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1.  Molecular Mechanisms of Drug Resistance in Clinical Candida Species Isolated from Tunisian Hospitals 
Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 μg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 μg/ml), voriconazole (MIC > 16 μg/ml), and amphotericin B (MIC > 32 μg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6β-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14α-demethylase and sterol Δ5,6-desaturase leading to azole-polyene cross-resistance.
PMCID: PMC3697321  PMID: 23629718
2.  Azole Affinity of Sterol 14α-Demethylase (CYP51) Enzymes from Candida albicans and Homo sapiens 
Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (Ks, 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [Kds], 42 to 131 nM) but bound fluconazole (Kd, ∼30,500 nM) and voriconazole (Kd, ∼2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (Kds, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (Kds, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (Kd, ∼40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC50) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC50, ∼150 μM) and did not significantly inhibit Δ60HsCYP51.
PMCID: PMC3591892  PMID: 23274672
3.  Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase 
Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.
PMCID: PMC3591943  PMID: 23275516
4.  Two Clinical Isolates of Candida glabrata Exhibiting Reduced Sensitivity to Amphotericin B Both Harbor Mutations in ERG2 
Antimicrobial Agents and Chemotherapy  2012;56(12):6417-6421.
Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 μg ml−1) were found to be ERG2 mutants, wherein Δ8-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr121 in ERG2; the corresponding residue (Thr119) in Saccharomyces cerevisiae is essential for sterol Δ8-Δ7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.
PMCID: PMC3497184  PMID: 23027188
5.  Facultative Sterol Uptake in an Ergosterol-Deficient Clinical Isolate of Candida glabrata Harboring a Missense Mutation in ERG11 and Exhibiting Cross-Resistance to Azoles and Amphotericin B 
We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 μg ml−1, respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium (glcYM). However, when grown on sterol-supplemented glcYM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ7-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented glcYM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using glcYM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using glcYM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 μg AMB ml−1, respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.
PMCID: PMC3421581  PMID: 22615281
6.  S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) Reduce In Vitro Inhibition by Fluconazole 
The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher Kd values observed.
PMCID: PMC3318376  PMID: 22252802
7.  Impact of Recently Emerged Sterol 14α-Demethylase (CYP51) Variants of Mycosphaerella graminicola on Azole Fungicide Sensitivity▿ 
Applied and Environmental Microbiology  2011;77(11):3830-3837.
The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
PMCID: PMC3127603  PMID: 21478305
8.  Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals ▿  
Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1 resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.
PMCID: PMC3067226  PMID: 21169436
9.  Complementation of a Saccharomyces cerevisiae ERG11/CYP51 (Sterol 14α-Demethylase) Doxycycline-Regulated Mutant and Screening of the Azole Sensitivity of Aspergillus fumigatus Isoenzymes CYP51A and CYP51B▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4920-4923.
Aspergillus fumigatus sterol 14α-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs showed identical sensitivity to itraconazole, posaconazole, clotrimazole, and voriconazole. Conversely, YUG37-pcyp51A withstood 16-fold-higher concentrations of fluconazole than YUG37-pcyp51B (8 and 0.5 μg ml−1, respectively).
PMCID: PMC2976139  PMID: 20733045
10.  Identification and Characterization of Four Azole-Resistant erg3 Mutants of Candida albicans▿  
Antimicrobial Agents and Chemotherapy  2010;54(11):4527-4533.
Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol Δ5,6-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ≥256, 16, 16, 8, and 1 μg ml−1, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 μg ml−1); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 μg ml−1). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.
PMCID: PMC2976150  PMID: 20733039
11.  Azole Binding Properties of Candida albicans Sterol 14-α Demethylase (CaCYP51)▿  
Antimicrobial Agents and Chemotherapy  2010;54(10):4235-4245.
Purified Candida albicans sterol 14-α demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with Ks values of 11 and 25 μM, respectively, and a Km value of 6 μM for lanosterol. Azole binding to CaCYP51 was “tight” with both the type II spectral intensity (ΔAmax) and the azole concentration required to obtain a half-ΔAmax 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 Kd values of 10 to 26 μM under oxidative conditions, compared with 47 μM 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.
PMCID: PMC2944560  PMID: 20625155
12.  Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B▿  
Antimicrobial Agents and Chemotherapy  2010;54(10):4225-4234.
Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (Ks, 8.6 μM) and eburicol (Ks, 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (Ks, 3.1 μM) and eburicol (Ks, 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the Kd (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a Kd value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with Kd values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.
PMCID: PMC2944604  PMID: 20660663
13.  A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B▿  
A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 μg ml−1, respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 μg ml−1 for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14α-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 μg ml−1). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.
PMCID: PMC2934972  PMID: 20547793
14.  Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae▿  
The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (Kd, 1.2 to 2.5 μM); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.
PMCID: PMC2650583  PMID: 19075057
15.  Discovery of a Novel Dual Fungal CYP51/Human 5-Lipoxygenase Inhibitor: Implications for Anti-Fungal Therapy 
PLoS ONE  2013;8(6):e65928.
We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.
PMCID: PMC3691235  PMID: 23826084
16.  Molecular Modelling of the Emergence of Azole Resistance in Mycosphaerella graminicola 
PLoS ONE  2011;6(6):e20973.
A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.
PMCID: PMC3124474  PMID: 21738598

Results 1-16 (16)