Artemisinin, an Endoperoxide Antimalarial, Disrupts the Hemoglobin Catabolism and Heme Detoxification Systems in Malarial Parasiteстатья из журнала
Аннотация: Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well asPlasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains. Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well asPlasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains. benzyloxycarbonyl aminomethylcoumarin phosphate-buffered saline Plasmodium falciparum histidine-rich protein II translationally controlled tumor protein The fast spreading resistance to commonly used quinoline antimalarials has made malaria a major global disease (1Su X. Kirkman L.A. Fujioka H. Wellems T.E. Cell. 1997; 91: 593-603Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Among the few alternative drugs available, artemisinin, an endoperoxide antimalarial drug, derived from an ancient Chinese herbal remedy, Qinghaosu, is the most promising (2Klayman D.L. Science. 1985; 228: 1049-1055Crossref PubMed Scopus (2045) Google Scholar, 3Hien T.T. White N.J. Lancet. 1993; 341: 603-608Abstract PubMed Scopus (544) Google Scholar). A preparation containing racemic mixture of α- and β-arteether (an analogue of artemisinin) has successfully completed clinical trials in India (4Mohapatra P.K. Khan A.M. Prakash A. Mahanta J. Srivastava V.K. Ind. J. Med. Res. 1996; 104: 284-287PubMed Google Scholar). However, very little is known about the molecular mechanism of action of these drugs. A two-step mechanism proposed recently by Meshnick et al.(5Meshnick S.R. Taylor T.E. Kamchonwongpaisan P. Microbiol. Rev. 1996; 60: 301-315Crossref PubMed Google Scholar) suggests the heme-catalyzed cleavage of the endoperoxide bridge forms a free radical followed by specific and selective alkylation of some malarial proteins (6Yang Y.Z. Asawamahasakda W. Meshnick S.R. Biochem. Pharmacol. 1993; 46: 336-339Crossref PubMed Scopus (78) Google Scholar). However, this does not explain the selective cytotoxic action of artemisinin on mature parasite stages (late stage trophozoites and schizonts) (7Geary T.G. Divo A.A. Jensen J.B. Am. J. Trop. Med. Hyg. 1989; 40: 240-244Crossref PubMed Scopus (97) Google Scholar, 8Caillard V. Beaute-Lafitte A. Chabaud A.G. Landau I. Exp. Parasitol. 1992; 75: 449-456Crossref PubMed Scopus (30) Google Scholar). The chloroquine-resistant strains of Plasmodium berghei that lack hemozoin are also resistant to artemisinin, indicating that the presence of preformed hemozoin may be necessary for antimalarial action of these drugs (9Peters W. Li Z.L. Robinson B.L. Warhurst D.C. Ann. Trop. Med. Parasitol. 1986; 80: 483-489Crossref PubMed Scopus (41) Google Scholar). During intraerythrocytic development and proliferation, hemoglobin is utilized as a major source of amino acids by the malarial parasite (10Foley M. Tilley L. Pharmacol. Ther. 1998; 79: 55-87Crossref PubMed Scopus (488) Google Scholar, 11Goldberg D.E. Slater A.F.G. Cerami A. Henderson G.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2931-2935Crossref PubMed Scopus (399) Google Scholar, 12Goldberg D.E. Slater A.F.G. Beavis R. Chait B. Cerami A. Henderson G.B. J. Exp. Med. 1991; 173: 961-969Crossref PubMed Scopus (232) Google Scholar). Constant degradation of hemoglobin inside the parasite food vacuole occurs through a sequentially ordered process that involves cysteine as well as aspartic acid proteases (13Francis S.E. Gluzman I.Y. Oksman A. Knickerboker A. Muller R. Bryant M.L. Sherman D.R. Russel D.G. Goldberg D.E. EMBO J. 1994; 13: 306-313Crossref PubMed Scopus (251) Google Scholar, 14Salas F. Fichmann J. Lee G.K. Scott M.D. Rosenthal P.J. Infect. Immun. 1995; 63: 2120-2125Crossref PubMed Google Scholar, 15Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar, 16Francis S.E. Sullivan Jr., D.J. Goldberg D.E. Annu. Rev. Microbiol. 