Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14alpha-demethylase (CYP51)

Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Sterol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14alpha-demethylase (CYP51). Structure-activity relationship study of the binding with the overexpressed human CYP51 indicates that the pyridine binds within the heme binding pocket in an analogy with the azoles.     

A relationship between the activities of hepatic lanosterol 14 alpha-demethylase and 3-hydroxy-3-methylglutaryl-CoA reductase.

At 1-2 h after intragastric administration of ketoconazole, a cytochrome P-450 inhibitor, to rats, there was a 50-60% decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Inhibition reached a maximum at 6-12 h after the drug was given, but after 24 h enzyme activity was stimulated by 60%. The rates of synthesis of hepatic non-saponifiable lipids in vivo showed a similar time-dependent pattern of change. During the first few hours after drug administration, the hepatic cytochrome P-450-dependent metabolism of lanosterol was suppressed in vivo. However, 24 h after treatment, this activity was stimulated, an effect which was also observed by pre-treatment of the rats with the drug for several days. Suppression of hepatic HMG-CoA reductase and lanosterol 14 alpha-demethylase activities was accompanied by a relative increase in the accumulation of labelled polar sterols in the liver in vivo. In the intestine, ketoconazole also resulted in a rapid decline in the rate of synthesis of non-saponifiable lipids and an inhibition of lanosterol 14 alpha-demethylation in vivo. However, in contrast with the liver, there was no stimulation of non-saponifiable lipid synthesis after 24 h.     

Non-sterol structural probes of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae

A number of non-sterol iron-liganding molecules were used to probe the active site of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae. Simple bi- and tricyclic aromatic amines were found to exhibit Type II binding spectra with the demethylase. Stereochemical and positional effects appear to play critical roles in the binding of these compounds to the demethylase. These compounds have been used to generate additional active-site structural information on this enzyme, currently a target for the development of new antifungal agents.     

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.     

Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol.

Male rats were fed a cholesterol-free diet or the same diet supplemented with either 0.05, 0.1, 0.25, 0.5, 1, or 2% C for 21 days to investigate the effects of cholesterol on secretion of very low density lipoprotein (VLDL). Cholesterol feeding increased plasma and hepatic concentrations of triglyceride (TG) and cholesteryl esters (CE) in a dose-dependent manner. Plasma VLDL and low density lipoprotein (LDL) lipids were elevated by cholesterol feeding, while the high density lipoprotein (HDL) lipids were reduced. The secretion of the VLDL by perfused livers from these cholesterol-fed rats was examined to establish the relationship between the accumulation of lipids in the liver and the concurrent hyperlipemia. Liver perfusions were carried out for 4 h with a medium containing bovine serum albumin (3% w/v), glucose (0.1% w/v), bovine erythrocytes (30% v/v), and a 10-mCi 3H2O initial pulse. Oleic acid was infused to maintain a concentration of 0.6 mM. Hepatic secretion of VLDL-TG, PL (phospholipid), free cholesterol (FC), and CE increased in proportion to dietary cholesterol and was maximal at 0.5% cholesterol in these experiments in which TG synthesis was stimulated by oleic acid. Secretion of VLDL protein and apoB by the perfused liver was also increased. The molar ratios of surface (sum of PL and cholesterol) to core (sum of TG and CE) lipid components of the secreted VLDL, regardless of cholesterol feeding, were the same, as were the mean diameters of the secreted particles. The molar ratios of surface to core lipid of VLDL isolated from the plasma also were not affected by cholesterol feeding. During perfusion with oleic acid of livers from the rats fed the higher levels of cholesterol, the hepatic concentration of CE decreased, while the level of TG was not changed. We conclude that the hypercholesterolemia and hypertriglyceridemia that occur in vivo from cholesterol feeding, concurrent with accumulation of CE and TG in the liver, must result, in part, from increased hepatic secretion of all VLDL lipids and apoB. The VLDL particles produced by the liver of the cholesterol-fed rat are assembled without modification of the surface lipid ratios (PL/FC), but contain a greater proportion of cholesteryl esters compared to triglyceride in the core, because of the stimulated transport of CE from the expanded pool in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction and substrate specificity of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae Y222.

The potential inducibility of the lanosterol 14 alpha-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37 degrees C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[a]pyrene.     

