Lanosterol demethylation

Blocks lanosterol demethylation to ergosterol, thus disrupting fungal membrane integrity. 

Aoyama Y, Y Yoshida, R Sato (1984) Yeast cytochrome P-450 catalyzing lanosterol 14a-demethylation. 11. Lanosterol metabolism by pnrified P-dSOj j and by intact micrososmes. J Biol Chem 259 1661-1666. 

Aoyama Y, Y Yoshida, Y Sonoda, Y Sato (1987) Metabolism of 32-hydroxy-24,25-dihydrolanosterol by pnrified cytochrome P-450j4]j]y[ from yeast. Evidence for contribntion of the cytochrome to whole process of lanosterol 14a-demethylation. J Biol Chem 262 1239-1243. 

Shyadehi AZ, DC Lamb, SL Kelly, DE Kelly, W-H Schunck, JN Wright, D Corina, M Akhtar (1996) The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14a-demethyl-ase of Candida albicans (other names are lanosterol 14a-demethylase, P-450]4p, and CYP51). J Biol Chem 271 12445-12450. 

C]-(35)-2,3-epoxysqualene and its racemate have been prepared by two routes in a metabolically non-labile position relative to the demethylation of lanosterol to cholesterol (equation 70 and 71). The racemic [24,30-14C]-2,3-epoxysqualene, 192, has been obtained163 by condensation of (35, 3/ )-2,3-epoxytrisnorsqualene aldehyde 193 with freshly prepared 14C-labelled isopropylidenephosphorane, 194 (equation 70). 

Figure 22-8 Steps in the demethylation of lanosterol. The most frequent sequence, labeled [1], begins with demethylation at C-14 by the action of a cytochrome P450 and is followed [2] by the successive demethylation of the 0C-CH3 and (S-CH3 at C-4 by an NADH-dependent oxygenase.

The last sequence of reactions in the biosynthesis of choles-terol involves approximately 20 enzymatic steps, starting with lanosterol. In mammals the major route involves a series of double-bond reductions and demethylations (fig. 20.10). The sequence of reactions involves reduction of the A24 double bond, the oxidation and removal of the 14a methyl group followed by the oxidation and removal of the two methyl groups at position 4 in the sterol. The final reaction is a reduction of the A7 double bond in 7-dehydro-cholesterol. An alternative pathway from lanosterol to cholesterol also exists. The enzymes involved in the transformation of lanosterol to cholesterol are all located on the endoplasmic reticulum. 

Fig. 12. Deformylation of C(19) 14a-demethylation of lanosterol 64 side chain cleavage of cholesterol 66...

Except for aromatization, the 14a-demethylation of lanosterol 64 seems to operate by the same sequence of events in order to remove the angular C(14) methyl group with concomitant introduction of the C(14) double bond to furnish 65 (Fig. 12). 

In ergosterol biosynthesis, side chain alkylation of lanosterol normally takes place to build 24-methylenedihydrolanosterol, which itself is then the substrate for demethylation reactions at and C. The C -demethylation has been studied in detail. It is an oxidative demethylation catalyzed by a cytochrome P -system. The first step involved in this reaction is the hydroxylation of the Cj -methy1-group to form the C -hydroxymethyl derivative. A second hydroxylation and loss of water lead to the C -formyl intermediate, which is hydroxylized a third time to form the corresponding carboxylic acid. Decarboxylation does not directly take place, but proceeds instead by abstraction of a proton from C, followed by elimination and formation of a A 4-double bond. The NADPH-dependent reduction of the A14 -double bond finishes the demethylation reaction. Subsequently, demethylation at has to take place twice, followed by a dehydrogenation reaction in A" -position and isomerization from A8 to A7 and A24(28) to A22. respectively. 

The first pathogen-specific reaction is the S-adenosylmethio-nine-dependent side chain aklylation of lanosterol. This is pathogen specific since in cholesterol synthesis, a side chain alkylation does not take place. Secondly, the demethylation reactions at C - and C -positions of 24-methylene-dihydrolanosterol are pathogen-specific as well. In mammals demethylation reactions take place, but the substrate is not side chain alkylated, so the corresponding enzyme should possess different binding sites for the different substrates. 

