Hugo Vanden Bossche

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.

Molecular mechanisms of drug resistance in fungi

Failures of treatment in fungal infections have drawn attention recently to the problem of antifungal resistance and its underlying mechanisms. The number of fungal isolates that are resistant to the orally active azole antifungals, especially fluconazole, is growing. Amphotericin-B-resistant isolates have been recovered during treatment of patients with candidiasis, and resistance to flucytosine is so common that this antifungal is no longer recommended as a single-drug therapy.

Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray

ABSTRACT The application of genome-wide expression profiling to determine how drugs achieve their therapeutic effect has provided the pharmaceutical industry with an exciting new tool for drug mode-of-action studies. We used DNA chip technology to study cellular responses to perturbations of ergosterol biosynthesis caused by the broad-spectrum antifungal agent itraconazole. Simultaneous examination of over 6,600 Candida albicans gene transcript levels, representing the entire genome, upon treatment of cells with 10 μM itraconazole revealed that 296 genes were responsive. For 116 genes transcript levels were decreased at least 2.5-fold, while for 180 transcript levels were similarly increased. A global upregulation ofERG genes in response to azole treatment was observed.ERG11 and ERG5 were found to be upregulated approximately 12-fold. In addition, a significant upregulation was observed for ERG6, ERG1, ERG3, ERG4, ERG10, ERG9, ERG26, ERG25, ERG2, IDII, HMGS, NCP1, and FEN2, all of which are genes known to be involved in ergosterol biosynthesis. The effects of itraconazole on a wide variety of known metabolic processes are discussed. As over 140 proteins with unknown function were responsive to itraconazole, our analysis might provide—in combination with phenotypic data—first hints of their potential function. The present report is the first to describe the application of DNA chip technology to study the response of a major human fungal pathogen to drug treatment.

Anti-Candida Drugs — The Biochemical Basis for Their Activity

The past years have seen a continuous effort toward the synthesis of new antifungal agents. Most of them belong to the N-substituted imidazoles and triazoles. Another interesting series of antifungals are the allylamines. Biochemically, both the azole derivatives and the allylamines belong to the class of ergosterol biosynthesis inhibitors and thus differ from the polyene macrolide antibiotics. Indeed, it is now believed that the antifungal action of the polyenes, nystatin and amphotericin B, is due to a direct interaction with ergosterol itself. A more detailed analysis of the ergosterol biosynthesis inhibitors revealed that ergosterol depletion is the consequence of the interaction of the azole derivatives, e.g., miconazole, ketoconazole, and itraconazole, with the cytochrome P-450 involved in the 14 alpha-demethylation of lanosterol. Both the accumulation of 14 alpha-methylsterols and the concomitant decreased ergosterol content affect the membranes and membrane-bound enzymes of yeast and fungi. The allylamines seem to act by inhibition of the squalene epoxidase resulting in ergosterol depletion and accumulation of squalene. The target for the fluorinated pyrimidine, flucytosine, is completely different. Its antifungal properties may result from its conversion to 5-fluorouracil. The latter is then phosphorylated and incorporated into RNA, thus disrupting the protein synthesis in the yeast cell. These different biochemical targets for the antifungals of use in candidosis are discussed in this paper.

Biochemical effects of miconazole on fungi—I: Effects on the uptake and/or utilization of purines, pyrimidines, nucleosides, amino acids and glucose by Candida albicans

Abstract The antifungal and antibacterial drug miconazole has been shown to inhibit, at concentrations lower than those affecting growth, the transport of adenine, guanine and hypoxanthine by Candida albicans in suspension culture. The decrease in the incorporation of purines into nucleic acids seems to be the consequence of an inhibitory effect on their uptake into the cells. When the purines were replaced by adenosine, deoxyadenosine and guanosine, miconazole increased the uptake and incorporation of the radioactivity derived from the nucleosides into macromolecules. The data suggest that the drug-induced increase of nucleoside incorporation into nucleic acids is secondary to enhanced nucleoside transport. Miconazole also slightly affected the uptake of orotic acid. The transport of glucose, glycine and leucine was not affected by miconazole whereas in some way the drug affected glutamine uptake. Studies on the distribution of miconazole and/or its metabolites in the Candida cell indicate that in log-phase cells most of the radioactivity was found in the fraction containing cell walls and plasmalemma. In stationary-phase cells the highest radioactivity was found in the fraction which contained the microsomes. Although more information will be needed, the data presented indicate that at low concentrations, miconazole acts primarily on the yeast cell membranes (cell wall and plasmalemma) resulting in a selective inhibition of the uptake of precursors of RNA and DNA (purines) and mucopolysaccharide (glutamine). Higher doses and longer incubation periods also alter the activities of microsomal membranes.

P450 inhibitors of use in medical treatment: focus on mechanisms of action.

