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

Abstract

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...

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

“…The modeling experiments suggested that lanosterol could fit into the active site, as supported by the type I binding spectra, but could not orientate viously calculated K s value of 11 M for eburicol binding to MgCYP51 by UV-visible spectroscopy (16) suggesting that the K m value is not solely dependent on the affinity of MgCYP51 for substrate. The MgCYP51 K m value for eburicol was comparable with that determined for human CYP51 of 30 M with lanosterol (30) but was 4-to 6-fold larger than K m values previously determined for C. albicans and S. cerevisiae CYP51 enzymes (15,31,32), suggesting a degree of variability with regard to substrate affinity among CYP51 enzymes.…”

Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase

“…L-Histidine was removed from the preparation by washing the enzyme with affinity chromatography medium using a 50-kDa-molecular-mass-cutoff Amicon Ultra-4 centrifugal filter (Millipore). Difference spectra were obtained by titrating affinity-purified ScErg11p6ϫHis and used to determine dissociation constant (K d ) values for the binding to the triazole drugs FLC and VCZ (30,31). Incremental additions of up to 7 l of triazole drug dissolved in dimethyl sulfoxide (DMSO) were added to a 0.5-ml sample of 1 M ScErg11p6ϫHis in affinity chromatography buffer in a 10-mm-pathlength cuvette.…”

Gain-of-Function Mutations in UPC2 Are a Frequent Cause of ERG11 Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

“…Overexpression of the efflux transporter genes CDR1, CDR2, and MDR1 is a common mechanism of drug resistance in this organism (10,17,26). Point mutations in the ERG11 gene result in reduced binding affinity of azoles to their target without precluding enzymatic function (31). In addition to point mutations, overexpression of ERG11 has also been shown to decrease fluconazole susceptibility.…”