A number of mechanisms of azole resistance in Candida albicans have already been described. One o... more A number of mechanisms of azole resistance in Candida albicans have already been described. One of these is increased synthesis of ergosterol in azole-resistant strains. This increase is thought to result from enhanced activity of the cytochrome P450 14 -demethylase and/or from increased amounts of cytochrome P450 in microsomal fractions. Lamb et al. have recently described mutations in this enzyme that reduce its affinity for azoles. Recent studies have also demonstrated an energy-dependent drug efflux mechanism in C. albicans similar to the mechanism described for resistance of cancer cells to anticancer agents. In tumour cells the multidrug resistance (MDR) phenotype is characterized by resistance to a broad spectrum of cytotoxic agents. This high level of crossresistance is due to increased expression of P-glycoprotein, a membrane glycoprotein. In humans this protein is encoded by the MDR1 gene; it pumps cytotoxic agents out of cells in an energy-dependent manner. A phenotype si...
The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matche... more The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378–2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14α-methyl-ergosta-8,24(28)-diene-3β,6α-diol (3,6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14α-methyl-ergosta-5,7,22,24(28)-tetraene-3βol, and 14α-methyl-fecosterol. In strains C48 and C56, no fu...
The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target... more 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 suscepti...
Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were pr... more Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and activity of the cytochrome P-450 14a-lanosterol demethylase which is the target for azole antifungals. The resistant isolate also had a lower intracellular level of fluconazole, but not of ketoconazole or itraconazole, than the susceptible isolate. In the present study a 3.7-fold increase in the copy number of the CYP51 gene, encoding the 14a-lanosterol demethylase, was found. The amount of CYP51 mRNA transcript in the resistant isolate was eight times greater than it was in the susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instability of the resistant isolate was demon-strated genotypically: a gradual loss of the duplicated chromosome was seen in successive subcultures of the isolate in flucon...
The many drugs that are available at present to treat fungal infections can be divided into four ... more The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allyalmines has not been reported from human pathogens, but, resistant phenotypes have ...
American Journal of Obstetrics and Gynecology, Oct 31, 1991
A large proportion of the presently available antifungal agents are claimed to derive their activ... more 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.
ABSTRACT The article gives an overview on the history of the discovery of P450 cytochromes and on... more ABSTRACT The article gives an overview on the history of the discovery of P450 cytochromes and on their occurrence in nature, especially on their interactions with metabolic pathways in fungi. The significance of the P450 cytochromes in the ergosterol synthesis as well as in the inhibitory mechanisms caused by imidazole and triazole antimycotics is described is detail.Zusammenfassung. Der Artikel gibt eine Übersicht über die Entdeckungsgeschichte der P450-Cytochrome und ihr Vorkommen in der Natur. Insbesondere wird eingegangen auf das Vorkommen der P450-Cytochrome in Pilzen und ihre Interaktionen mit Stoffwechselprozessen der Pilzzellen. Die Bedeutung der P450-Cytochrome für die Ergosterol-Synthese der Pilze und für deren Hemmechanismen durch Imidazol- und Triazol-Antimykotika wird im Detail dargestellt.
The many drugs that are available at present to treat fungal infections can be divided into four ... more The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allylamines has not been reported from human pathogens, but, resistant phenotypes have ...