1997; 51: 97-123Crossref PubMed Scopus (662) Google Scholar). The toxic free heme, which is generated due to digestion of hemoglobin, is simultaneously detoxified by the malarial parasite through a specific mechanism of heme polymerization (17Schwarzer E. Turrini F. Ulliers D. Giribaldi G. Ginsburg H. Arese P. J. Exp. Med. 1992; 176: 1033-1041Crossref PubMed Scopus (260) Google Scholar, 18Vander Jagt D.L. Hunsaker L.A. Campose N.M. Mol. Biochem. Parasitol. 1986; 18: 389-400Crossref PubMed Scopus (90) Google Scholar, 19Vander Jagt D.L. Hunsaker L.A. Campos N.M. Scaletti J.V. Biochim. Biophys. Acta. 1992; 1122: 256-264Crossref PubMed Scopus (47) Google Scholar, 20Sherry B.A. Alava G. Tracey K.J. Martiney J. Cerami A. Slater A.F.G. J. Inflamm. 1995; 45: 85-96PubMed Google Scholar). The polymerized heme commonly referred to as “hemozoin” or “malaria pigment” accumulates in the form of a crystalline, insoluble, black-brown pigment (21Fitch C.D. Kanjananggulpan P. J. Biol. Chem. 1987; 262: 15552-15555Abstract Full Text PDF PubMed Google Scholar, 22Slater A.F. Swiggard W.J. Orton B.R. Flitter W.D. Goldberg D.E. Cerami A. Henderson G.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 325-329Crossref PubMed Scopus (483) Google Scholar, 23Bohle D.S. Dinnebier R.E. Madsen S.K. Stephens P.W. J. Biol. Chem. 1997; 272: 713-716Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Once the parasite life cycle is complete, this pigment is sequestered to various tissues of the host (24Pandey A.V. Tekwani B.L. Pandey V.C. Biomed. Res. 1995; 16: 115-120Crossref Scopus (22) Google Scholar). The heme polymerization pathway is specific to the malarial parasite and offers a potential biochemical target for the design of antimalarials (25Pandey A.V. Chauhan V.S. Curr. Sci. 1998; 75: 911-918Google Scholar). An enzyme “heme polymerase” was initially described, which could promote hemozoin formation (26Slater A.F.G. Cerami A. Nature. 1992; 355: 167-169Crossref PubMed Scopus (538) Google Scholar, 27Chou A.C. Fitch C.D. Life Sci. 1992; 51: 2073-2078Crossref PubMed Scopus (70) Google Scholar, 28Wellems T.E. Nature. 1992; 355: 108-109Crossref PubMed Scopus (43) Google Scholar), but the molecular mechanisms of this process are still under debate (29Pandey A.V. Joshi R.M. Tekwani B.L. Singh R.L. Chauhan V.S. Mol. Biochem. Parasitol. 1997; 90: 281-287Crossref PubMed Scopus (22) Google Scholar, 30Ridley R.G. Dorn A. Vippagunta S.R. Vennerstrom J.L. Ann. Trop. Med. Parasitol. 1997; 91: 559-566Crossref PubMed Scopus (60) Google Scholar, 31Egan T.J. Ross D.C. Adams P.A. FEBS Lett. 1994; 352: 54-57Crossref PubMed Scopus (356) Google Scholar, 32Adams P.A. Egan T.J. Ross D.C. Silver J. Marsh P.J. Biochem. J. 1996; 318: 25-27Crossref PubMed Scopus (43) Google Scholar, 33Dorn A. Stoffel R. Matile H. Bubendorf R. Ridley R.G. Nature. 1995; 374: 269-271Crossref PubMed Scopus (362) Google Scholar, 34Pandey A.V. Tekwani B.L. FEBS Lett. 1996; 393: 189-192Crossref PubMed Scopus (61) Google Scholar, 35Ignatushchenko M.V. Winter R.W. Bachinger H.P. Hinrichs D.J. Riscoe M.K. FEBS Lett. 1997; 409: 67-73Crossref PubMed Scopus (108) Google Scholar, 36Fitch C.D. Chou A.C. Mol. Biochem. Parasitol. 1996; 82: 261-264Crossref PubMed Scopus (16) Google Scholar, 37Sullivan Jr., D.J. Gluzman I.Y. Russell D.G. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11865-11870Crossref PubMed Scopus (444) Google Scholar). Recent studies have shown that artemisinin taken up by the malarial parasite growingin vitro was selectively concentrated in the parasite food vacuole and was associated with hemozoin (38Hong Y.L. Yang Y.Z. Meshnick S.R. Mol. Biochem. Parasitol. 1994; 63: 121-128Crossref PubMed Scopus (176) Google Scholar). Artemisinin also interacts with heme, forming covalent adducts (38Hong Y.L. Yang Y.Z. Meshnick S.R. Mol. Biochem. Parasitol. 