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At least 19 recordsLinked to original sources
PubMed1992

Plant sterol biosynthesis: novel potent and selective inhibitors of cytochrome P450-dependent obtusifoliol 14 alpha-methyl demethylase.

The R-(-) isomer of methyl 1-(2,2-dimethylindan-1-yl)imidazole-5-carboxylate (CGA 214372; 2) strongly inhibited P450-dependent obtusifoliol 14 alpha-demethylase (P450OBT.14DM) (I50 = 8 x 10(-9) M, I50/Km = 5 x 10(-5) in a maize (Zea mays) microsomal preparation. Kinetic studies indicated uncompetitive inhibition with respect to obtusifoliol. The corresponding S-(+) isomer was a 20-fold weaker inhibitor for P450OBT.14DM. The molecular features of a variety of analogues of 2 were related to their potency as inhibitors of P450OBT.14DM in vitro, allowing delineation of the key structural requirements governing inhibition of the demethylase. CGA 214372 proved to have a high degree of selectivity for P450OBT.14DM. This allowed easy distinction of this activity from other P450-dependent activities present in the maize microsomal preparation and gave strong evidence that P450OBT.14DM is a herbicidal target. Microsomal maize P450OBT.14DM and yeast P450LAN.14DM, the only known examples of P450-dependent enzymes carrying out an identical metabolic function in different eukaryotes, showed distinct inhibition patterns with CGA 214372 and ketoconazole, a substituted imidazole anti-mycotic.

Azoles↗
PubMed1992

A new series of natural antifungals that inhibit P450 lanosterol C-14 demethylase. II. Mode of action.

From a Penicillium sp. we identified a new series of antifungals having a tetrahydropyran skeleton with an alkenyl side chain. We elucidated the mode of action of Ro 09-1470, the most active compound of the series. Treatment of Candida albicans with the compound caused an accumulation of C-14 methyl intermediates of ergosterol at concentrations of which no significant interference with the biosyntheses of other macromolecules and respiration was observed. P450 lanosterol C-14 demethylase (P450(14DM)) activity was inhibited and furthermore, the binding of Ro 09-1470 to the heme of the enzyme was demonstrated by a difference spectrum. We conclude that Ro 09-1470 is the first natural antifungal that inhibits the P450(14DM) of fungi.

Candida albicans↗
PubMed1991

Effects of a novel lanosterol 14 alpha-demethylase inhibitor on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hep G2 cells.

A 32-carboxylic acid derivative of lanosterol (SKF 104976) was found to be a potent inhibitor of lanosterol 14 alpha-demethylase (14 alpha DM). 14 alpha DM activity in a Hep G2 cell extract was inhibited 50% by 2 nM SKF 104976. Exposure of intact cells to similar concentrations of the compound resulted in the inhibition of incorporation of [14C]acetate into cholesterol with concomitant accumulation of lanosterol as well as a 40-70% decrease in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity. SKF 104976 did not effect low density lipoprotein uptake and degradation in Hep G2 cells, suggesting that HMGR and low density lipoprotein receptor activity were not coordinately regulated under these conditions. Reduction of the flux of carbon units in the sterol synthetic pathway by as much as 80% did not alter the suppressing effect of SKF 104976 on HMGR activity. However, under conditions where sterol synthesis was almost completely blocked by lovastatin, HMGR activity was not suppressed by SKF 104976. Mevalonate, at concentrations that did not decrease HMGR activity, was able to restore the inhibiting effect of SKF 104976 on HMGR activity. The rapid inhibition (2-3 h) of HMGR activity by SKF 104976 to 30-60% of the level in controls was not dependent on the initial amount of HMGR enzyme present. These findings suggest that upon inhibition of 14 alpha DM by SKF 104976, a mevalonate-derived precursor regulates HMGR activity, even when the sterol synthetic rate is considerably reduced and when HMGR protein levels are very high. In Hep G2 cells, formation of oxylanostenols from [3H]mevalonate reached a maximum between 1 and 10 nM SKF 104976 and was negligible at higher concentrations. This result suggests that oxylanostenols are not the key mediators of the modulation of HMGR in Hep G2 cells upon 14 alpha DM inhibition.

Carcinoma, Hepatocellular↗
PubMed1992

Factors affecting homologous overexpression of the Saccharomyces cerevisiae lanosterol 14 alpha-demethylase gene.

The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S. cerevisiae using promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values. With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.

Base Sequence↗
PubMed1991

CYP1 (HAP1) is a determinant effector of alternative expression of heme-dependent transcribed genes in yeast [corrected].