Mode of action Ketoconazole interacts with C-14 a-demethylase (a cytochrome P-450 enzyme) to block demethylation of lanosterol to ergosterol, the principal sterol of fungal membranes Figure 34.4). This inhibition disrupts membrane function and increases permeability. Ketoconazole acts in an additive manner with flucytosine against Candida, but antagonizes amphotericin B s antifungal activity. 

In contrast to the above results the substrates (23) and (25) were transformed enzymically by 2,3-oxidosqualene sterol cyclase to the corresponding lanosterol derivatives (27) and (28). In addition, 6-demethyl-2,3-oxidosqualene underwent enzymic cyclization to 19-norlanosterol (29). Van Tamelen and Freed ... 

Cyclization of Squalene.—Marked differences have been found between specir mens of the enzyme 2,3-oxidosqualene cyclase isolated from yeast and hog liver. In the former organism the enzyme was in the soluble fraction, whereas in the latter it was associated with the microsomes. Further studies of unnatural precursors showed that a preformed steroidal ring-D (65) gave in low yield the corresponding lanosterol analogue (68). Similarly, 6-demethyl-2,3-oxido-squalene (66) was efficiently used and gave 19-norlanosterol (69). ... 

In our laboratory, the major sterol biosynthesised in untreated extracts was the triene (VIII) (1J), rather than ergosterol Itself which is, of course, the end product of the pathway in intact cells. It should be noted that VIII arises directly from 14-demethylation of lanosterol. In the presence of 0.1 yM prochloraz (or even 0.01 pM prochloraz in some experiments) the concentration of VIII was significantly reduced, while the level of lanosterol Increased (4) indicating clearly that prochloraz inhibited 14-demethylation. At higher fungicide concentrations both the triene and ergosterol were totally absent and only lanosterol was present. 

Since ergosterol is used in the formation of the leishmanial cell membrane, inhibition of ergosterol biosynthesis has been considered as a useful target for chemotherapeutic attack. Allylamines (eg. terbinafine) and imidazole antifungals (eg. ketoconazole) have been found to interfere with different steps in the biosynthetic pathway of C28 sterols in leishmania and fungi. Allylamines inhibit the microsomal squalene 2,3-epoxidase and, therefore, inhibit the synthesis of squalene epoxide, the precursor of lanosterol. Imidazoles, on other hand, inhibit cytochrome P-450 dependent C-14 demethylation of lanosterol leading to decreased or no synthesis of ergosterol [30]. 

Azoles are fungicidal and interfere with the synthesis of ergosterol by inhibiting the P450-dependent 14 alpha-demethylation of its precursor molecule, lanosterol. 

As indicated, the oxidative demethylation of lanosterol in rat liver preparations is inhibited by CO. However, if 32-hydroxylanosterol is used as substrate, CO no longer inhibits. This indicates that in animals cytochrome P-450 catalyzes only the first oxidation and that other cytochromes are used in the subsequent steps. Apparently, the yeast and liver systems are different [5]. 

Lanosterol is converted to cholesterol by a series of oxidations, reductions, and demethylations. The required enzyme reactions have been defined and metabolic intermediates identified however, the precise sequence of reactions between lanosterol and cholesterol remains to be established [7] (Fig. 4). There is evidence for two alternative pathways that differ in the point at which the A24 double bond is reduced (Section 2.3). Both 7-dehydrocholesterol (DHC) and desmosterol have been postulated to be the... 

Lanosterol is converted into cholesterol in a series of nineteen enzyme reactions [18]. The production of cholesterol from lanosterol involves the reduction of the double bond at C-24, demethylations of ge/w-dimethyl at C-4 and a tertiary methyl at C-l4, and isomerization of the double bond from C-8 to C-l. Two major pathways involving the same enzymes have been proposed [16-18]. Cholesterol biosynthetic pathway is outlined in... 

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