1994; 63: 121-128Crossref PubMed Scopus (176) Google Scholar, 39Meshnick S.R. Thomas A. Ranz A. Xu C.M. Pan H.Z. Mol. Biochem. Parasitol. 1991; 49: 181-189Crossref PubMed Scopus (259) Google Scholar). In this report, we describe the inhibition of hemoglobin breakdown and heme polymerization by artemisinin and one of its analogues α-/β-arteether. Our studies provide evidence that the antimalarial effect of artemisinin may at least partly be due to inhibition of malarial hemoglobin degradation pathway and heme detoxification system. [14C]Leucine was a kind gift from Prof. O. P. Shukla (Central Drug Research Institute, Lucknow, India). Peptide substrate Z1-FR-AMC was purchased from Bachem. Artemisinin and α-/β-arteether were kindly provided by Dr. S. K. Puri. Bovine serum albumin standard solution was from Pierce, and Ni2+-NTA-Sepharose was from Amersham Pharmacia Biotech (Sweden). Hemin, SDS, dithiothreitol, isopropyl-1-thio-β-d-galactopyranoside, E-64, pepstatin A, LB media, and all other chemicals were from Sigma. Male Swiss albino mice (obtained from the Division of Laboratory Animals, Central Drug Research Institute, Lucknow, India) weighing 15–20 g were infected with Plasmodium yoelii nigeriensis by intraperitoneal passage of 1 × 107 infected erythrocytes. Parasitemia was monitored by microscopic examination of Giemsa-stained thin blood smears. Blood was collected at high levels of parasitemia (>60%) in sterile acid/citrate/dextrose. Animals were housed in the animal house at the Institute, and care was provided as per the guidelines laid down by ethics committee. Swiss albino mice were injected with phenylhydrazine (40 mg/kg body weight) to induce reticulocytes. Blood was collected 3 days post-phenylhydrazine injection. 50 μCi of [14C]leucine was added to this and incubated at 37 °C for 10 h in a conical flask rotating at 60 rpm. Erythrocytes were pelleted by centrifugation; leukocytes were removed, and hemoglobin was purified as described previously (12Goldberg D.E. Slater A.F.G. Beavis R. Chait B. Cerami A. Henderson G.B. J. Exp. Med. 1991; 173: 961-969Crossref PubMed Scopus (232) Google Scholar). Blood from 30 mice infected with P. yoelii was pooled and centrifuged at 500 ×g for 10 min at 4 °C. The plasma and buffy coat were removed, and the erythrocyte pellet was suspended in phosphate-buffered saline (PBS) and passed through a CF-11 column to remove leukocytes. Food vacuole preparation was done as described previously (12Goldberg D.E. Slater A.F.G. Beavis R. Chait B. Cerami A. Henderson G.B. J. Exp. Med. 1991; 173: 961-969Crossref PubMed Scopus (232) Google Scholar). Purified vacuoles were resuspended in acetate buffer (pH 5.0, 100 mm) and homogenized by a Polytron homogenizer at 6000 rpm. This vacuole extract was used for the protease assay. Proteolytic activity was measured using 14C-labeled bovine hemoglobin as a substrate. Reactions were performed in a total volume of 1.0 ml containing 50 μl of vacuolar lysate, 100 μg of hemoglobin, 1 mm CaCl2, 1 mm dithiothreitol in 100 mm acetate buffer (pH 5.0). The reaction was stopped by adding 500 μl of chilled 15% trichloroacetic acid, and the radioactivity in trichloroacetic acid-soluble fractions was monitored as a measure of proteolytic activity. Fluorimetric assay of cysteine protease was performed on an LS 50B spectrofluorimeter from Perkin-Elmer using Z-Phe-Arg-AMC as substrate as described previously (14Salas F. Fichmann J. Lee G.K. Scott M.D. Rosenthal P.J. Infect. Immun. 