The CYP1 (HAP1) gene of Saccharomyces cerevisiae is known to activate a number of target genes in response to the presence of heme. Several features of the protein, deduced from the sequence of the gene, suggest that CYP1 is a general sensor of the redox state of the cell. To investigate further the function of CYP1, we analysed its effects on the transcription of two genes, HEM13 and 14DM, which are preferentially expressed in anaerobiosis. HEM13 encodes coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway and 14DM encodes lanosterol-14-demethylase which is involved in sterol biosynthesis and is a member of the cytochrome P450 family. Isogenic CYP1+ and cyp1 degree deleted strains, either heme-sufficient or heme-deficient (HEM1 disrupted), were grown in aerobic or anaerobic conditions, and transcripts of HEM13 and 14DM were analysed on Northern blots. The results show that in anaerobic and in heme-deficient cells, CYP1 activates the transcription of HEM13 and inhibits that of 14DM. Opposite effects of CYP1 are observed in aerobic, heme-sufficient cells. We concluded that: (i) CYP1 is an efficient activator especially in heme-depleted cells; (ii) CYP1 exerts both positive and negative regulatory effects; (iii) the nature of the regulatory function of CYP1 depends on the target gene; and (iv) for a given gene, the presence or absence of heme or oxygen reverses the sense of CYP1-dependent regulation.

Anaerobiosis↗
PubMed1991

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.

Catalysis↗
PubMed1992

Plant sterol biosynthesis: 7-oxo-obtusifoliol analogues as potential selective inhibitors of cytochrome P-450 dependent obtusifoliol 14 alpha-demethylase.

A series of 7-oxo-obtusifoliol analogues have been synthetized and investigated as potential inhibitors of cytochrome P-450 dependent obtusifoliol 14 alpha-demethylase (P-450OBT.14DM) from higher plant microsomes. 7-Oxo-24 xi(24')-dihydro-obtusifoliol and 7-oxo-24(25)-dihydro-29-nor-lanosterol were potent competitive inhibitors for P-450OBT.14DM, binding 125-200 times more tightly than the substrates obtusifoliol and 24(25)-dihydro-29-nor-lanosterol. Inhibition of P-450OBT.14DM by these analogues showed strict structural requirements including the 8-en-7-one system which was compulsory for binding. 7-Oxo-24(25)-dihydro-lanosterol possessing an additional 4 beta-methyl substituent, did not have such inhibitory effects. Treatment of cultures of suspended bramble cells with 7-oxo-24(25)-dihydro-29-nor-lanosterol resulted in a strong decrease of [14C]acetate incorporation into the demethylsterols fraction and in an accumulation of [14C]obtusifoliol. This confirms that P-450OBT.14DM is the main in vivo target of 7-oxo-24(25)-dihydro-29-nor-lanosterol in the sterol-biosynthetic pathway.

Cells, Cultured↗
PubMed1991

Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol.

The purified lanosterol 14a-demethylase (P-45014DM) of S. cerevisiae catalyzed the 14a-demethylation of 24-methylene-24,25-dihydrolanosterol (24-methylenelanost-8-en-3 beta-ol, 24-methylene-DHL), the natural substrate of the demethylase of filamentous fungi, as well as its natural substrate, lanosterol. Lanosterol 14a-demethylase of rat liver microsomes also catalyzed the 14a-demethylation of 24-methylene-DHL, but the activity was considerably lower than that for lanosterol. The activity of the rat liver enzyme for 24-methylene-DHL was also lower than that for 24,25-dihydrolanosterol (DHL), while the activity of yeast P-45014DM for 24-methylene-DHL was considerably higher than that for DHL. Since 24-substituted sterols are not found in mammals and DHL is not an intermediate of ergosterol biosynthesis by yeast, above-mentioned different substrate specificities between the yeast and the mammalian 14a-demethylases may reflect certain evolutional alteration in their active sites in relation to the difference in their sterol biosynthetic pathways.

Animals↗
PubMed1991

Identification of lanosterol 14 alpha-methyl demethylase in human tissues.

Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite. The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation. Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied. Kinetic parameters were determined and shown to differ between two separate liver samples. Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite. The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene. The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.

Adult↗
PubMed1992

Inhibition by a novel azole antifungal agent with a geranyl group on lanosterol 14 alpha-demethylase of yeast.

AFK-108 (1-[2-(2,4-dichlorophenyl)-2-((2E)-3,7-dimethylocta-2,6- dienyloxy)ethyl]-1H-imidazole) is a new imidazole derivative characterized by a geranyl substituent showing strong antifungal activity. Azole antifungal agents are known to be potent inhibitors of lanosterol 14 alpha-demethylase (P450(14)DM) of fungi. The role of the geranyl group of AFK-108 on interaction of AFK-108 with the target was studied by using Saccharomyces cerevisiae P450(14)DM as the model enzyme. AFK-108 and some of its derivatives bound to oxidized P450(14)DM with one-to-one stoichiometry and inhibited the demethylase activity. AFK-108 derivatives having the longer farnesyl or the shorter prenyl group showed lower affinity than AFK-108 for the enzyme. AFK-108 caused 100% inhibition at the equivalent concentration to P450(14)DM in the reaction mixture (0.07 microM), while the farnesyl derivative inhibited the activity by 60% at the same concentration. AFK-108 interfered with the binding of CO to the ferrous P450(14)DM. However, the interfering effect of the prenyl derivative was lower than that of AFK-108. Another AFK-108 derivative having the saturated 3,7-dimethyloctyl group was also a weaker inhibitor than AFK-108. These experiments suggest that the geranyl group of AFK-108 interacts with the substrate binding site of P450(14)DM that recognises the side chain of the substrate. AFK-108 is the first example of an azole derivative interacting with the side chain recognising region of the substrate binding site of P450(14)DM.

