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New Azasterols Against Trypanosoma Brucei: Role of 24-Sterol

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New Azasterols Against Trypanosoma Brucei: Role of 24-Sterol ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2006, p. 2595–2601 Vol. 50, No. 8 0066-4804/06/$08.00ϩ0 doi:10.1128/AAC.01508-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved. New Azasterols against Trypanosoma brucei: Role of 24-Sterol Methyltransferase in Inhibitor Action Ludovic Gros,1 Victor Manuel Castillo-Acosta,2 Carmen Jime´nez Jime´nez,2 Marco Sealey-Cardona,2 Sofia Vargas,2 Antonio Manuel Este´vez,2 Vanessa Yardley,3 Lauren Rattray,3 Simon L. Croft,3 Luis M. Ruiz-Perez,2 Julio A. Urbina,4 Ian H. Gilbert,1 and Dolores Gonza´lez-Pacanowska2* Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, United Kingdom1; Instituto de Parasitologı´a y Biomedicina Lo´pez-Neyra, Parque Tecnolo´gico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain2; Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom3; and Laboratorio de Quı´mica Biolo´gica, Centro de Bioquı´mica y Biofı´sica, Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Altos de Pipe, Downloaded from Km. 11, Carretera Panamericana, Caracas 1020, Venezuela4 Received 23 November 2005/Returned for modification 3 January 2006/Accepted 5 May 2006 A series of azasterol derivatives, designed as potential inhibitors of the ⌬24-sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nano- molar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense blood- aac.asm.org stream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component at UNIVERSITY OF PENNSYLVANIA LIBRARY on July 12, 2007 of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei. Diseases caused by parasitic protozoa affect many people in ation of the 24 position of the side chain, a step not found in large areas of the world (24). Among diseases caused by the cholesterol biosynthesis. This step is catalyzed by S-adenosyl- 24 protozoa of the Trypanosomatidae family, Chagas’ disease L-methionine:⌬ -sterol methyltransferase (24-SMT), which is a (American trypanosomiasis), sleeping sickness (human African key difference between cholesterol and ergosterol biosynthesis. trypanosomiasis), and leishmaniasis are of importance. The Azasterols have been shown to cause inhibition of 24-SMT; ex- cure of these diseases depends largely on chemotherapy (4). amples are given in references 10, 11, 13, 16, 20, and 22. The Although several antiparasitic drugs are available, toxic side supposed mechanism of action of 24-SMT (18) goes through a effects and development of drug resistance are often associated carbocationic intermediate which could be mimicked by azaster- with them. Therefore, new, effective drugs have to be found. ols protonated at physiological pH (10). However, some azaster- The sterol biosynthetic pathway presents opportunities for ols have also been reported to inhibit the sterol 24-reductase, an the design of anti-infective agents (1, 15, 19). Ergosterol and enzyme involved in biosynthesis of cholesterol. This latter effect 24-alkylated sterols are major components in the cell mem- leads to a buildup of steroid precursors to toxic concentrations branes of fungi and plants and also of Leishmania spp. and (17). Trypanosoma cruzi. This contrasts to the situation in mamma- Previously, we (9, 16, 21–23) and others (5) reported the lian cells, where cholesterol is the principle steroid found. In activity of 22,26-azasterol (AZA) (Fig. 1) as an agent against the case of Trypanosoma brucei, the procyclic form of the Leishmania and T. cruzi. AZA was found to be active against parasite has been reported to biosynthesize ergosterol and both promastigote/epimastigote and intracellular amastigote other 24-alkylated sterols (2, 3), while in the bloodstream form forms of the parasites and has been shown to give parasitolog- (BSF), the parasite is reported to scavenge cholesterol from ical cure in a rodent model of Chagas’ disease (21). Encour- the host with uptake from LDL receptors. These differences in aged by those findings, we decided to extend the range of sterol metabolism between the parasites and mammalian cells azasterols; some of these compounds were active against are clearly important and could be exploited as an antiparasitic bloodstream form Trypanosoma brucei subsp. rhodesiense, the drug target. causative organism of human African trypanosomiasis (7, 8). Biosynthesis of ergosterol and related sterols requires alkyl- In the present paper, we report some new analogues, their biological evaluation, and studies on