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: Ka = 6.54 × 108m−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 = ½), 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. 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: Ka = 6.54 × 108m−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 = ½), 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. chloroquine clotrimazole electron spin resonance histidine-rich protein 2 ferric protoporphyrin IX mesoprotoporphyrin IX Malaria has become a key global threat due to quickly spreading resistance to quinoline-based antimalarial drugs such as quinine, chloroquine (CQ),1 and mefloquine (1Trigg P.I. Kondrachine A.V. Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. ASM press (American Society for Microbiology), Washington, D. C.1998: 11-22Google Scholar). Furthermore, artemisinin-resistant strains ofPlasmodium falciparum have been developed in the laboratory (2Inselburg J. Am. J. Trop. Med. Hyg. 1985; 34: 417-418Crossref PubMed Scopus (37) Google Scholar). Therefore, there has been extensive research into a new series of antimalarial drugs. The antifungal agent clotrimazole (CLT) (Fig.1) inhibits the growth of chloroquine-resistant P. falciparum strains in vitro (3Saliba K.J. Kirk K. Trans. R. Soc. Trop. Med. Hyg. 1998; 92: 666-667Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 4Tiffert T. Ginsburg H. Krugliak M. Elford C. Lew V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 331-336Crossref PubMed Scopus (67) Google Scholar). Mechanisms of the antimalarial activity of CLT have been proposed in relation to Ca2+ ions; CLT inhibits the sarcoplasmic reticulum Ca2+ pump and capacitative Ca2+channels of malaria-infected red blood cells, causing the depletion of intracellular Ca2+ stores (5Benzaquen L.R. Brugnara C. Byers H.R. Gattoni-Celli S. Halperin J.A. Nat. Med. 1995; 1: 534-540Crossref PubMed Scopus (145) Google Scholar, 6Tiffert T. Staines H.M. Ellory J.C. Lew V.L. J. Physiol. 2000; 525: 125-134Crossref PubMed Scopus (39) Google Scholar). Depletion of intracellular Ca2+ induces the activation of protein kinase R and phosphorylation of eukaryotic translation initiation factor 2α, thereby inhibiting protein synthesis in the parasite (7Aktas H. Fluckiger R. Acosta J.A. Salvage J.M. Palakurthi S.S. Halperin J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8280-8285Crossref PubMed Scopus (123) Google Scholar). However, the actual mechanism of CLT antimalarial action at the molecular level remains equivocal. During development and proliferation in human erythrocytes, malaria degrades hemoglobin to use as a major source of amino acids, accompanied by the release of free heme. As free heme is highly toxic to the malarial parasite, the parasite has developed a means of detoxifying heme through polymerization to non-toxic, insoluble hemozoin (8Francis S.E. Sullivan D.J. Goldberg D.E. Annu. Rev. Microbiol. 1997; 51: 97-123Crossref PubMed Scopus (656) Google Scholar) or by degradation with GSH (9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 10Ginsburg H. Famin O. Zhang J. Krugliak M. Biochem. Pharmacol. 1998; 56: 1305-1313Crossref PubMed Scopus (269) Google Scholar, 11Platel D.F.N. Mangou F. Tribouley-Duret J. Mol. Biochem. Parasitol. 1999; 98: 215-223Crossref PubMed Scopus (59) Google Scholar), which is found at millimolar concentrations in red blood cells and parasite compartments (12Atamna H. Ginsburg H. Eur. J. Biochem. 1997; 250: 670-679Crossref PubMed Scopus (135) Google Scholar, 13Luersen K. Walter R.D. Muller S. Biochem. J. 2000; 346: 545-552Crossref PubMed Scopus (89) Google Scholar). About 30–50% of free heme is detoxified by polymerization at the trophozoite stage (10Ginsburg H. Famin O. Zhang J. Krugliak M. Biochem. Pharmacol. 