A number of cytochrome P450s are targets for compounds that are clinically used or under clinical evaluation for treatment of patients with mycotic infections, such as dermatophytosis, superficial and systemic candidiasis, cryptococcosis and aspergillosis, with skin diseases, such as psoriasis or ichthyosis, and other retinoid-sensitive malignancies, e.g., neuro-ectodermal glioma. Some of the P450 inhibitors are candidates for the treatment of hirsutism or prostate cancer, others are potent inhibitors of the P450 isomerase involved in the synthesis of thromboxane A2, a potent platelet aggregation inducer and vasoconstrictor.

Mebendazole and Related Anthelmintics

Publisher Summary This chapter discusses the mebendazole and related anthelmintics. Ascaris lumbribcoides has been estimated to infect one-quarter of the worlds population. In a review on costs, prevalence, and approaches for control of Ascaris infection in Kenya, Stephenson et al. collected evidence indicating that even light Ascaris infections may have detrimental effects on the growth of undernourished “preschool” children. These authors proved that it is simply not true that Ascaris infection is harmless in most cases as often considered by other investigators. Ascariasis is linked not only to poor growth and protein-caloric malnutrition but also to malabsorption of macronutrients and vitamin A. Ascaris affects the growth of pigs. Spindler (1947) artificially infected pigs with Ascaris suum eggs and determined 126 days later the weight increase in both infected and uninfected animals. A negative correlation was found between the number of Ascaris and weight increase.

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.

Aromatase inhibitors — mechanisms for non-steroidal inhibitors

SummaryThe conversion of androgens to estrogens occurs in a variety of cells and tissues, such as ovarian granulosa and testicular cells, placenta, adipose tissue, and various sites of the brain. The extragonadal synthesis of estrogens has great pathophysiological importance. Estrogens produced by, for example, adipose tissue have a role in the pathogenesis of certain forms of breast cancer and endometrial adenocarcinoma. The biosynthesis of estrogens is catalyzed by the aromatase, an enzyme localized in the endoplasmic reticulum that consists of two components: a cytochrome P450 (P450 Arom, P450 19 product of theCYP 19 gene) and the NADPH cytochrome P450 reductase. The alignment of the amino acid sequences of human P450 19 with other mammalian P450s shows little sequence similarity, which indicates not only that P450 19 is a unique form of the P450 superfamily but also that the aromatase may be a good target for the development of selective P450 inhibitors.Aminoglutethimide (AG) is the pioneer drug of the reversible competitive nonsteroidal aromatase inhibitors. Since AG is a nonspecific aromatase inhibitor and presents some problems with tolerability, a number of structural analogues have been synthesized. For example, rogletimide is slightly less potent than AG but has the advantage of not inhibiting the cholesterol side-chain cleavage and is devoid of sedative action. Elongation of the ethyl substituent of AG and rogletimide leads to an increase in aromatase inhibition. Further studies led to the discovery of a new generation of much more potent aromatase inhibitors. An example is fadrozole. However, although fadrozole is a poor inhibitor of the cholesterol side-chain cleavage, it suppresses aldosterone release by ACTH-stimulated human adrenocortical cells. More selective aromatase inhibitors are the triazole derivatives. Examples are CGS 20267, CGS 47645, R 76 713, and ICI D1033.R 76 713s aromatase inhibitory effect is largely due to its (+)-S-enantiomer, vorozole. Computer modeling studies of the interaction of vorozole with part of the “I-helix” of P450 19 suggest that the chlorine-substituted phenyl ring of vorozole interacts with the gamma-carbonyl group of Glu-302. Thr-310, which corresponds to the highly conserved Thr-252 in P450 101, interacts with vorozoles triazole ring, and the 1-methyl-benzotriazole moiety binds near Asp-309.

The biochemical mechanism of action of the antinematodal drug tetramisole

Abstract The effect of tetramisole was studied on succinate dehydrogenase activity of the nematodes Ascaris suum, Ascaridia galli, Toxocara cati and Dictyocaulus viviparus , the cestodes Taenia taeniaeformis. Taenia pisiformis and Dipylidium caninum, and also on rat liver and pigeon breast muscle. Administered to nematode preparations at low concentrations, it reduced the succinate, NAD and ATP levels and increased the fumarate, NADH 2 and inorganic phosphate levels. In all experiments the laevo-isomer was a more potent inhibitor of the fumarate-succinate system than the dextro-isomer. Although tetramisole inhibited NADH 2 oxidation in coenzyme-free pulps of nematodes, there was no effect upon NADH 2 oxidation in cestodes under the same conditions. Similarly, although tetramisole inhibited succinate oxidation in coenzymefree pulps of nematodes, there was no effect upon suceinate oxidation in pigeon breast muscle at the drug-concentrations tested. Inhibition of succinate oxidation was observed in rat liver, but at drug-concentrations fifty times higher than those necessary to inhibit fumarate reduction to the same extent in the nematodes studied. The experiments suggest that at low concentrations tetramisole inhibited stereospecifically “fumarate reductase” activity in nematodes, although succinate dehydrogenase activity was unaffected in the other organisms tested.