-b]benzothiazine S,S-dioxide analogs

An improved procedure for the synthesis of 3-amino-9-arylsubstituted-thieno[3,2-b]benzothiazine S,S-dioxide 2-decarboxylated is reported. Thieno-[3,2-b]benzothiazine S,S-dioxide derivatives were investigated for their abilities to inhibit beta-hematin formation, hemoglobin hydrolysis and in vivo for their efficacy in rodent Plasmodium berghei. Compounds 5j-o were the most promising as inhibitors of hemoglobin hydrolysis, however, the compounds are not as efficient as chloroquine. A structure-activity relationship (SAR) study was carried out in this series. Our results allow us to determine the minimal structural requirements to produce the biological response.     

Design, synthesis and antimalarial activity of a glyoxylylhydrazone library

Synthesis of a new family of quinolylhydrazone derivatives and evaluation of their activity against a chloroquine-resistant strain of Plasmodium falciparum are described. The best compound displayed an activity 6-fold higher than chloroquine. None of the active compounds were found to inhibit beta-hematin formation in vitro in the same range as chloroquine and five among them displayed lower calculated vacuolar accumulation ratios, suggesting the implication of a different mechanism of action.     

Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update

Digestion of hemoglobin in the food vacuole of the malaria parasite produces very high quantities of redox active toxic free heme. Hemozoin (beta-hematin) formation is a unique process adopted by Plasmodium sp. to detoxify free heme. Hemozoin formation is a validated target for most of the well-known existing antimalarial drugs and considered to be a suitable target to develop new antimalarials. Here we discuss the possible mechanisms of free heme detoxification in the malaria parasite and the mechanistic details of compounds, which offer antimalarial activity by inhibiting hemozoin formation. The chemical nature of new antimalarial compounds showing antimalarial activity through the inhibition of hemozoin formation has also been incorporated, which may help to design future antimalarials with therapeutic potential against multi-drug resistant malaria.     

Design and synthesis of new antimalarial agents from 4-aminoquinoline

This study describes the synthesis of new 4-aminoquinoline derivatives and evaluation of their activity against a chloroquine sensitive strain of P. falciparum in vitro and chloroquine resistant N-67 strain of P. yoelii in vivo. All the analogues were found to form strong complex with hematin and inhibit the beta-hematin formation in vitro. These results suggest that these compounds act on heme polymerization target.     

Beta-hematin interaction with the hemopexin domain of gelatinase B/MMP-9 provokes autocatalytic processing of the propeptide, thereby priming activation by MMP-3

Gelatinase B or matrix metalloproteinase-9 is involved in inflammation and in autoimmune and vascular diseases. In contrast to the constitutive and homeostatic matrix metalloproteinase-2, matrix metalloproteinase-9 is an inducible enzyme. Furthermore, it needs tight regulation, and a major control mechanism of its enzymatic activity is the activation of the latent enzyme by proteolysis of the 87 residue propeptide. Activated matrix metalloproteinase-9 is detected in many vascular or hematological disease states, including in an experimental model for cerebral malaria with Plasmodium berghei ANKA. However, insight into its activation mechanism is incomplete. In view of the association with hemorrhagic and hemolytic diseases, it was studied whether and how hemoglobin and its derivatives might activate pro-matrix metalloproteinase-9. Incubation of matrix metalloproteinase-9 with hemin or beta-hematin, the core constituent of hemozoin or malaria pigment, leads to differential autocatalysis of the propeptide, mediated by allosteric interaction with the hemopexin domain. The cleavage catalyzed by beta-hematin coincides with the first cleavage by stromelysin-1/matrix metalloproteinase-3, and preincubation of matrix metalloproteinase-9 with beta-hematin enhances the activation rate by matrix metalloproteinase-3 at least 6-fold. These findings suggest that reduction of hemorrhage and hemolysis might prevent matrix metalloproteinase-9-mediated inflammatory and vascular damages.     

Inhibition assay of beta-hematin formation initiated by lecithin for screening new antimalarial drugs

Measurement of heme crystallization provides a tool for screening new antimalarial drugs. Current assays for heme crystallization have employed initiators such as thermo, histidine-rich proteins, and lipids extracted from parasites and infected plasma. These initiators are unnatural or require laborious steps to prepare. In this study, we used a commercially available lipid, lecithin, a kind of phospholipid containing about 50% unsaturated fatty acids, as an initiator for heme crystal (beta-hematin) formation. We demonstrated that the inhibition of lecithin-based beta-hematin formation by antimalarial drugs is highly correlated with the preformed beta-hematin-based method. In addition, the lecithin-based assay is sensitive and convenient for large-scale screening of new novel antimalarials. We also indicated that dimethyl sulfoxide is an ideal solvent for preparation of heme stock solution, which is stable and can be used for 1 month.     

Metalloporphyrin probes for antimalarial drug action

Metal-substituted protoporphyrin IXs (Co(III)PPIX (1), Cr(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6) and Sn(IV)PPIX (7)), phthalocyanine tetrasulfonates (PcS (8) and Ni(II)PcS (9)), and anionic and cationic porphyrins (meso-tetra(4-sulfonatophenyl)porphine (TPPS4, 10), meso-tetra(4-carboxyphenyl)porphine (TPPC4, 11), tetrakis(4-N-trimethylaminophenyl)porphine (TMAP, 12) and meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4, 13)) have been used as probes to compare two different assays for the inhibition of beta-hematin formation. The results demonstrate that the efficacy of these probes in either the beta-hematin inhibition assay (9, 7, 6, 5>4>11, 3>10, 8>2, 1; 12 and 13 did not inhibit.) or the bionucleating template assay (8>1>11>9, 2>4>3>7>10>5>6; 12 and 13 did not inhibit.) differ significantly. These differences are examined in light of possible interactions between the inhibitor probes, heme, beta-hematin and the bionucleating template. This detailed analysis highlights the fact that while dominant modes of interactions may be occasionally identified, the precise mechanism of inhibition undoubtedly consists of the interplay between multiple interactions.     

Physico-chemical aspects of hemozoin (malaria pigment) structure and formation

Recent literature dealing with the chemical composition, structure and mechanism of formation of hemozoin and its synthetic counterpart, beta-hematin, is reviewed.     

Evidences for the formation of bisbenzamidine-heme complexes in cell-free systems

Infrared and colorimetry data suggest that bisbenzamidines connected by various rigid or flexible linkers are able to interact with heme in cell-free systems. At pH 5.0 the inhibition of formation of beta-hematin could be ascertained by infrared spectroscopy whereas at pH 7.0 the interaction yielded insoluble complexes for which a sandwich-type structure of stoichiometry 2:1, heme-drug, is tentatively proposed.     

Alcohol-water as a novel medium for beta-hematin preparation.

It is shown that alcohol-water mixtures (e.g., 43 wt% ethanol) provide a suitable medium for the efficient production in high yield of beta-hematin from ferriprotoporphyrin IX (FP), as measured by infrared spectroscopy. Previous and present light-absorption data of FP, obtained under specific acid conditions in an ethanol-water medium, suggest the presence of FP monomers, which are considered to promote the reaction leading to beta-hematin. Other aspects of the mechanism of reaction are discussed.     

Purification and characterization of a serine protease secreted by Brevibacillus sp. KH3 for reducing waste activated sludge and biofilm formation

A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 °C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50 °C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.