Demethylation, ergosterol

Triadimefon (Triazole) C —O - CH—COC(CHj)3. N, N— Steroid demethylation (ergosterol biosynthesis) inhibitor... 

Although termed "SI" or "EBI" compounds, the latter referring to ergosterol biosythesis inhibitors, these compounds do not all inhibit ergosterol biosynthesis at the same metabolic site (Fig. 2). For instance, the fungicide tridemorph, unlike most EBI compounds, does not inhibit demethylation at C-14 but rather it apparently prevents the A A isomerization resulting in the accumulation of A containing sterols in treated cells (3). 

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 biosynthetic pathway of ergosterol is shown in Figure 15 to explain the steps of inhibition by morpholines the accumulating sterols are framed. Inhibition of the A -reduction by fenpropimorph should lead to an accumulation of 4,4-dimethyl- A8, -ergo-statriene-ol. However, C -demethylation and side chain hydrogenation are obviously able to occur with the accumulating sterol as substrate... 

When Candida albicans was treated with bifonazole or clotrimazole, the expected accumulation of the ergosterol precursor 24-methy-lenedihydrolanosterol was observed (Figure 17). This was an indication that both compounds inhibit the cytochrome P -dependent C -demethylation. Using 2.5 ug /ml clotrimazole, dihydrolanosterol accumulates, which is known to regulate the HMG-CoA-reductase by a... 

Sterol biosynthesis inhibiting insecticides. The earliest examples of sterol biosynthesis inhibitors were triadimefon, imazalil, and triarimol. These compounds act by blocking the C14 alpha demethylation step in ergosterol biosynthesis [54], Further work in this area has resulted in numerous fungicides, a number of them containing aromatic fluorine, perfluoroalkyl ethers, and the trifluoromethylphenyl group (Fig. 11). In this section, we will discuss the trifluoromethylphenyl sterol biosynthesis inhibitors, such as triflumizole and fluotrimazole. 

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. 

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

Rahier and his co-workers also characterized the activities of a sterol C-4 methyl oxidase (SMO), a 4-carboxysterol-3-hydroxysteroid dehydrogenase/ C-4 decarboxylase (3-HSD/D) and an NADPH-dependent 3-oxosteroid reductase in order to define the steps involved in C-4 demethylation in plants (Pascal et al, 1993 Rondet et al, 1999). Only recently, they have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3-HSD/D. Transformation of a yeast ergosterol auxotroph mutant, which lacks 3-HSD/D activity, with either of these cDNAs restored ergosterol biosynthesis in the yeast mutant (Rahier et al, 2006). 

Triadimefon binds to hepatic cytochrome P450 and inhibits microsomal enzyme activities. It inhibits sterol demethylation and thus sterol synthesis. Fungi sensitive to triadimefon utilize ergosterol as the primary sterol, the production of which is inhibited. It is also thought that triadimefon may have actions similar to those caused by indirect-acting dopamine agonists. 

These 1,2,4-triazole fungicides share a common mode of action by inhibiting the C-14a-demethylation step in ergosterol biosynthesis (Figure 1) ( 3 ) between 24-methylenedihydrolanosterol and 4,4-dimethylergosta-8,14,24(28)-trienol. 

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. 

In summary, prochloraz, like other azoles, is an ergosterol biosynthesis inhibitor (i.e. it is an EB1) and it does this by blocking 14-demethylation (such compounds are sometimes referred to as demethylation inhibitors, or DMIs). As is now well known, these molecules bind to the sterol binding site of the demethylase enzyme in such a way as to allow the azole to bind to an iron atom in the active site (see below). The normal physiological reaction is therefore prevented. 

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]. 

Many fungicides [15] act by inhibiting sterol biosynthesis. More specifically, they block the C-14a demethylation step in ergosterol biosynthesis [16]. In many of these compounds (Flutriafol [17], Flusilazole [18], Triflumizole [19], Fluotri-... 

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

Itraconazole is a triazole antifungal agent that inhibits the C-demethylation step in the synthesis of ergosterol, which is a vital component of fungal cell membranes. Injection used in treatment of aspergillosis, blastomycosis, histoplasmosis, and the empiric treatment of febrile neutropenic patients with suspected fungal infections. Capsules used in treatment of asper-gillosis, blastomycosis, histoplasmosis, and... 

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