Emergence of Fluconazole-Resistant Sterol 14-Demethylase P450 (CYP51) in Candida albicans Is a Model Demonstrating the Diversification Mechanism of P450

Aoyama, Yuri; Kudo, Makiko; Asai, Kentaro; Okonogi, Kenji; Horiuchi, Tadao; Gotoh, Osamu; Yoshida, Yasuhiko

doi:10.1006/abbi.2000.1852

Cited by 10 publications

(10 citation statements)

References 5 publications

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“…Accordingly, crystal-structure determination of the MT 14DM fluconazole-bound form showed no interaction between F89 and fluconazole (26). In CA 14DM, Y132H mutant showed no effect on fluconazole binding but F145L substitution resulted in a 5-fold increase of IC 50 , a value similar to the one reported for the CA 14DM clone from the DUMC136 isolate (having both Y132H and F145L substitutions) when expressed in E. coli (27). This suggests that F145L but not Y132H is responsible for fluconazole resistance.…”

Section: Discussionsupporting

confidence: 68%

“…It is possible that the differences in fluconazole sensitivity of CA 14 DM mutants, seen between results using E. coli expression (data herein and Ref. 27) and fungal microsomal fractions (16,17) may be due to differences in the lipid composition (DLPC in the reconstituted system and E. coli membranes versus CA and S. cerevisiae microsomes). Unexpectedly, the H1 4DM Y145H substitution resulted in a dramatic decrease in the IC 50 value (three orders of magnitude), suggesting a possible interaction of a histidine at this position with the fluconazole, leading to better positioning of the azole toward the heme center and therefore greater sensitivity for fluconazole of the human Y145H versus CA Y132H during the catalytic cycle.…”

Section: Discussionmentioning

confidence: 73%

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Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Bellamine

Lepesheva

Waterman

2004

Journal of Lipid Research

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14 ␣ -Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azoleresistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC 50 for fluconazole, suggesting that F145 (conserved only in fungal 14 ␣ -demethylases) interacts with this azole. In C. albicans , F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.-Bellamine, A, G. I. Lepesheva, and M. R. Waterman. Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology.

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 73%

Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Bellamine

Lepesheva

Waterman

2004

Journal of Lipid Research

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“…Fungal resistance to azole drugs is frequently associated with CYP51 mutations that reduce affinity of the altered proteins for the drugs (Aoyama et al, 2000). A rationale for the use of azoles as antibacterials has been absent, due to assumptions that bacteria generally do not possess sterol biosynthetic pathways.…”

Section: Discussionmentioning

confidence: 99%

Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes

et al. 2002

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The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14α-sterol demethylases, the targets for the azole class of drugs in yeast and fungi. Previously, this type of P450 was considered to be absent from bacteria. When present at low concentrations in growth medium, azole antifungal drugs were shown to be potent inhibitors of the growth of Mycobacterium smegmatis and of Streptomyces strains, indicating that one or more of the P450s in these bacteria were viable drug targets. The drugs econazole and clotrimazole were most effective against M. smegmatis (MIC values of T02 and 03 µM, respectively) and were superior inhibitors of mycobacterial growth compared to rifampicin and isoniazid (which had MIC values of 12 and 365 µM, respectively). In contrast to their effects on the actinomycetes, the azoles showed minimal effects on the growth of Escherichia coli, which is devoid of P450s. Azole drugs coordinated tightly to the haem iron in M. tuberculosis H37Rv P450s encoded by genes Rv0764c (the sterol demethylase CYP51) and Rv2276 (CYP121). However, the azoles had a higher affinity for M. tuberculosis CYP121, with K d values broadly in line with the MIC values for M. smegmatis. This suggested that CYP121 may be a more realistic target enzyme for the azole drugs than CYP51, particularly in light of the fact that an S. coelicolor ∆CYP51 strain was viable and showed little difference in its sensitivity to azole drugs compared to the wild-type. If the azole drugs prove to inhibit a number of important P450s in M. smegmatis and S. coelicolor, then the likelihood of drug resistance developing in these species should be minimal. This suggests that azole drug therapy may provide a novel antibiotic strategy against strains of M. tuberculosis that have already developed resistance to isoniazid and other front-line drugs.

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“…Alterations of the Cyp51p leads to azole resistance in other Candida species such as C. albicans [14][15][16]. Occasionally, high level resistance to azoles is reported to be due to the combined effect of drug efflux and drug target modification [17,18].…”

Section: Introductionmentioning

confidence: 99%

Molecular Characterization and In Vitro Antifungal Susceptibility of Candida Glabrata Clinical Isolates with Reduced Echinocandin Susceptibility and High Level Multi-Azole Resistance

Vázquez¹,

Baxa²,

Wierman³

et al. 2014

Clin Microbial

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Candida glabrata is the second most commonly isolated yeast recovered from blood cultures in the United States. We characterized 85 C. glabrata clinical isolates recovered from various clinical specimens obtained from immunocompromised individuals. This collection was unique because it included a series of isolates recovered from the blood of a patient who only partially responded to antifungal therapy. In vitro activity of caspofungin, micafungin, anidulafungin, fluconazole, voriconazole and amphotericin B was evaluated. Most of the isolates were susceptible to the echinocandins, triazoles and amphotericin B. The geometric mean MIC of the antifungals for the susceptible isolates (n=79) were as follows: caspofungin, 0.061315 ± 0.076934; micafungin, 0.123521 ± 0.457202; anidulafungin, 0.044158 ± 0.895249; fluconazole, 7.013461 ± 20.56794; voriconazole, 0.324939 ± 1.051247; amphotericin B, 0.474923 ± 0.162994. Five of the six serial blood isolates showed a reduced echinocandin susceptibility (RES) to the echinocandins and the triazoles. Characterization of the hot spot 1 region of FKS1, FKS2 and FKS3 showed no amino acid alterations. However, the genes coding for the drug efflux proteins CgCDR1, CgCDR2, CgSNQ2, as well as Cgcyp51 were over-expressed in the isolates with RES and azole resistance compared to the susceptible isolates, indicating that the upregulation of the synthesis of efflux proteins and the drug target is responsible for conferring resistance to triazoles in these isolates. These results demonstrate that multiechinocandin and multi-azole resistant C. glabrata clinical isolates can emerge under the selection pressure imposed by specific drug therapy over a relatively short period of time.

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Emergence of Fluconazole-Resistant Sterol 14-Demethylase P450 (CYP51) in Candida albicans Is a Model Demonstrating the Diversification Mechanism of P450

Cited by 10 publications

References 5 publications

Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes

Molecular Characterization and In Vitro Antifungal Susceptibility of Candida Glabrata Clinical Isolates with Reduced Echinocandin Susceptibility and High Level Multi-Azole Resistance

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