Clotrimazole Binds to Heme and Enhances Heme-dependent Hemolysis

Abstract

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K a ‫؍‬ 6.54 ؋ 10 8 M ؊2 ). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S ‫؍‬ 1 ⁄2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite.

Simultaneous determination of 24 personal care products in fish muscle and liver tissues using QuEChERS extraction coupled with ultra pressure liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometer analyses

“…In particular, the quantitation of clotrimazole in liver was intensively disturbed by a strong adjacent interfering peak. As a potent antifungal agent, clotrimazole has a high binding affinity for heme to form a stable and soluble complex [58], which may result in its failure of extraction from liver samples. Figures 3 and 4 exhibit the LC-MS/MS and GC-MS chromatograms of the analytes in fish samples for the recovery experiment, with spiking concentrations of 10 and 40 ng/g Fig.…”

Enhancement of Heme-Induced Membrane Damage by the Anti-malarial Clotrimazole: the Role of Colloid-Osmotic Forces

“…18) Therefore, the much higher anti-malarial activity of CLT compared to 2-chlorophenyl-bis-phenyl methanol might be due to the formation of an imidazole group-mediated heme-CLT complex.…”

“…2), and the absorption spectrum of heme-CLT complex did not change upon the addition of GSH. 18) Because an excess of GSH was used, CLT suppression of GSH-dependent heme degradation was not caused by the direct interaction of CLT and GSH.…”

“…Heme-loaded membrane or heme-CLT complex-loaded membrane was incubated with GSH in the presence or absence of CLT, and then centrifuged to separate membrane and unbound heme. Because absorption of heme decreases when decomposed by GSH, 13,18) we measured the absorption at 402 nm after membrane lysis in 2.5% SDS to determine the amounts of heme or heme-CLT complex that had not been degraded by GSH in the membrane. Figure 2 shows that approximately 66% of the heme that had been loaded in membrane was degraded following a 2 h incubation with 10 mM GSH in absence of CLT (Fig.…”

“…At the end of the incubation, the percentage of hemolysis was evaluated as described in our previous report. 18) Briefly, the erythrocyte suspension was centrifuged at 1500ϫg for 3 min to collect non-hemolyzed erythrocytes, and the amount of hemoglobin in the supernatant was determined by absorbance at 578 nm. 24) The hemoglobin content in the sedimented non-hemolyzed erythrocytes was measured following lysis in distilled water.…”