1995; 63: 2120-2125Crossref PubMed Google Scholar). Fluorescence of aminomethylcoumarin (AMC) released by proteolysis was used to monitor the activity. All assays using peptide substrates were performed at 25 °C in a continuous manner for 3 min total time. Artemisinin was incubated with hemoglobin to study the drug substrate interaction. Hemoglobin was extensively dialyzed after artemisinin exposure and checked by electrophoresis. The drug was incubated with infected erythrocytes for 0–6 h at 37 °C. After the incubation erythrocytes were washed with PBS, and parasites were collected by saponin lysis. Parasites were lysed by the addition of SDS-polyacrylamide gel electrophoresis sample buffer and boiling for 3 min and kept at −20 °C until further use. Electrophoresis was carried out according to standard protocols. Gels contained 15% acrylamide and were stained with Coomassie Blue for protein visualization. Bovine hemoglobin was used as a control. Hemozoin was purified from the erythrocytes of the mice infected with P. yoelii according to the method described earlier (24Pandey A.V. Tekwani B.L. Pandey V.C. Biomed. Res. 1995; 16: 115-120Crossref Scopus (22) Google Scholar). Plasmid containing the gene encoding P. falciparumHRP II (PfHRP II) in a pET 3d vector (Novagen) was a kind gift from Dr. D. E. Goldberg (Washington University, St. Louis, MO). Plasmids were transformed into Escherichia coli strain BL21(DE3).E. coli cells containing the plasmid were grown at 37 °C until A 600 reached between 0.4 and 0.6 and were cooled to 30 °C, and PfHRP II expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.4 mm. Cells were allowed to grow for 8 h with shaking at 30 °C. Purification of PfHRP II was performed by metal chelate chromatography on Ni2+-NTA-Sepharose followed by HPLC (Waters) on a reverse phase C18 μBondapak column. Heme polymerization activity was assayed using P. yoelii lysates or PfHRP II (40Sullivan D.J. Gluzman I.Y. Goldberg D.E. Science. 1996; 271: 216-221Crossref PubMed Scopus (351) Google Scholar, 41Pandey A.V. Singh N. Tekwani B.L. Puri S.K. Chauhan V.S. J. Pharm. Biomed. Anal. 1999; 20: 203-207Crossref PubMed Scopus (55) Google Scholar). For preparing parasite lysate, plasma and buffy coat were removed from the infected blood, and the erythrocyte pellet was washed once with PBS and suspended in 4 volumes of PBS containing glucose (0.9% w/v). The lysate was prepared by freezing the suspension in liquid nitrogen. Frozen droplets of the lysate were stored in aliquots at −70 °C until further use. When required, an aliquot of the lysate was thawed and centrifuged at 16,000 × g for 20 min at 4 °C. The pellet was resuspended in acetate buffer (100 mm, pH 5.0) by brief sonication and used for heme polymerization assay. The assay mixture contained 50 μl of the parasite extract, 100 μm hemin as the substrate and acetate buffer (100 mm, pH 5.0) in a total volume of 1.0 ml. Two controls, one lacking the substrate and the other lacking the parasite extract, but containing only 100 μm hemin in the acetate buffer, were run simultaneously. Each assay was set up in triplicate and incubated at 37 °C for 4 h (16 h for assays using PfHRP II) in a constantly shaking water bath. The reaction was stopped by centrifugation at 16,000 × g for 5 min, and pellets were resuspended in Tris-HCl (100 mm, pH 7.4) containing 2.5% SDS. The pellets were washed twice with same buffer and once with sodium bicarbonate buffer (100 mm, pH 9.0). The final pellet thus obtained was of polymerized heme (hemozoin). For quantitation of the hemozoin, the pellets were solubilized in 50 μl of 2 n NaOH, and spectra were recorded, using 2.5% SDS as solvent and blank, in the range of 360–700 nm on a Hitachi-557 double beam spectrophotometer. An extinction coefficient of 91,000m−1 cm−1 at 400 nm was used to quantitate the hemozoin in the form of heme as described previously (42Pandey A.V. Joshi S.K. Tekwani B.L. Chauhan V.S. Anal. Biochem. 