Beta hematin inhibition: evaluating the mechanism of action of some selected antimalarial plants (original) (raw)
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Experimental Parasitology, 2000
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.
Analytical Biochemistry, 2005
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 (b-hematin) formation. Aqueous pyridine (5% v/v, pH 7.5) forms a low-spin complex with hematin but not with b-hematin. Its absorbance obeys BeerÕs law, making it useful for quantitating hematin concentration in hematin/b-hematin mixtures, allowing compounds to be investigated for inhibition of b-hematin formation. The assay is rapid (60 min incubation) and requires no centrifugation. The b-hematin inhibition data show good agreement with alternative assay methods reported by four laboratories. The assay was adapted for highthroughput colorimetric screening, allowing visual identification of b-hematin inhibitors. In this mode, the assay successfully detected all 18 b-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 b-hematin inhibition values determined using the assay.
2019
The search for antimalarials from plant sources has yield significant success in drug discovery approaches. The heme polymerization inhibitory activity as well as the antimalarial activity of Senna occidentalis, a local medicinal plant used for malaria therapy in Hausa folk medicine in Northern Nigeria was evaluated in in vitro assays. Results obtained revealed a good inhibition of β-hematin formation (83.08% and 83.97%) by the methanolic and aqueous leaves extracts of S. occidentalis at 500μg/mL as against the 54.92% inhibition exhibited by hexane extract at the same concentration. Findings of the in vitro antimalarial studies revealed a dose dependent suppression of plasmodium growth. At a concentration of 6.25 μg/ml, 73% suppression of parasite growth was observed for the hexane extract. This suppression of plasmodial growth attains 84.43% at 50 μg/ml with an IC50 of 3.47 μg/ml. Secondary metabolites such as anthraquinones, phenols, tannins, alkaloids and flavonoids were detected...
Screening of guava (Psidium guajava) leaves extracts against β-hematin formation
Pharmacy & Pharmacology International Journal, 2021
The present work aimed to determine the in-vitro inhibitory influence of guava leaves infusion water and ethanol extracts on the β-hematin formation using a semi-quantitative screening method. Moreover, the principal polar secondary metabolites in guava leaves that are extracted out of the infusion process are scanned and identified in an effort to discover novel antimalarial leads. Materials and methods Acetonitrile (ACN) and ultra-pure water (H 2 O, prepared by using a Millipore Milli-Q plus water purification system), formic acid and ammonium formate were all of LC-MS grades. Dimethyl sulfoxide (DMSO), chloroquine diphosphate salt (CQ), morin, quercetin, catechin and quercitrin hydrate analytical standards, sodium acetate, and hemin chloride were all purchased from Sigma-Aldrich. Glacial acetic acid was purchased from Fluka. Ethanol (EtOH) was from Merck. Guava leaves (Psidium guajava) was collected in 2020 from Qalqiliya city at northern part of Palestine. The leaves dried under shade and stored at in amber bottle and kept in the fridge until used. Preparation of guava leaves infusion Preparation of the guava extracts-Method A Air-dried guava (P. guajava) leaves were grinded into coarse powder; extracted by soaking 2-grams in 150ml of distilled hot water at 90°C for 20 minutes.
Antimicrobial Agents and Chemotherapy, 2000
Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of 14 C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100,000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.
Pharmacy & Pharmacology International Journal
The effect of wild Pomegranate (Punica granatum) juice, peels, leaves and membranes water infusion extracts on β-hematin inhibition was investigated in-vitro using semi-quantitative method. Reversed phase HPLC coupled with photodiode array detector and mass spectrometer (RP-LC-PDA-MS) was used to screen the major phenolic phytochemical(s) that may play a key role in the mechanism of β-hematin inhibition. Eighteen compounds were detected of which six are present in major quantity namely corilagin, brevifolin carboxylic acid, galloyl-HHDP-hexoside, granatin B, ellagic acid and eschweilenol C. The leaves, peels and membranes extracts have shown significant antimalarial potential when compared to chloroquine positive control. However, plain pomegranate juice which was not further enriched was inactive.
Antimalarial Hemozoin Inhibitors (β-Hematin Formation Inhibition): Latest Updates
Combinatorial Chemistry & High Throughput Screening, 2022
The above article has been published, as is, ahead-of-print, to provide early visibility but is not the final version. Major publication processes like copyediting, proofing, typesetting and further review are still to be done and may lead to changes in the final published version, if it is eventually published. All legal disclaimers that apply to the final published article also apply to this ahead-of-print version.
Antimalarial and β-hematin formation inhibitory activities of chromone derivatives
ScienceAsia
A series of chromone compounds were evaluated as new potential antimalarial agents using in vitro antimalarial activity assay. The most potent compound 36 with an IC 50 of 0.95 µM was shown to be more potent than primaquine and tafenoquine (IC 50 2.41 and 1.95 µM, respectively). β-Hematin formation inhibitory activity test and stoichiometry determination have also been performed to investigate the preliminary mechanism of antimalarial activity of the studied compounds.
Combinatorial Chemistry & High Throughput Screening, 2005
Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts' erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. The malaria parasite digests hemoglobin within the digestive vacuole through a sequential metabolic process involving multiple proteases. Massive degradation of hemoglobin generates large amount of toxic heme. Malaria parasite, however, has evolved a distinct mechanism for detoxification of heme through its conversion into an insoluble crystalline pigment, known as hemozoin. Hemozoin is identical to β-hematin, which is constituted of cyclic heme dimers arranged in an ordered crystalline structure through intermolecular hydrogen bonding. The exact mechanism of biogenesis of hemozoin in malaria is still obscure and is the subject of intense debate. Hemozoin synthesis is an indispensable process for the parasite and is the target for action of several known antimalarials. The pathway has therefore attracted significant interest for new antimalarial drug discovery research. Formation of β-hematin may be achieved in vitro under specific chemical and physiochemical conditions through a biocrystallization process. Based on these methods several experimental approaches have been described for the assay of formation of β-hematin in vitro and screening of compounds as inhibitors of hemozoin synthesis. These assays are primarily based on differential solubility and spectral characteristics of monomeric heme and β-hematin. Different factors viz., the malaria parasite lysate, lipids extracts, preformed β-hematin, malarial histidine rich protein II and some unsaturated lipids have been employed for promoting β-hematin formation in these assays. The assays based on spectrophotometric quantification of β-hematin or incorporation of 14 C-heme yield reproducible results and have been applied to high throughput screening. Several novel antimalarial pharmacophores have been discovered through these assays.
Inhibition assay of β-hematin formation initiated by lecithin for screening new antimalarial drugs
Analytical Biochemistry, 2006
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 ( -hematin) formation. We demonstrated that the inhibition of lecithin-based -hematin formation by antimalarial drugs is highly correlated with the preformed -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.