Antifungal Agents↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1992

Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast.

The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14)DM (lanosterol 14 alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14)DM from Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of 26-hydroxylanosterol and 25-hydroxy-24,25-dihydrolanosterol with a lower activity than lanosterol and 24,25-dihydrolanosterol. This enzyme demethylated the (Z)-24-ethylidene-24,25-dihydrolanosterol with a low rate, but did not metabolize the E-isomer. The apparent Km of 26-hydroxylanosterol was 10.8 microM, which was higher than that of lanosterol, but lower than that of 24,25-dihydrolanosterol. On the other hand, competition experiments suggested that the affinity of 25-hydroxy-24,25-dihydrolanosterol and (Z)-24-ethylidene-24,25-dihydrolanosterol for P-450(14)DM was significantly lower than that of 24,25-dihydrolanosterol. Integration of the present results with the preceding ones (Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1991) Biochim. Biophys. Acta, 1081, 262-266 and Aoyama, Y. and Yoshida, Y. (1991) Biochem. Biophys. Res. Commun., 178, 1064-1071) suggests that yeast P-450(14)DM recognizes two parts of the side-chain, the structure around C-24 and the terminal fork consisting of C-25, C-26 and C-27.

Binding Sites↗
PubMed1991

Mode of action of anti-Candida drugs: focus on terconazole and other ergosterol biosynthesis inhibitors.

A large proportion of the presently available antifungal agents are claimed to derive their activity from interaction with the biosynthesis of ergosterol, the key sterol in most pathogenic fungi. An important target for the allylamines, naftifine and terbinafine, is the squalene epoxidase. Interaction with the epoxidation step results in a decreased availability of ergosterol and an accumulation of squalene. Although the squalene epoxidase is clearly the primary target for this class of antifungals, it still remains an open question whether the fungistatic or fungicidal effects originate from a decrease in ergosterol or squalene accumulation. Indeed, preliminary evidence suggests that squalene does not change the physicochemical properties of membranes. Much more is known about the primary and secondary effects of the azole antifungals, such as miconazole, ketoconazole, terconazole, and itraconazole. Most of the imidazole and triazole derivatives are highly potent and selective inhibitors of the cytochrome P-450-dependent 14 alpha-demethylation of lanosterol (P-45014DM). Their potency and selectivity are determined by the nitrogen heterocycle and to a much greater extent by the hydrophobic N-1 substituent. The triazole antifungals, terconazole and itraconazole, combine a high affinity for Candida P-45014DM with an exceptionally low effect on mammalian cytochrome P-450.

Antifungal Agents↗
PubMed1992

The 4 beta-methyl group of substrate does not affect the activity of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast: difference between the substrate recognition by yeast and plant sterol 14 alpha-demethylases.

Interaction of obtusifoliol and 24,28-dihydroobtusifoliol with yeast lanosterol 14 alpha-demethylase (P-450(14)DM) was studied to elucidate the role of the 4 beta-methyl group of substrate. P-450(14)DM of Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of obtusifoliol. Apparent Vmax of obtusifoliol demethylation (15.4 nmol/min/nmol P-450) was similar to that of 24-methylene-24,25-dihydrolanosterol demethylation and was a little higher than those of lanosterol and 24,25-dihydrolanosterol demethylations. Apparent Km for obtusifoliol (12.0 microM) was higher than those for lanosterol and 24-methylene-24,25-dihydrolanosterol but was lower than that for 24,25-dihydrolanosterol. 24,28-Dihydroobtusifoliol was a very poor substrate for yeast P-450(14)DM. These facts suggest that the 4 beta-methyl group of sterol slightly affects the activity of yeast P-450(14)DM, while hydrogenation of a double bond in the sterol side-chain considerably impairs the activity. This finding is a contrast to the fact that the plant P-450(14)DM could not catalyze demethylation of sterols having 4 beta-methyl group, but favorably interacts with sterols having saturated side chain.

Cholestadienols↗
PubMed1991

Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 alpha-demethylase from maize (Zea mays) seedlings.

The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined. In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum. P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol. The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol. The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM. They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction. Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast. This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.

Cholestadienols↗