The squalene-2,3-epoxide cyclase as a model for the development of new drugs. The azole drugs (itraconazole, ketoconazole, etc.) target the lanosterol C14-demethylase enzyme in the ergosterol biosynthesis pathway (Fig. ?(Fig.1).1). Azole drugs cause the accumulation of 14-methylsterols and decreased production of ergosterol (25). The allylamine antifungal CHMFL-KIT-033 drug terbinafine inhibits squalene epoxidase in the sterol biosynthesis pathway (Fig. ?(Fig.1).1). Terbinafine was shown to be synergistic with ketoconazole against cultures of (17, 24). The polyene antifungal drug amphotericin B works by directly associating with ergosterol to disrupt the integrity of the cell membrane. Amphotericin B and its liposomal preparations have potent anti-activities (14, 31). Open in a separate windows FIG. 1 Biosynthesis of ergosterol as characterized in yeast. The pathway shows the major intermediates (strong character types) and enzymes by their yeast designations (ERG1, squalene epoxidase; ERG7, oxidosqualene cyclase; ERG11, C14-demethylase). Drug classes that take action on selected sites in the pathway are CHMFL-KIT-033 shown in italics. The synthesis of lanosterol is an essential step in the production of mature sterols. In yeast and higher eukaryotes (including humans), oxidosqualene cyclase (OSC) directly catalyzes the synthesis of lanosterol from 2,3-oxidosqualene (Fig. ?(Fig.2).2). This is a remarkably complex cyclization-rearrangement reaction involving the formation of a total of six new carbon-carbon bonds by a single enzyme (2). Inhibitors of OSC are under investigation as potential antifungal drugs (12) and cholesterol-lowering drugs (1, 6). One series of OSC inhibitors was designed as electron-poor aromatic mimics of a monocyclized transition state or high-energy intermediate formed from oxidosqualene (21). In this paper, we report that these compounds have potent activities against and inhibit sterol biosynthesis in these organisms. Open in a separate windows FIG. 2 Conversion of 2,3-oxidosqualene to lanosterol. MATERIALS AND METHODS Test compounds. Benznidazole (Rochagan; Roche Pharmaceuticals, Rio de Janeiro, Brazil) was extracted from tablets with methanol-CHCl3 (1:1) and further purified via flash silica gel chromatography. OSC inhibitor no. 1 ((22) that expresses the -galactosidase gene was described previously (5). Peru and Sonya strains of (20) were kindly provided by S. Croft (London School of Hygiene and Tropical Medicine, London, United Kingdom). The VL2067 strain (from Minas Gerais, Brazil) was kindly provided by J. Peralta (Federal University, Rio de Janeiro, Brazil). Epimastigotes were grown in liver infusion tryptone broth with 10% fetal bovine CHMFL-KIT-033 serum, penicillin, and streptomycin as previously described (27). Amastigotes were produced at 37C in monolayers of murine 3T3 fibroblasts in RPMI 1640 (Biowhittaker Inc., Walkersville, Md.) with 10% fetal bovine serum, penicillin, streptomycin, and glutamine as previously described (27). Transfection and cloning of parasites. In order to perform drug screening assays using a colorimetric method (5), we transfected the Peru, Sonya, and VL2067 strains with the gene. The plasmid (pBS:CL-Neo-01-BC-LacZ-10) was first linearized for integrative transformation, and 5 g of DNA was electroporated into epimastigotes as previously described (7). Transfectants were selected by growth in G418 (Gibco BRL, Rockville, Md.) at 500 g/ml. The drug-resistant populace of epimastigotes was cloned by limiting dilution. Clones were tested for the ability to catalyze the colorimetric reaction with the substrate, chlorophenolred–d-galactopyranoside (CPRG; Boehringer-Mannheim, Indianapolis, Ind.) (5). The clones were transformed into mammalian-stage parasites by inoculation of the culture onto monolayers of 3T3 fibroblasts at 37C. RAB7B After approximately one week, intracellular amastigotes were visible microscopically, and shortly thereafter the host cells spontaneously lysed and released trypomastigotes. These trypomastigotes were used to infect subsequent monolayers of fibroblasts. -Galactosidase expressing clones that were observed to have essentially the same growth rate in fibroblasts as untransfected parasites were used for subsequent experiments. Growth inhibition assay of mammalian stages of was performed as described previously (5). The assays were performed in 96-well tissue culture plates (Costar, Cambridge, Mass.). 3T3 fibroblasts were inoculated at 103/well using RPMI 1640 without phenol red (Biowhittaker Inc.) plus 10% fetal bovine serum and glutamine. The next day, the plates were seeded with 3T3-derived trypomastigotes at 104/well. After 4 h, drugs were added in serial dilutions to give a final volume of 200 l/well. The plates were incubated at 37C in 5% CO2 atmosphere for 7 days. At this time, CPRG (100 M final) and Nonidet P-40 (0.1% final) (Sigma Chemical Co., St. Louis, Mo.) were added and the plates were incubated at 37C for approximately 4 h. Wells with -galactosidase activity switched the media from yellow to red, and this was quantified on an enzyme-linked immunosorbent assay reader at epimastigotes (1 107 in 1 ml of culture medium) were incubated for 24 h with 100 Ci of RS[5-3H]mevalonolactone (American Radiolabel Chemicals, St. Louis, Mo.) with or without inhibitors. The cells were centrifuged and washed once in phosphate-buffered saline..