1999; 268: 159-161Crossref PubMed Scopus (31) Google Scholar). In some experiments instead of parasite lysate, recombinant PfHRP II was used as a template for hemozoin formation. Interaction of drugs with malarial hemozoin was studied as described previously (43Pandey A.V. Tekwani B.L. FEBS Lett. 1997; 402: 236-240Crossref PubMed Scopus (25) Google Scholar). A suspension of fine crystals of purified hemozoin was prepared by sonication. Hemozoin was incubated in acetate buffer (pH 5.0, 100 mm, final volume 1.0 ml) for 4 h at 37 °C with the specified concentration of artemisinin or α-/β-arteether. Controls without the drug were also run simultaneously, and each assay was run in triplicate. At the end of the incubation period, the suspensions were centrifuged at 10,000 ×g for 5 min. The hemozoin pellets were washed once with ethanol to remove the drugs and once with alkaline bicarbonate buffer (pH 9.0, 100 mm). The amount of hemozoin remaining was quantified as described previously (27Chou A.C. Fitch C.D. Life Sci. 1992; 51: 2073-2078Crossref PubMed Scopus (70) Google Scholar). The difference between amount of hemozoin in the incubation mixtures without and with the drug gave the amount of hemozoin breakdown due to the endoperoxide antimalarials. Protein was estimated by the method of Lowry et al. (44Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. To study the effect of artemisinin and its analogue arteether on the hemoglobin catabolic process of the malarial parasite, we used purified digestive vacuoles from P. yoelii as the source of proteolytic activity. We found that artemisinin inhibited the proteolytic activity of the digestive vacuole lysate in an in vitro assay, involving degradation of 14C-labeled hemoglobin (TableI). Around 70% of the proteolytic activity in malaria food vacuole is due to aspartic acid proteases (13Francis S.E. Gluzman I.Y. Oksman A. Knickerboker A. Muller R. Bryant M.L. Sherman D.R. Russel D.G. Goldberg D.E. EMBO J. 1994; 13: 306-313Crossref PubMed Scopus (251) Google Scholar). After treatment of the vacuolar lysate with pepstatin A (a specific inhibitor of aspartic acid proteases), ∼71% inhibition of the proteolytic activity was observed. The remaining proteolytic activity (∼29%) in the reaction mixture could be inhibited by E-64 (a highly specific cysteine protease inhibitor). Artemisinin treatment also resulted in similar inhibition. Maximum inhibition caused by artemisinin was comparable to that achieved by E-64, suggesting the specific inhibition of cysteine protease by this drug. Addition of artemisinin in reaction mixture after E-64 treatment did not result in any significant addition into the inhibition of proteolysis as compared with E-64 alone. This indicated that the specific protease targets of both E-64 and artemisinin might be same. On the other hand, combination of either pepstatin A and E-64 or pepstatin A and artemisinin resulted in almost complete inhibition of the vacuolar proteolytic activity. This was expected as both aspartic and cysteine proteases would be blocked by these inhibitors. These results suggested that artemisinin may be involved in specific inhibition of malarial cysteine protease activity.Table IInhibition of P. yoelii vacuolar proteolytic activity by artemisinin using denatured hemoglobin as substrateSubstrate no.SampledpmInhibition%1Control1750 ± 3452Artemisinin1243 ± 19229 ± 113Arteether1195 ± 25332 ± 144E-641298 ± 21726 ± 125Heme1125 ± 30936 ± 186E-64 + artemisinin1147 ± 26635 ± 157Pepstatin A507 ± 19871 ± 118Pepstatin A + artemisinin93 ± 5995 ± 39Pepstatin A + E-6471 ± 6396 ± 4Concentration of inhibitors was as follows: artemisinin 200 μm, E-64 100 μm, pepstatin A 100 μm, heme 20 μm. Assay was performed as described under “Experimental Procedures.” Inhibitors were incubated with enzyme for 1 h before addition of substrate. Values are mean ± S.D. of triplicate observations. Open table in a new tab Concentration of inhibitors was as follows: artemisinin 200 μm, E-64 100 μm, pepstatin A 100 μm, heme 20 μm. Assay was performed as described under “Experimental Procedures.” Inhibitors were incubated with enzyme for 1 