1998; 56: 1305-1313Crossref PubMed Scopus (269) Google Scholar, 14Wood P.A. Eton J.W. Am. J. Trop. Med. Hyg. 1993; 48: 465-472Crossref PubMed Scopus (20) Google Scholar, 15Slater A.F.G. Pharmacol. Ther. 1993; 57: 203-235Crossref PubMed Scopus (311) Google Scholar), and the remainder is detoxified by GSH-dependent degradation. The two detoxification processes of free heme are initiated by heme histidine-rich protein (HRP) 2 and heme-GSH complex formation, respectively. Antimalarials such as quinine and CQ also bind to free heme and inhibit its degradation. Furthermore, the imidazole moiety of CLT behaves as an axial ligand, binding free heme. We therefore considered that CLT exerts antimalarial activity by forming complexes with heme, similar to the heme-binding antimalarials, CQ and quinine. In this study, the coordination reaction between CLT and heme was investigated by optical absorption spectroscopy and electron spin resonance (ESR) spectroscopy. The structure of the heme-CLT complex was characterized based on spectroscopic evidence. Furthermore, we compared the effects of CLT and CQ on GSH-dependent heme degradation and heme-induced hemolysis, and we propose a mechanism of antimalarial CLT action. GSH, CLT, CQ, imidazole, and hemin (heme) were from Sigma. Mesoheme was from Porphyrin Products Inc. (Logan, UT). Human blood was drawn from healthy volunteers. Dimethyl sulfoxide (Me2SO) was purchased from Wako Pure Chemicals (Osaka, Japan). All other chemicals were of the highest commercially available grade. At the start of each experiment, a stock heme solution was prepared by dissolving hemin chloride in 20 mm NaOH and then removing the remaining hemin crystals by centrifugation for 10 min at 15,000 rpm. The heme concentration was estimated from absorbance at 385 nm (εmm = 58,400) in 100 mm NaOH (16Shaklai N. Shviro Y. Rabizadeh E. Kirschner-Zilber I. Biochim. Biophys. Acta. 1985; 821: 355-366Crossref PubMed Scopus (91) Google Scholar) and adjusted to 1.0 mm. This stock reagent was stored in the dark on ice and used within 24 h. All absorption spectra were recorded on a Hitachi U-3300 double-beam spectrophotometer (Tokyo, Japan) using a 1.0-cm light-path quartz cuvette at 23 °C. A solution of 17 μm heme in 40% Me2SO and 20 mmHEPES buffer (pH 7.4) revealed Soret at 401 nm and Q band absorption maxima at 493 and 616 nm, and the ratio of absorption of the Soret (401 nm) and Q band (616 nm) was 28.72, indicating that the heme in the present system exists as a monomeric mode (17Beaven G.H. Chen S. D'Albis A. Gratm W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (254) Google Scholar, 18Collier G.S. De Pratt J.M. Wet R. Tshabalala C.F. Biochem. J. 1979; 179: 281-289Crossref PubMed Scopus (60) Google Scholar, 19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar). The optical absorption spectra of the heme-CLT and heme-imidazole complexes were recorded 5 min after adding CLT (final 100 and imidazole (final to heme (final 17 in 40% Me2SO with 20 mm (pH The of the reaction was 1.0 absorption spectra were on a Hitachi U-3300 spectrophotometer as The CLT or CQ, was to a cuvette heme The two an heme solution to which a buffer other than the was and the other a solution heme and the of the In the of CLT the cuvette and the cuvette 17 μm heme in 40% Me2SO by 20 mm (pH and the cuvette the solution heme. of CLT μm in Me2SO were with the of the cuvette and the in which the of the reaction was at 1.0 adding CQ, the cuvette and the cuvette 5 μm heme in 40% Me2SO by 20 mm (pH and the cuvette the solution heme. During the of complex formation, of CQ μm in μm were to the and the the of the reaction was 1.0 the All spectra were recorded at between and nm, and the concentrations of heme-CLT and complexes were based on absorbance at and 401 nm, respectively. The binding mode of CLT and CQ to heme was in of Scholar, D. M. Biochem. Biophys. 1974; 57: PubMed Scopus Google Scholar) and N. Y. Acad. Sci. 51: Scopus Google Scholar, H. Scholar) The equilibrium for the formation of heme-CLT and as as the of ligands that bind to heme, were from using and using the 2 and A are the absorbance of the and is the or is the of that bind to and Ka is the equilibrium of the were for heme-CLT complexes that were formed in Me2SO at of heme to CLT of and an at the of heme-CLT complex was recorded at by a with The the of each The was by of in = was used as the The were and using a system The for were as to and degradation by GSH was by as described by and Ginsburg (9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). GSH stock solution was prepared in buffer mm mm and mm 7.4) R. PubMed Scopus Google Scholar, R. Y. 1974; PubMed Scopus Google Scholar). (final and GSH (final 2 were in buffer (pH 7.4) and at °C. spectra nm) were recorded at after using the The and GSH-dependent heme degradation in the of CLT and CQ were from the of absorbance at nm in of reaction In the of CLT or CQ the were by the CLT in Me2SO and CQ in buffer 7.4) were prepared as GSH and CLT or CQ were in buffer (pH 7.4) and at °C. In the experiment, Me2SO (final was to the of heme and GSH of CLT and The of absorbance at nm was recorded as an of heme degradation. blood from healthy was of to The were from by centrifugation at × min and with the effects of CLT and CQ on by heme were in cell in were with concentrations of heme and CLT or CQ and 10 at for min at were then by centrifugation at × for and the of hemoglobin from the into the was by absorbance at nm Y. N. Biochem. Pharmacol. PubMed Scopus Google Scholar). the were with and to the the hemoglobin in was as absorbance at The of was from the ratio of hemoglobin from by heme to the heme of the Y. N. Biochem. Pharmacol. PubMed Scopus Google Scholar). using erythrocytes, of red blood cells in were with 10 μm heme at for 10 The was by centrifugation at for and the was with buffer to free heme, A of of a of was prepared in Me2SO CLT CQ or GSH was then and the was at for 2 h. The was from such Soret and Q band absorption at and 616 nm by heme in 40% which is of high spin ferric complexes a structure M. Biochem. Biophys. PubMed Scopus Google J. Am. Chem. Soc. Scopus Google Scholar) with axial such as or chloride CLT (final 100 was to the the Soret red at nm, and Q band absorption was at and nm, as shown in The was into a ferric complex having nitrogenous ligands at axial In the spectroscopic with of similar of heme and imidazole Q and nm) shown in Furthermore, complexes have similar spectra 1985; PubMed Scopus Google Scholar, M. K. T. Y. F. Biochem. Biophys. PubMed Scopus Google Scholar), as in I. results the that CLT, imidazole, has affinity for the heme is that the imidazole moiety of CLT is the nitrogenous maxima for protoporphyrin with CLT, CQ, imidazole, or two 20 mm buffer at 20 mm buffer at 20 mm buffer at mm mm mm mm 1985; PubMed Scopus Google M. K. T. Y. F. Biochem. Biophys. PubMed Scopus Google 40% 20 mm buffer at 20 mm in a new The heme-CLT complex in the of Me2SO a similar absorption as by a Soret band at nm and Q at and nm In the of the heme-imidazole complex in the of Me2SO a at nm which is derived from the of heme W.B. Biochem. J. PubMed Scopus Google Scholar, on and in of Scholar). The heme-CLT prepared in buffer in the of was stable The of the absorption maxima of the heme-CLT complex in Me2SO and in buffer and heme was in results potent of the of CLT to the monomeric of heme and to inhibit the formation of heme in aqueous the binding of heme with CLT, heme was by the spectra between heme and heme-CLT complex at CLT As described by 20 mm was used heme in this solution a at concentrations to μm (17Beaven G.H. Chen S. D'Albis A. Gratm W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (254) Google Scholar, 18Collier G.S. De Pratt J.M. Wet R. Tshabalala C.F. Biochem. J. 1979; 179: 281-289Crossref PubMed Scopus (60) Google Scholar, 19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar). that CLT the of heme, indicating between the and heme. of CLT into a heme solution of the heme to a with Soret absorption and a Soret to a The absorption spectra as the CLT concentration through at and nm, indicating that two are present in the reaction During complex formation, the was accompanied by a in the of monomeric heme at the Soret and Q as described by T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar), indicating between CQ and heme. The effects of heme with CLT and CQ on were using Scholar, D. M. Biochem. Biophys. 1974; 57: PubMed Scopus Google Scholar) to the of to heme in aqueous of binding in heme-CLT complexes at 17 μm heme and complexes at 5 μm heme. The of are 2 and within The are in in N. Y. Acad. Sci. 51: Scopus Google Scholar, H. Scholar). The the of between heme and CQ, a is for heme-CLT indicating and the of binding The using the that heme two CLT with an of 6.54 × heme of CQ with a Ka of × in aqueous 40% Me2SO at We the and coordination of the heme-CLT complex by spectroscopy. We recorded spectra at for heme-CLT complexes prepared in as described adding CLT, the of heme the and = and = of the ferric high spin (S into M. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). A is at = which be due to from heme K. Inorg. Acta. Scopus Google Scholar) or in the and adding CLT (final 100 to the reaction the of the high spin with the formation of a new with a 2 and = = and = which was of ferric low spin complex (S = having axial ligands at axial the of CLT, the ratio of and the of the new was recorded that CLT to the equilibrium to the low spin ferric The of the low spin was similar to that recorded for a of heme-imidazole complex In the of complexes and This that heme-CLT and heme-imidazole have similar ligands at the axial and by using J. of heme-CLT complex and ferric mesoprotoporphyrin IX mesoprotoporphyrin IX 1985; PubMed Scopus Google protoporphyrin IX 1985; PubMed Scopus Google dimethyl M. Biochim. Biophys. Acta. PubMed Scopus Google in a new The absorption of heme in buffer 7.4) two Soret at 385 and nm, the and M. E. C. Biophys. Chem. PubMed Scopus Google Scholar), the of heme in 40% Me2SO revealed Soret band at 401 nm, indicating the of heme (17Beaven G.H. Chen S. D'Albis A. Gratm W.B. Eur. J. Biochem. 1974; 41: 539-546Crossref PubMed Scopus (254) Google Scholar, 18Collier G.S. De Pratt J.M. Wet R. Tshabalala C.F. Biochem. J. 1979; 179: 281-289Crossref PubMed Scopus (60) Google Scholar, 19Egan T.J. Mavuso W.W. Ross D.C. Marques H.M. J. Inorg. Biochem. 1997; 68: 137-145Crossref PubMed Scopus (150) Google Scholar). The absorption of the Soret of heme was to nm after adding GSH as described in H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, due to the formation of that the Soret absorption of heme with GSH as described in H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, indicating the degradation of heme by The and of the heme degradation were from to the reaction as × and in buffer at The absorption spectra of heme-CLT and of complexes the of GSH, indicating that complex with GSH The GSH-dependent degradation of heme in the of CLT or CQ was as a of absorbance at nm heme-CLT complex and complex have similar molecular absorption at The results shown in indicate that CLT and CQ inhibit GSH-dependent heme degradation. were using blood cells as described to in in which heme was The by the of heme was by CLT as as by CQ and the effects on the concentrations of heme and the We also that CLT at to 20 on in the of heme Therefore, the of of be by the formation of heme-CLT The of by CQ was to that in a P. J. Pharmacol. Ther. Google Scholar). The similar of by CLT and CQ that have the at high concentrations of heme as shown in However, at heme concentration CLT was more than CQ this of CLT in we with 10 μm heme by and then the The in this were as that were by heme which to heme (9Atamna H. Ginsburg H. J. Biol. Chem. 1995; 270: 24876-24883Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). were with CLT or CQ to the of drugs on the of to that CLT CQ the of erythrocytes, Me2SO and GSH We propose a mechanism for the antimalarial activity of CLT based on the results of spectroscopic and We investigated between heme and CLT using optical absorption and electron spin The optical and of the heme-CLT complex and were compared with of complexes to the axial of the The and optical absorption spectra complexes having nitrogenous ligands at axial of heme, as has been for heme and complexes 1985; PubMed Scopus Google Scholar, M. K. T. Y. F. Biochem. Biophys. PubMed Scopus Google Scholar). spectroscopic of indicate that the heme has two moieties at axial 1985; PubMed Scopus Google Scholar). In the spectroscopic of heme-CLT and heme-imidazole complexes This that the imidazole moiety of CLT is to be the axial of the The and of the heme-CLT complex and complexes were in of J. Scholar). The of heme-CLT complexes with of heme-imidazole as in the of the results from and optical as as from the we that the axial of the heme-CLT complex is therefore the nitrogenous derived from the imidazole moiety of CLT The proposed coordination structure of heme-CLT complex is with the results of the in that the heme-CLT complex two of CLT at axial The equilibrium for the formation of the heme-CLT complex was 6.54 × In with of heme-imidazole R. The Porphyrin Scholar), CLT is of the axial ligands for heme. We CLT has affinity for heme reaction However, the of CLT, with a axial to heme. into the results of the and we found that CLT a stable complex with a high equilibrium The malaria parasite has developed processes to free heme as a of hemoglobin of the free heme is to hemozoin (3Saliba K.J. Kirk K. Trans. R. Soc. Trop. Med. Hyg. 1998; 92: 666-667Abstract Full Text PDF PubMed Scopus (30) Google Scholar) through the formation of complex with malarial The remaining free heme through the membrane of and also the of the parasite F. U. PubMed Scopus Google Scholar, J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar, H. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, H. Chem. PubMed Scopus Google Scholar), free heme be by reaction with GSH (10Ginsburg H. Famin O. Zhang J. Krugliak M. Biochem. Pharmacol. 1998; 56: 1305-1313Crossref PubMed Scopus (269) Google Scholar, 11Platel D.F.N. Mangou F. Tribouley-Duret J. Mol. Biochem. Parasitol. 1999; 98: 215-223Crossref PubMed Scopus (59) Google Scholar). is that CLT of was a concentration in of 2 μm within of C. M. N. O. J. Pharmacol. Ther. 1995; Google Scholar) and into C. M. N. O. J. Pharmacol. Ther. 1995; Google Scholar, C. S. M. N. J. 97: PubMed Scopus Google Scholar). Therefore, CLT be to the parasite of stable heme-CLT complexes in and the of GSH to heme, the Soret absorption to nm due to the formation of heme-GSH and the GSH-dependent heme degradation quickly the GSH in the heme-CLT complex and the Soret absorption of the complex the CLT GSH-dependent heme degradation. The concentration of GSH in red blood cells and in the parasite millimolar (12Atamna H. Ginsburg H. Eur. J. Biochem. 1997; 250: 670-679Crossref PubMed Scopus (135) Google Scholar, 13Luersen K. Walter R.D. Muller S. Biochem. J. 2000; 346: 545-552Crossref PubMed Scopus (89) Google Scholar). the high equilibrium of CLT × the heme-CLT complex to in the and malaria parasite. Furthermore, free heme, which bind to malarial heme polymerization into is also to heme-CLT complex through the axial reaction by of CLT in vitro. The due to heme-CLT complex was recorded after a of CLT to a solution of the amino complex in which two are at the axial This means that the heme, heme to be to heme-CLT complex in the of indicate that the formation of heme-CLT complex malarial toxic free heme, heme polymerization by and heme by into the molecular structure of CLT, the heme-CLT complex in which two CLT bind to axial is considered to be and The and to in the of the its the that in the and of CLT, we found that the free was by the of CLT to free heme and The and the molecular of the heme-CLT complex therefore be for the of heme-CLT complexes malaria-infected are be causing the malarial to In heme-CLT complexes in the malarial membrane the the malaria parasite. Furthermore, CLT was found to be more than CQ in The shown in indicate that CQ has affinity to heme than CLT with an concentration of CQ for in 40% Me2SO Therefore, we are to CLT demonstrated a to However, in other we found that the concentrations of CLT and CQ for for binding with 10 μm heme were 10 μm and in This that CLT has high affinity to heme similar to CQ the in the effects of between CLT and CQ might have due to the of and structure of complexes with heme due to a in the affinity to heme. In we have also found that CLT intracellular heme-induced T. K. R. and S. for We have found that CLT from to in in which free heme been from hemoglobin by with on results and the that CLT has the to with and GSH, we that CLT at concentrations in and a complex with heme from and cell The of the membrane by the heme-CLT complex is to an in the antimalarial action of that of in of CLT the imidazole, has antimalarial activity growth concentration of than CLT of (3Saliba K.J. Kirk K. Trans. R. Soc. Trop. Med. Hyg. 1998; 92: 666-667Abstract Full Text PDF PubMed Scopus (30) Google Scholar). This that the imidazole is is in the antimalarial activity of The antimalarial mechanism of might be due to of the Ca2+ pump and Ca2+ (5Benzaquen L.R. Brugnara C. Byers H.R. Gattoni-Celli S. Halperin J.A. Nat. Med. 1995; 1: 534-540Crossref PubMed Scopus (145) Google Scholar, 6Tiffert T. Staines H.M. Ellory J.C. Lew V.L. J. Physiol. 2000; 525: 125-134Crossref PubMed Scopus (39) Google Scholar, C. M. N. O. J. Pharmacol. Ther. 1995; Google Scholar). However, the antimalarial activity of CLT than of might be by the formation of complexes with heme through an imidazole of the antimalarial mechanism of CLT malarial heme (3Saliba K.J. Kirk K. Trans. R. Soc. Trop. Med. Hyg. 1998; 92: 666-667Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 4Tiffert T. Ginsburg H. Krugliak M. Elford C. Lew V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 331-336Crossref PubMed Scopus (67) Google Scholar) indicate that CLT inhibits the growth of strains of Furthermore, the low of CLT malaria a antimalarial a CLT concentration of 2 μm is with an of Therefore, CLT is as a new antimalarial