their mode of action. * Corresponding author. Mailing address: Instituto de Parasitologı´a y Biomedicina Lo´pez-Neyra, Consejo Superior de Investigaciones Cien- MATERIALS AND METHODS tı´ficas, Avda. del Conocimiento s/n, Parque Tecnolo´gico de Ciencias Materials. Chemicals, reagents, and solvents were purchased from Aldrich, de la Salud, 18100 Armilla, Granada, Spain. Phone: 34 958 181621. Sigma, Lancaster, Merck, Fisher, or Steraloids and employed without further Fax: 34 958 181633. E-mail: [email protected]. purification, unless otherwise stated. Genomic DNA from the Trypanosoma 2595 2596 GROS ET AL. ANTIMICROB.AGENTS CHEMOTHER. BL21(DE3) cells, which were plated on LB agar plates containing 34 ␮gof kanamycin per ml. Enzyme activity assays. In assays of inhibition of 24-SMT, soluble protein extracts from E. coli BL21(DE3)/pET28TbSMT cells were used. Trypanosoma brucei supsp. brucei recombinant 24-SMT is produced as a His-tagged fusion protein and is overexpressed when induced with 1 mM IPTG (isopropyl-␤-D- thiogalactopyranoside) for 4 h. Cells were disrupted by sonication in a buffer containing 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 4 mM CHAPS {3-[(3- cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 0.5% (vol/vol) Tween FIG. 1. Structure of 22,26-azasterol (AZA). 80, and protease inhibitors. The sonicate was centrifuged at 12,000 rpm for 30 min at 4°C to obtain the soluble fraction, which contained the active form of the enzyme. When parasite extracts were analyzed, bloodstream forms were collected at 2 ϫ 106 6 brucei subsp. brucei 427 strain was used for 24-SMT cloning. Fetal calf serum was cells per ml, while procyclic forms were collected at 7.5 ϫ 10 cells per ml. Cultures obtained from Gibco. Trypanosoma brucei subsp. rhodesiense STIB900 and were centrifuged at 2,600 rpm for 10 min at 4°C; parasites were washed twice with Trypanosoma brucei subsp. brucei 427 were used in growth inhibition assays. PBS (pH 7.2) and lysed by sonication in the same buffer as that used for bacterial Inhibitor synthesis. Nuclear magnetic resonance spectra were obtained with a cells. Total extracts obtained after sonication were used in the enzymatic assay. A Bruker Avance DPX 300-MHz spectrometer at 300 MHz for 1H and 75 MHz for standard 24-SMT activity assay contained 1.7 mg/ml of bacterial soluble extracts or Downloaded from 13C. Mass spectra and exact mass measurements were performed on a Waters 3.3 mg/ml of parasitic total extracts in buffer containing 50 mM Tris-HCl (pH 7.4), ␮ ZQ4000 and a Finnigan MAT 95XP, respectively. Precoated Merck silica gel 2 mM MgCl2, 4 mM CHAPS, 0.5% (vol/vol) Tween 80, 100 M desmosterol, and 14 5 6 F254 plates were used for thin-layer chromatography, and spots were examined 200 ␮M[ C]S-adenosyl-L-methionine (6 ϫ 10 dpm or 1 ϫ 10 dpm per reaction). with phosphomolybdic acid (0.5% in ethanol) solution. Column chromatography Desmosterol was dissolved in chloroform, which was evaporated before the rest of was performed on silica gel 60 (0.035 to 0.070 mm). The 1H and 13C nuclear the components were added. The inhibitor was resuspended first in a minimal magnetic resonance spectra allowed the characterization of all purified interme- volume of its corresponding solvent and later added to the reaction mixture as an diates in the synthesis and final products. The full synthetic details are described aqueous solution. The reaction was started with the enzyme. Incubations were aac.asm.org elsewhere (4a). performed at 30°C for 45 min and terminated with 0.5 ml of 10% KOH dissolved in Growth inhibition of Trypanosoma brucei subsp. rhodesiense and Trypanosoma 80% (vol/vol) methanol. To quantify the efficiency of the extraction, [3H]cholesterol brucei subsp. brucei. Trypanosoma brucei subsp. rhodesiense STIB900 BSF trypo- (3 mg, 30,000 dpm per reaction) was added as an internal standard. The methylated mastigotes were maintained in HMI-18 medium (6) with 15% heat-inactivated sterol product was extracted three times with 1 ml of hexane and the resulting 14 L at UNIVERSITY OF PENNSYLVANIA LIBRARY on July 12, 2007 fetal calf serum (Harlan-SeraLab, United Kingdom) at 37°C in a 5% CO2–95% organic layer washed once with Tris-HCl buffer to remove the [ C]S-adenosyl- - air mixture. Trypomastigotes were washed and resuspended in fresh medium at methionine that was not incorporated. One milliliter of the organic layer was added a concentration of 2 ϫ 105/ml. The top concentration for the test compounds was to 10 ml of hydrofluor (National Diagnostics), and the radioactivity was measured in 30 ␮g/ml.
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