Regulation of guinea pig hepatic acyl-coa:cholesterol acyltransferase activity by dietary fat saturation and cholesterol

We measured the interactive effects of dietary cholesterol and fat on the regulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity and its relationship to hepatic microsomal lipid composition in guinea pigs fed 15 g/100 g (w/w) fat diets (corn oil, olive oil, or lard) with 0.01, 0.08, 0.17, or 0.33 g/100 g (w/w) added cholesterol. Guinea pigs exhibited a dose dependent increase in hepatic microsomal ACAT activity, with increasing levels of cholesterol intake (P < 0.001) in all dietary fat groups. Animals fed monounsaturated olive oil had the highest hepatic ACAT activity with the exception of the 0.33 g/100 g cholesterol diet (P < 0.001). There were no differences in ACAT activity with intake of polyunsaturated corn oil or saturated lard. Dietary cholesterol resulted in increased microsomal free cholesterol (FC) concentrations in a dose dependent manner but had no effects on microsomal phosphatidylcholine (PC) concentrations. Guinea pigs fed olive oil generally had the highest microsomal FC/PC molar ratios, and hepatic ACAT activities correlated significantly with this parameter. After modification of the lipid compositions of the microsomes from guinea pigs fed the 12 test diets with FC/PC liposome treatment, microsomal ACAT activities remained significantly related to the microsomal FC/PC molar ratios, and dietary fat type did not affect this correlation. Our findings do not support the hypothesis that the stimulation of hepatic ACAT activity with cholesterol intake is enhanced by polyunsaturated fat intake. The data demonstrate that although dietary fat type and cholesterol amount have differential effects on hepatic ACAT activity, substrate availability, expressed as microsomal FC/PC molar ratio, is a major regulator of hepatic microsomal ACAT activity.     

Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11).

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.     

Purification and characterization of cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from pig liver microsomes.

Cytochrome P-45014DM, which catalyzes lanosterol 14 alpha-demethylation, from pig liver microsomes was purified to a state of virtually homogeneous by gel electrophoresis. Its apparent monomeric molecular weight was estimated to be 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the amino-terminal amino acid sequence was Gly-Leu-Leu-Thr-Gly(Leu)-Asp-Leu-Leu-Gly-Ile. When reconstituted with NADPH-cytochrome P-450-reductase, the enzyme showed a high activity for lanosterol and 24,25-dihydrolanosterol 14 alpha-demethylation. Furthermore, the oxygenated intermediates of 24,25-dihydrolanosterol 14 alpha-demethylation, 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol, were converted to the 32-nor compound, 4,4-dimethylcholesta-8,14-dien-3 beta-ol, by the reconstituted enzyme system.     

Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes

Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14 alpha-demethylase and cholesterol 7 alpha-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel and adsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mg of protein and a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 51,000. A Soret maximum for the reduced/CO binding complex at 448 nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochrome-c reductase, dilaurylphosphatidylcholine, NADPH, and O2 supports the demethylation process which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediates with single catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P-450 is responsible for all three oxidations and the lyase activity involved in the lanosterol C-32 demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterol substrates is observed for the purified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutive P-450 involved in anabolic biochemical processes.     

Role of the side chain of lanosterol in substrate recognition and catalytic activity of lanosterol 14 alpha-demethylase (cytochrome P-450 (14DM)) of yeast

The 14 alpha-demethylation of 24,25-dihydrolanosterol (DHL) derivatives having trimmed side chains, 27-nor-DHL, 26,27-dinor-DHL, 25,26,27-trinor-DHL, 24,25,26,27-tetranor-DHL, 23,24,25,26,27-pentanor-DHL and 22,23,24,25,26,27-hexanor-DHL, was studied with the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-450(14DM) and NADPH-cytochrome P-450 reductase both purified from yeast microsomes. The demethylase catalyzed the 14 alpha-demethylation of the derivatives having the side chains longer than tetranor but the activities for the trinor- and tetranor-derivatives were lower. Kinetic analysis indicated that affinity of the trinor-derivative for the demethylase was considerably higher than that of DHL. The affinities of the 27-nor- and dinor-derivatives were increased by this order and were the intermediates of DHL and the trinor derivative. On the other hand, Vmax values of the demethylase for the DHL derivatives were decreased depending on their side-chain lengths, and the substrate-dependent reduction rate of cytochrome P-450(14DM) was also decreased in the same manner. Based on these observations, it was concluded that interaction of the side chain of lanosterol especially C-25, 26 and 27 with the substrate site of lanosterol 14 alpha-demethylase was necessary for enhancing the catalytic activity of the enzyme. However, this interaction was considered not to be essential for substrate binding.     

Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans

The Saccharomyces cerevisiae cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase)-coding gene was used as a hybridization probe to isolate two HindIII fragments of 2.5 kb and 6.85 kb from a phage lambda library of Candida albicans nucleotide sequences. Restriction endonuclease mapping and Southern blot hybridization experiments indicated that these fragments represent two allelic forms of the same gene. This cloned sequence, when introduced into S. cerevisiae or C. albicans on a multiple copy vector, produced an increase in cytochrome P450 content and resistance to imidazole antifungal agents which are inhibitors of cytochrome P450 L1A1. In addition, the cloned sequence was able to complement a cytochrome P450 L1A1 gene disruption when introduced into S. cerevisiae. These data indicate that the cloned sequence codes for the lanosterol 14 alpha-demethylase cytochrome P450 L1A1 from C. albicans.     

Deformylation of 32-oxo-24,25-dihydrolanosterol by the purified cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from yeast evidence confirming the intermediate step of lanosterol 14 alpha-demethylation

32-Oxo-24,25-dihydrolanosterol (32-oxo-DHL) was deformylated to 4,4-dimethylcholesta-8,14-dien-3 beta-ol, the product of 14 alpha-demethylation of 24,25-dihydro-lanosterol (DHL), by the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-45014DM and NADPH-cytochrome P-450 reductase of yeast. Affinity of 32-oxo-DHL to the cytochrome was considerably higher than those of lanosterol and DHL, and the rate of deformylation of 32-oxo-DHL was faster than the rate of demethylation of lanosterol and DHL. Spectral analysis of the 32-oxo-DHL complex of cytochrome P-45014DM suggested the interaction between the 32-aldehyde group and the heme iron. These observations, together with our preceding findings on the metabolism of 32-hydroxy-24,25-dihydrolanosterol (Aoyama, Y., Yoshida, Y., Sonoda, Y., and Sato, Y. (1987) J. Biol. Chem. 262, 1239-1243), indicate that the 14 alpha-demethylation of lanosterol catalyzed by cytochrome P-45014DM proceeds with three step monooxygenations via the 32-hydroxy and 32-oxo intermediates, and the cytochrome mediates this sequential reaction without releasing the intermediates.     

Expression of microsomal lanosterol 14alpha-demethylase (CYP51) in an engineered soluble monomeric form

We prepared a soluble monomeric form of bovine cytochrome P450 lanosterol 14α-demethylase (CYP51), which in mammals is a ubiquitously expressed membrane protein of the endoplasmic reticulum. We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains. Both of these were expressed in Escherichia coli at levels of 500 nmol/l. The protein variants were purified and tested for the solubility in the absence of detergent. Variant bCYP51-d1 exhibited ∼10-fold better solubility over variant bCYT51-d2. The bCYP51-d1 eluted as a single peak in size-exclusion chromatography, corresponding to its monomeric form. The activity of bCYP51-d1 is similar to that of recombinant human CYP51 with a non-truncated membrane-spanning region. High solubility and low tendency to non-specific oligomer formation make bCYP51-d1 a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme.     

Homology modeling of lanosterol 14alpha-demethylase of Candida albicans and Aspergillus fumigatus and insights into the enzyme-substrate Interactions

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MT_14DM) provides a good template for modeling the three dimensional structure of lanosterol 14alpha-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14alpha-demethylase from Candida albicans (CA_14DM) and Aspergillus fumigatus (AF_14DM) were built on the basis of the crystal coordinates of MT_14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandran plots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P450(14DM). The overall structures of the resulting CA_14DM model and AF_14DM model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues, the residues lining the substrate access channel, and residues in active site were identified from the model. To explore the binding mode of the substrate with the two models, 24(28)-methylene-24,25-dihydrolanosterol was docked into the active site of the two models and hydrophobic interaction and hydrogen-bonding were found to play an important role in substrate recognition and orientation. These results provided a basis for experiments to probe structure-function relationships in the P450(14DM). Although CA_14DM and AF_14DM shared similar core structural character, the active site of the two models were quite different, thus allowing the rational design of specific inhibitors to the target enzyme and the discovery of novel antifungal agents with broad spectrum.