h before addition of substrate. Values are mean ± S.D. of triplicate observations. We next investigated whether the observed inhibition could be due to the specific interaction of artemisinin with the parasite cysteine protease. For this a fluorogenic peptide substrate of malarial cysteine protease, Z-Phe-Arg-AMC, was used to study the mechanism of protease inhibition by artemisinin. In a continuous fluorometric assay when artemisinin was incubated with the parasite lysate for 1 h prior to the assay, significant decrease in the proteolytic cleavage of peptide substrate was observed indicating that the drug could be making some modifications in the enzyme (TableII). The inhibition was significantly higher when heme was included in the reaction mixture along with artemisinin. No change in the protease activity was observed when artemisinin was added just before the start.Table IIInhibition of P. yoelii cysteine protease activity by artemisininSubstrate no.SampleΔ Fluorescence intensity(arbitrary units)/min1Control35 ± 52Artemisinin (preincubated with enzyme)19 ± 33Artemisinin (added before start)34 ± 64Artemisinin + heme (preincubation with enzyme) (heme 0.001 mm)11 ± 55Heme (preincubated with enzyme) (heme 0.001 mm)32 ± 66E-640.07Pepstatin A34 ± 5Activity was measured by degradation of specific peptide for plasmodial cysteine protease. Concentration of inhibitors was as follows: artemisinin 50 μm, E-64 100 μm, pepstatin A 100 μm, heme 1 μm. Assay was performed as described under “Experimental Procedures.” Inhibitors were incubated with enzyme for 1 h before addition of substrate. Open table in a new tab Activity was measured by degradation of specific peptide for plasmodial cysteine protease. Concentration of inhibitors was as follows: artemisinin 50 μm, E-64 100 μm, pepstatin A 100 μm, heme 1 μm. Assay was performed as described under “Experimental Procedures.” Inhibitors were incubated with enzyme for 1 h before addition of substrate. To confirm whether artemisinin could inhibit the hemoglobin degradation inside the malarial parasite, artemisinin was incubated in vitro with P. yoelii-infected erythrocytes for 0–6 h, and the hemoglobin levels in the cell-free parasite preparations were monitored by SDS-polyacrylamide gel electrophoresis. We observed that artemisinin-treated parasites showed a higher level of hemoglobin compared with the untreated parasites (Fig.1). These results suggest that degradation of hemoglobin in the digestive vacuole of the malarial parasite may have been inhibited by artemisinin treatment. When free heme is incubated with the particulate fraction of the cell-freeP. yoelii under conditions similar to that of the parasite food vacuole, a fraction of heme is polymerized, which can be identified as a product insoluble in SDS (2.5% w/v) and alkaline bicarbonate solution (41Pandey A.V. Singh N. Tekwani B.L. Puri S.K. Chauhan V.S. J. Pharm. Biomed. Anal. 1999; 20: 203-207Crossref PubMed Scopus (55) Google Scholar). However, no such product was formed when heme alone (without any parasite extract) was processed in a similar manner (29Pandey A.V. Joshi R.M. Tekwani B.L. Singh R.L. Chauhan V.S. Mol. Biochem. Parasitol. 1997; 90: 281-287Crossref PubMed Scopus (22) Google Scholar). We found that the formation of hemozoin by P. yoelii extract is dependent on incubation time, amount of protein, and concentration of free heme (41Pandey A.V. Singh N. Tekwani B.L. Puri S.K. Chauhan V.S. J. Pharm. Biomed. Anal. 1999; 20: 203-207Crossref PubMed Scopus (55) Google Scholar). A similar pattern has been observed by earlier workers (26Slater A.F.G. Cerami A. Nature. 1992; 355: 167-169Crossref PubMed Scopus (538) Google Scholar, 27Chou A.C. Fitch C.D. Life Sci. 1992; 51: 2073-2078Crossref PubMed Scopus (70) Google Scholar) for heme polymerization byP. falciparum and P. berghei. Recently, a histidine-rich protein (PfHRP II) from P. falciparum has been shown to catalyze polymerization of heme (40Sullivan D.J. Gluzman I.Y. Goldberg D.E. Science. 