ABSTRACT : A system is described consisting of mixed cultures of human fibroblasts and fungi that... more ABSTRACT : A system is described consisting of mixed cultures of human fibroblasts and fungi that can be used on a large scale for the semiquantitative determination of the fungistatic and cytotoxic activity of chemotherapeutic compounds.In this system ketoconazole strongly inhibits the growth of Candida albicans at a concentration of 0,01 μg/ml while only being toxic for the mammalian cells at 100 μg/ml. This high “therapeutic ratio” corresponds to its in vivo effectiveness and lack of toxicity. At the same low concentration (0.01 μg/ml) the compound completely inhibits the formation of mycelia which is the predominant form in control cultures. The remaining population consists solely of clusters of mutually attached yeast cells. It is believed that the serious growth inhibitory effect at very low concentrations and specially the inhibition of mycelium formation explains the in vivo activity of the compound where the host phagocytic cells are left with a small population of more easily removable pathogens.Trichophyton mentagrophytes and Microsporum canis are as sensitive to ketoconazole as C. albicans.The activity of ketoconazole against Aspergillus fumigatus is much less pronounced. A concentration of 10 μg/ml is needed to inhibit its growth. This also correlates with the in vivo observations.Zusammenfassung: Ein Verfahren wird beschrieben, bei dem Mischkulturen von menschlichen Fibroblasten mit Pilzen zur semiquantitativen Bestimmung der fungistatischen sowie zytotoxischen Wirksamkeit von Chemotherapeutika angewandt werden.Bei einer Konzentration von 0,01 μg/ml hemmt Ketoconazol unter unseren Versuchs-bedingungen erheblich das Wachstum von C. albicans, während erst bei 100 μg/ml eine toxische Wirkung auf Säugetierzellen beobachtet wird. Diesem hohen therapeutischen Index entsprechen die in vivo Wirksamkeit und das Fehlen toxischer Effekte. Bei derselben niedrigen Konzentration (0,01 μg/ml) hemmt die Substanz die Myzelbildung, die auf den Kontrollkuren bei weitem vorherrscht, völlig. Die übrige Population besteht lediglich aus Anhäufungen von miteinander verbundenen Hefezellen. Man nimmt an, daß die starke Wachstumshemmung durch sehr niedrige Konzentrationen und besonders die Hemmung der Myzelbildung die in vivo Wirksamkeit des Ketoconazols erklären. Dabei wird nur eine kleine Zahl leicht zu beseitigender Pathogene den phagozytierenden Wirtzellen hinterlassen.Trichophyton mentagrophytes und Microsporum canis sind ebenso empfindlich gegen Ketoconazol wie C. albicans.Die Wirksamkeit gegenüber Aspergillus fumigatus ist viel weniger ausgeprägt. Eine Wachstumshemmung wird bei 10 μg/ml erzielt. Dies entspricht den Beobachtungen in vivo.
1. Med Mycol. 1998;36 Suppl 1:119-28. Antifungal drug resistance in pathogenic fungi. Vanden Boss... more 1. Med Mycol. 1998;36 Suppl 1:119-28. Antifungal drug resistance in pathogenic fungi. Vanden Bossche H, Dromer F, Improvisi I, Lozano-Chiu M, Rex JH, Sanglard D. Janssen Research Foundation, Beerse, Belgium. hvbossch ...
A number of mechanisms of azole resistance in Candida albicans have already been described. One o... more A number of mechanisms of azole resistance in Candida albicans have already been described. One of these is increased synthesis of ergosterol in azole-resistant strains. This increase is thought to result from enhanced activity of the cytochrome P450 14 -demethylase and/or from increased amounts of cytochrome P450 in microsomal fractions. Lamb et al. have recently described mutations in this enzyme that reduce its affinity for azoles. Recent studies have also demonstrated an energy-dependent drug efflux mechanism in C. albicans similar to the mechanism described for resistance of cancer cells to anticancer agents. In tumour cells the multidrug resistance (MDR) phenotype is characterized by resistance to a broad spectrum of cytotoxic agents. This high level of crossresistance is due to increased expression of P-glycoprotein, a membrane glycoprotein. In humans this protein is encoded by the MDR1 gene; it pumps cytotoxic agents out of cells in an energy-dependent manner. A phenotype si...
The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matche... more The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378–2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14α-methyl-ergosta-8,24(28)-diene-3β,6α-diol (3,6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14α-methyl-ergosta-5,7,22,24(28)-tetraene-3βol, and 14α-methyl-fecosterol. In strains C48 and C56, no fu...
The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target... more 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 suscepti...
Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were pr... more Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and activity of the cytochrome P-450 14a-lanosterol demethylase which is the target for azole antifungals. The resistant isolate also had a lower intracellular level of fluconazole, but not of ketoconazole or itraconazole, than the susceptible isolate. In the present study a 3.7-fold increase in the copy number of the CYP51 gene, encoding the 14a-lanosterol demethylase, was found. The amount of CYP51 mRNA transcript in the resistant isolate was eight times greater than it was in the susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instability of the resistant isolate was demon-strated genotypically: a gradual loss of the duplicated chromosome was seen in successive subcultures of the isolate in flucon...
The many drugs that are available at present to treat fungal infections can be divided into four ... more The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allyalmines has not been reported from human pathogens, but, resistant phenotypes have ...
American Journal of Obstetrics and Gynecology, Oct 31, 1991
A large proportion of the presently available antifungal agents are claimed to derive their activ... more 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.
ABSTRACT The article gives an overview on the history of the discovery of P450 cytochromes and on... more ABSTRACT The article gives an overview on the history of the discovery of P450 cytochromes and on their occurrence in nature, especially on their interactions with metabolic pathways in fungi. The significance of the P450 cytochromes in the ergosterol synthesis as well as in the inhibitory mechanisms caused by imidazole and triazole antimycotics is described is detail.Zusammenfassung. Der Artikel gibt eine Übersicht über die Entdeckungsgeschichte der P450-Cytochrome und ihr Vorkommen in der Natur. Insbesondere wird eingegangen auf das Vorkommen der P450-Cytochrome in Pilzen und ihre Interaktionen mit Stoffwechselprozessen der Pilzzellen. Die Bedeutung der P450-Cytochrome für die Ergosterol-Synthese der Pilze und für deren Hemmechanismen durch Imidazol- und Triazol-Antimykotika wird im Detail dargestellt.
