Danny Bellens

Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans

The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

Biochemical Approaches to Selective Antifungal Activity. Focus on Azole Antifungals

Summary: Azole antifungals (e.g. the imida‐zoles: miconazole, clotrimazole, bifona‐zole, imazalil, ketoconazole, and the tria‐zoles: diniconazole, triadimenol, propico‐nazole, fluconazole and itraconazole) inhibit in fungal cells the 14α‐demethylation of lanosterol or 24–methylenedihydro‐lanosterol. The consequent inhibition of ergosterol synthesis originates from binding of the unsubstituted nitrogen (N‐3 or N‐4) of their imidazole or triazole moiety to the heme iron and from binding of their N‐1 substituent to the apoprotein of a cytochrome P‐450 (P‐45014DM) of the endo‐plasmic reticulum.

Effects of etomidate on steroid biosynthesis in subcellular fractions of bovine adrenals

The imidazole derivative, etomidate, inhibits the 11 beta-hydroxylase in cell-free systems and mitochondria isolated from bovine adrenal cortex. Fifty per cent inhibition is achieved at 3.10(-7) M. The less active hypnotic L-enantiomer is also a less potent inhibitor of the 11-hydroxylation. At a 2 times higher concentration, etomidate affects the cholesterol side chain cleavage. The inhibition of both steroidogenic enzyme systems may be due to binding of the unhindered nitrogen of the imidazole ring of etomidate to the heme iron atom of the adrenal cortex mitochondrial cytochrome P-450 species.

R 76713 and enantiomers: Selective, nonsteroidal inhibitors of the cytochrome P450-dependent oestrogen synthesis

The triazole derivative, R 76713 and its enantiomers R 83839(-) and R 83842(+) are effective inhibitors of the aromatization of androstenedione. For human placental microsomes, the (+) enantiomer (R 83824) is about 1.9- and 32-times more active than the racemate (IC50 2.6 nM) and the (-) enantiomer, respectively. R 83842 is about 30- and 1029-times more active than 4-hydroxyandrostene-3,17-dione and aminoglutethimide. This potency might originate from its high affinity for the microsomal cytochrome P450 (P450). Indeed, R 83842, compared to R 76713 and R 83839, forms a more stable P450-drug complex. Difference spectral measurements indicate that the triazole nitrogen N-4 coordinates to the haem iron. The reversed type 1 spectral changes suggest that R 76713 is able to displace the substrate from its binding place and the stable complex formed in particular with the (+) enantiomer suggests that its N-1-substituent occupies a lipophilic region of the apoprotein moiety. Kinetic analysis implies that there is a competitive part in the inhibition of the human placental aromatase by R 76713. The Ki values for R 76713, R 83842 and R 83839 are 1.3 nM, 0.7 nM and 18 nM, respectively. These results are indicative of stereospecificity for binding. Up to 10 microM, R 76713 and its enantiomers have no statistically significant effect on the regio- and stereoselective oxidations of testosterone in male rat liver microsomes. All three compounds have no effect on the P450-dependent cholesterol synthesis, cholesterol side-chain cleavage and 7 alpha-hydroxylation and 21-hydroxylase. At 10 microM, R 76713 has a slight effect on the bovine adrenal 11 beta-hydroxylase. This effect originates mainly from R 83839, the less potent aromatase inhibitor. On the other hand, the inhibition of the 17,20-lyase of rat testis observed at concentrations greater than or equal to 0.5 microM, originates rather from R 83842. However, 50% inhibition is only achieved at 1.8 microM R 83842, i.e. at a concentration about 1300-times higher than that needed to reach 50% inhibition of the human placental aromatase.

Saperconazole: a selective inhibitor of the cytochrome P-450-dependent ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes.