1996; 271: 216-221Crossref PubMed Scopus (351) Google Scholar). We also used recombinant PfHRP II as a source of heme polymerization activity in one of the experiments to study the effect of artemisinin on hemozoin formation in vitro. Artemisinin as well as α-/β-arteether caused marked inhibition of heme polymerization mediated by the parasite lysate (Fig.2 A) as well as PfHRP II (Fig.2 B). Inhibition of hemozoin formation by α-/β-arteether (IC50 value 7.3 μm) was slightly higher than that by artemisinin (IC50 value 11.5 μm) (Fig. 2 A). The effect of increasing substrate (Fig.3 A) and inhibitor (Fig.3 B) concentration on inhibition of heme polymerization was also studied. Normally polymerization of heme in the presence of parasite extract or PfHRP II is a saturable reaction following a hyperbolic pattern (41Pandey A.V. Singh N. Tekwani B.L. Puri S.K. Chauhan V.S. J. Pharm. Biomed. Anal. 1999; 20: 203-207Crossref PubMed Scopus (55) Google Scholar). However, the inhibition of heme polymerization by artemisinin was not affected by the presence of high concentrations of heme to any significant extent; following an initial fall in the percent inhibition between 5 and 10 μm, the inhibition potency of drug remained almost constant up to 200 μmheme (Fig. 3 A). The pattern of inhibition was qualitatively similar at 1 and 10 μm concentrations of artemisinin with the expected quantitative increase for higher drug concentration. AK i value of 12.0 μm was obtained for inhibition of P. yoelii-mediated heme polymerization by artemisinin, from the Dixon plot (Fig. 3 B).Figure 3Inhibition of heme polymerase activity ofP. yoelii extracts by artemisinin at varying substrate concentrations (A) and varying concentration of the drug (B). A, Lineweaver-Burk plot of the reaction containing 0 (○), 1.0 μm (●), and 10.0 μm (♦) inhibitor (artemisinin) and variable concentrations of the substrate (heme). B, Dixon plot of inhibition containing 50 μm (●) or 100 μm(○) of substrate (heme) and variable concentrations of inhibitor (artemisinin). The values are mean ± S.D. of triplicate observations.View Large Image Figure ViewerDownload (PPT) Studies on the interaction of purified hemozoin with artemisinin and α-/β-arteether revealed a novel reaction. The incubation of purified hemozoin with artemisinin or α-/β-arteether under acidic conditions (acetate buffer, 100 mm, pH 5.0) resulted in the loss of hemozoin contents as compared with control, indicating that hemozoin may be disrupted as a result of its interaction with the endoperoxide. This hemozoin disruption increased with the increasing concentration of the drug (Fig.4). Interaction of hemozoin with the drug may result in breakdown of the hemozoin pigment which could then form a complex with the heme units (38Hong Y.L. Yang Y.Z. Meshnick S.R. Mol. Biochem. Parasitol. 1994; 63: 121-128Crossref PubMed Scopus (176) Google Scholar). Earlier Meshnick et al. (5Meshnick S.R. Taylor T.E. Kamchonwongpaisan P. Microbiol. Rev. 1996; 60: 301-315Crossref PubMed Google Scholar) proposed a two-step mechanism for the antimalarial action of artemisinin and other related endoperoxides. In the first step, the endoperoxide bridge in artemisinin is cleaved by free heme or iron, leading to the generation of an unstable radical of the drug. This subsequently causes selective alkylation of malarial proteins, leading to death of the parasite. However, the transient source of free heme or iron proposed in this mechanism has n
Год издания: 1999
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Malaria Research and Control, Computational Drug Discovery Methods, Drug Transport and Resistance Mechanisms
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