The many drugs that are available at present to treat fungal infections can be divided into four ... more The many drugs that are available at present to treat fungal infections can be divided into four broad groups on the basis of their mechanism of action. These antifungal agents either inhibit macromolecule synthesis (flucytosine), impair membrane barrier function (polyenes), inhibit ergosterol synthesis (allylamines, thiocarbamates, azole derivatives, morpholines), or interact with microtubules (griseofulvin). Drug resistance has been identified as the major cause of treatment failure among patients treated with flucytosine. A lesion in the UMP-pyrophosphorylase is the most frequent clinical determinant of resistance to 5FC in Candida albicans. Despite extensive use of polyene antibiotics for more than 30 years, emergence of acquired resistance seems not be a significant clinical problem. Polyene-resistant Candida isolates have a marked decrease in their ergosterol content. Acquired resistance to allylamines has not been reported from human pathogens, but, resistant phenotypes have ...
ABSTRACT : A system is described consisting of mixed cultures of human fibroblasts and fungi that... more ABSTRACT : A system is described consisting of mixed cultures of human fibroblasts and fungi that can be used on a large scale for the semiquantitative determination of the fungistatic and cytotoxic activity of chemotherapeutic compounds.In this system ketoconazole strongly inhibits the growth of Candida albicans at a concentration of 0,01 μg/ml while only being toxic for the mammalian cells at 100 μg/ml. This high “therapeutic ratio” corresponds to its in vivo effectiveness and lack of toxicity. At the same low concentration (0.01 μg/ml) the compound completely inhibits the formation of mycelia which is the predominant form in control cultures. The remaining population consists solely of clusters of mutually attached yeast cells. It is believed that the serious growth inhibitory effect at very low concentrations and specially the inhibition of mycelium formation explains the in vivo activity of the compound where the host phagocytic cells are left with a small population of more easily removable pathogens.Trichophyton mentagrophytes and Microsporum canis are as sensitive to ketoconazole as C. albicans.The activity of ketoconazole against Aspergillus fumigatus is much less pronounced. A concentration of 10 μg/ml is needed to inhibit its growth. This also correlates with the in vivo observations.Zusammenfassung: Ein Verfahren wird beschrieben, bei dem Mischkulturen von menschlichen Fibroblasten mit Pilzen zur semiquantitativen Bestimmung der fungistatischen sowie zytotoxischen Wirksamkeit von Chemotherapeutika angewandt werden.Bei einer Konzentration von 0,01 μg/ml hemmt Ketoconazol unter unseren Versuchs-bedingungen erheblich das Wachstum von C. albicans, während erst bei 100 μg/ml eine toxische Wirkung auf Säugetierzellen beobachtet wird. Diesem hohen therapeutischen Index entsprechen die in vivo Wirksamkeit und das Fehlen toxischer Effekte. Bei derselben niedrigen Konzentration (0,01 μg/ml) hemmt die Substanz die Myzelbildung, die auf den Kontrollkuren bei weitem vorherrscht, völlig. Die übrige Population besteht lediglich aus Anhäufungen von miteinander verbundenen Hefezellen. Man nimmt an, daß die starke Wachstumshemmung durch sehr niedrige Konzentrationen und besonders die Hemmung der Myzelbildung die in vivo Wirksamkeit des Ketoconazols erklären. Dabei wird nur eine kleine Zahl leicht zu beseitigender Pathogene den phagozytierenden Wirtzellen hinterlassen.Trichophyton mentagrophytes und Microsporum canis sind ebenso empfindlich gegen Ketoconazol wie C. albicans.Die Wirksamkeit gegenüber Aspergillus fumigatus ist viel weniger ausgeprägt. Eine Wachstumshemmung wird bei 10 μg/ml erzielt. Dies entspricht den Beobachtungen in vivo.
1. Med Mycol. 1998;36 Suppl 1:119-28. Antifungal drug resistance in pathogenic fungi. Vanden Boss... more 1. Med Mycol. 1998;36 Suppl 1:119-28. Antifungal drug resistance in pathogenic fungi. Vanden Bossche H, Dromer F, Improvisi I, Lozano-Chiu M, Rex JH, Sanglard D. Janssen Research Foundation, Beerse, Belgium. hvbossch ...
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