The N‐1‐substituted triazole antifungal, saperconazole, is a potent inhibitor of ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. Fifty % inhibition is already achieved at nanomolar concentrations. The saperconazole‐induced inhibition of ergosterol synthesis coincides with an accumulation of 14‐methylated sterols, such as 24‐methylenedihydrolanosterol, lanosterol, obtusifoliol, 14α‐methylfecosterol, 14α‐methylergosta‐8,24(28)‐dien‐3,β‐6α‐diol and 14α‐methylergosta‐5,7,22,24(28)‐tetraenol. This indicates that saperconazole interferes with the cytochrome P‐450 (P‐450)‐dependent 14α‐demethylation of lanosterol and/or 24‐methylenedihydrolanosterol. Saperconazole forms stable drug‐P‐450‐complexes by binding via its free triazole nitrogen to the heme iron and via its N‐1 substituent to the apoprotein moiety. The triazole derivative is a highly selective inhibitor of the 14α‐demethylase in fungal cells. It is a poor inhibitor of the 14α‐demethylation of lanosterol in rat and human liver cells. Saperconazole is, at concentrations as high as 10 µM, devoid of effects on the P‐450‐dependent cholesterol side‐chain cleavage and 11β‐hydroxylase, 17,20‐lyase, 21‐hydroxylase and aromatase. Saperconazole does not interfere with the 2α, 6α‐, 6β‐ and 7α‐hydroxylations of testosterone in microsomes from male rat liver. At high concentrations (> 5 µM) an inhibition of the 16β‐+hydroxylations is seen.

Mode of Action of Antifungals of Use in Immunocompromised Patients. Focus on Candida Glabrata and Histoplasma Capsulatum

During recent years considerable advances have been made in the identification of potential targets for antifungal agents. The most important antifungals for use in immunocompromised patients interfere with targets in the plasma membrane (polyenes), nucleus (5-fluorocytosine) or smooth endoplasmic reticulum (allylamines, morpholines, azole derivatives).

Ridogrel: A selective inhibitor of the cytochrome P450-dependent thromboxane synthesis

Ridogrel [(E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl] methylene]amino]oxy] pentanoic acid] is a potent inhibitor of the P450-dependent human platelet thromboxane A2 (TxA2) synthase. Fifty percent inhibition is already achieved at 5.0 +/- 0.37 nM. This IC50 value is close to half the P450 concentration used, i.e. 10.7 nM. Ridogrel binds to human platelet microsomal P450 as proven by the type II spectral changes induced by the addition of increasing concentrations of ridogrel to solubilized microsomes. The calculated half-maximal spectral change (SC50 value) is 3.78 +/- 1.79 nM. These results indicate that ridogrel binds stoichiometrically and suggest that inhibition of thromboxane synthesis may originate from liganding of its basic nitrogen to the haem-iron of P450 and from the attachment of the hydrophobic carboxylic side chain to or near the substrate binding place. Ridogrel is a selective inhibitor of the TxA2 synthase. At a high concentration (10 microM), ridogrel has a slight, if any, effect on the P450-mediated cholesterol synthesis in human liver and hepatoma cells and androgen synthesis from 17 alpha-hydroxy-20-dihydroprogesterone or pregnenolone in subcellular fractions from rat testes. These results indicate that ridogrel is a poor inhibitor of the P450-dependent 14 alpha-demethylase, 17 alpha-hydroxylase and 17,20-lyase. It has, up to 10 microM, no effect on the adrenal mitochondrial 11 beta-hydroxylase and cholesterol side-chain cleavage enzyme and does not inhibit aromatase activity in human placental microsomes. Ridogrel has no significant effect on the regio- and stereoselective P450-dependent oxidations of testosterone in liver microsomes from unpretreated or from 5-pregnen-3 beta-ol-20-one-16 alpha-carbonitrile-, phenobarbital- or 3-methylcholanthrene-pretreated male and female Sprague-Dawley rats. It does not interfere with the reduction of testosterone into 5 alpha-dihydrotestosterone and 5 alpha androstane 3 beta, 17 beta-diol.