Effects of Y132H and F145L Substitutions on the Activity, Azole Resistance and Spectral Properties of Candida albicans Sterol 14-Demethylase P450 (CYP51): A Live Example Showing the Selection of Altered P450 through Interaction with Environmental Compounds

Kudo, Makiko; Ohi, Miwa; Aoyama, Yuri; Nitahara, Yuko; Chung, Sung‐Kee; Yoshida, Yasuhiko

doi:10.1093/jb/mvi073

Cited by 21 publications

(16 citation statements)

References 27 publications

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“…IC 50 s of 1.2 to 1.3 M for both fluconazole and itraconazole (half of the CaCYP51 concentration present) were obtained, with total inhibition at 2.5 M azole. These results are in agreement with previous in vitro CYP51 activity studies using purified CaCYP51 and microsomal protein fractions (4,19,22,23,50). However, MIC values for CYP51 activity using C. albicans cell extracts (51) previously indicated that itraconazole was 4-fold more effective as an inhibitor of CYP51 activity than fluconazole, in contrast to the 2-fold differences in K d values (Table 2) and the similar IC 50 s from the CYP51 reconstitution assays (Fig.…”

Section: Resultssupporting

confidence: 91%

“…Even where purified C. albicans CYP51 protein was used (4,19), analysis of the azole binding data was by the Michaelis-Menten or Hill equations, which is applicable for systems where the K d for the ligand is at least 5-fold higher than the concentration of enzyme (6). Previous work with S. cerevisiae microsomes containing wild-type C. albicans CYP51 gave [azole] 0.5 values approximately half the P450 concentration used, which is compatible with the binding observed being "tight" for fluconazole, itraconazole, and ketoconazole (22,23).…”

Section: Resultsmentioning

confidence: 99%

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Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

Warrilow

Martel

Parker

et al. 2010

Antimicrob Agents Chemother

103

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Purified Candida albicans sterol 14-␣ demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K s values of 11 and 25 M, respectively, and a K m value of 6 M for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (⌬A max ) and the azole concentration required to obtain a half-⌬A max being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K d values of 10 to 26 M under oxidative conditions, compared with 47 M for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4-and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min ؊1 ), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.Fungal sterol 14-␣ demethylase (CYP51) is required for ergosterol biosynthesis, an ancestral activity in the cytochrome P450 (CYP) superfamily of hemoproteins, and is the main target for azole antifungal drugs (15). CYP51 has been shown to be essential for viability in Saccharomyces cerevisiae (14). Azole inhibitors that are selective for the fungal enzyme over the human homologue have been developed and are commonly used to treat fungal infections, including those caused by Candida albicans (23,39). The mode of action of azole antifungal drugs involves the selective inhibition of the fungal CYP51, involving the nucleophilic nitrogen of the azole heterocyclic ring coordinating as the sixth ligand of the heme iron in the ferric state and the azole drug side chains interacting with the polypeptide structure (12, 57). However, due to prolonged and prophylactic use of azole drugs in the clinic, the emergence of azole-resistant C. albicans strains and other Candida species has become an increasing problem, especially among hospitalized immunocompromised patients, such as HIV and AIDS, cancer, and transplant patients, leading to a growing need to develop new effective antifungal strategies against drug-resistant stra...

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

Warrilow

Martel

Parker

et al. 2010

Antimicrob Agents Chemother

103

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“…Similarly, a Histoplasma capsulatum isolate with an Y136F substitution (Y136 is analogous to Y132 in C. albicans) displayed reduced susceptibility to fluconazole but not posaconazole (16). Finally, a C. albicans CYP51 protein harboring Y132H and F145L substitutions was significantly less susceptible to inhibition by fluconazole (10). Together these data suggest that substitutions at Y132 and F145 can strongly impact susceptibility to fluconazole without impacting either posaconazole or itraconazole.…”

mentioning

confidence: 89%

Molecular Basis for Enhanced Activity of Posaconazole against Absidia corymbifera and Rhizopus oryzae

Chau¹,

Chen²,

McNicholas³

et al. 2006

Antimicrob Agents Chemother

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“…The CYP51 BC-loop residue 105 (numbering according to T. cruzi and T. brucei CYP51) is indispensable in the discrimination of the species-specific sterol substrates in T. cruzi and T. brucei [19], [21]. Also, a critical mutation hot spot [22], the well conserved BC-loop residue Y116 was reported to be involved in fungal drug resistance, inhibitor binding, and the catalytic function of CYP51 in Candida albicans (Y132, according to C. albicans numbering) [22]–[27], Histoplasma capsulatum (Y136, according to H. capsulatum numbering) [28], and in the causative agents of zygomycosis in humans, Rhizopus oryzae and Absidia corymbifera [29]. It may therefore play a similar role in T. cruzi .…”

Section: Introductionmentioning

confidence: 99%

Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole

et al. 2010

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BackgroundChagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding.Methodology/Principal FindingsWe have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 Å and 2.27 Å), and from the related pathogen T. brucei (resolutions of 2.7 Å and 2.6 Å), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180° depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target.Conclusions/SignificanceThe structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.

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Effects of Y132H and F145L Substitutions on the Activity, Azole Resistance and Spectral Properties of Candida albicans Sterol 14-Demethylase P450 (CYP51): A Live Example Showing the Selection of Altered P450 through Interaction with Environmental Compounds

Cited by 21 publications

References 27 publications

Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)

Molecular Basis for Enhanced Activity of Posaconazole against Absidia corymbifera and Rhizopus oryzae

Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole

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