title: Selective inhibition of mammalian lanosterol 14 alpha-demethylase by RS-21607 in vitro and in vivo.
abstract: The discovery of selective lanosterol 14 alpha-demethylase inhibitors may lead to novel hypolipidemic drugs. RS-21607, (2S,4S)-cis-2[1H-imidazol-1-yl)methyl]-2-[2-(4-chlorophenyl)ethyl]-4- [[(4-aminophenyl)thio]methyl]-1,3-dioxolane, was characterized as a tight-binding, competitive inhibitor of lanosterol 14 alpha-demethylase purified from rat liver. The apparent Ki was determined to be 840 pM and found to be similar in hepatic microsomes from human, rat, and hamster. RS-21607, which contains two chiral centers, was a more effective lanosterol 14 alpha-demethylase inhibitor than its three stereoisomers. In vitro, RS-21607 had a greater affinity for lanosterol 14 alpha-demethylase than the other cytochromes P450 evaluated: CYP7, CYP27, CYP11A1, CYP19, CYP17, CYP11B1, CYP21, CYP3A4, CYP4A, CYP2D6, CYP1A2, CYP2C9, and 27-hydroxycholesterol 7 alpha-hydroxylase. The other stereoisomers were not as selective as RS-21607. Doses of 3-30 mg/kg RS-21607 given orally to hamsters caused a dose-dependent decrease in cholesterol biosynthesis with a corresponding accumulation of 24,25-dihydrolanosterol. RS-21607 inhibited the enzyme and cholesterol biosynthesis in hamster liver by 50% at 18 h following a 30 mg/kg oral dose. This was interpreted to indicate that RS-21607 is able to distribute to the site of action in hamsters and inhibit the target enzyme. In the same dose range, the plasma concentrations of testosterone, corticosterone, and progesterone, the endpoints for the cytochromes P450 involved in steroid biosynthesis, were relatively unaffected. These data show RS-21607 to be an effective and selective inhibitor of lanosterol 14 alpha-demethylase, both in vivo and in vitro. RS-21607 interacted with the purified enzyme to produce a type II binding spectrum, consistent with an interaction between the imidazole moiety and the heme. The electrostatic contribution of the imidazole binding was investigated using the desimidazole analog of RS-21607. The apparent Ki for the desimidazole compound (65 microM) was similar to the apparent Km for the substrate DHL (79 microM). Together, these data confirm that the ligand attached to the imidazole in RS-21607 is a good non-sterol substitute for DHL, i.e., binding to the enzyme with similar affinity, and that the coordination of the imidazole to the heme provides a major electrostatic contribution for the inhibition of lanosterol 14 alpha-demethylase by RS-21607. RS-21607 was also observed to increase the accumulation of 3 beta-hydroxy-24,25-dihydrolanost-8-en-32-al, the second intermediate in the multistep oxidation, but not the first intermediate. 24,25-dihydrolanost-8-ene-3 beta,32-diol.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Syntex Discovery Research, Palo Alto, California 94304.
authors.forename: D C
authors.initials: DC
authors.lastname: Swinney
authors.forename: O Y
authors.initials: OY
authors.lastname: So
authors.forename: D M
authors.initials: DM
authors.lastname: Watson
authors.forename: P W
authors.initials: PW
authors.lastname: Berry
authors.forename: A S
authors.initials: AS
authors.lastname: Webb
authors.forename: D J
authors.initials: DJ
authors.lastname: Kertesz
authors.forename: E J
authors.initials: EJ
authors.lastname: Shelton
authors.forename: P M
authors.initials: PM
authors.lastname: Burton
authors.forename: K A
authors.initials: KA
authors.lastname: Walker
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002784:Cholesterol / Q000096:biosynthesis; D006224:Cricetinae; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D007810:Lanosterol / Q000031:analogs & derivatives / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D013045:Species Specificity; D013237:Stereoisomerism; D058886:Sterol 14-Demethylase; D013440:Sulfides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014316:Tritium
chemical_list: D000814:Aniline Compounds; C548418:CYP51A1 protein, human; C548417:Cyp51 protein, rat; D065607:Cytochrome P-450 Enzyme Inhibitors; D013440:Sulfides; D014316:Tritium; D007810:Lanosterol; C082410:azalanstat; D007987:Gonadotropin-Releasing Hormone; C015316:lanostenol; D000324:Adrenocorticotropic Hormone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D007654:Ketoconazole