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1.  Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51) 
Applied and Environmental Microbiology  2014;80(19):6154-6166.
A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml−1). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml−1) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml−1). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml−1) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.
PMCID: PMC4178690  PMID: 25085484
2.  Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms 
The Biochemical journal  2012;443(1):267-277.
Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14-demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate–mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control <[2-13C]leucine<[2-13C]acetate<[1-13C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.
PMCID: PMC3491665  PMID: 22176028
[1-13C]glucose; ergosterol biosynthesis; sterol C24-methyltransferase; sterol C14-demethylase; Trypanosoma brucei; trypanosome
3.  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
4.  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
5.  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
6.  C. neoformans Site-2 protease is required for virulence and survival in the presence of azole drugs 
Molecular microbiology  2009;74(3):672-690.
In the human fungal pathogen Cryptococcus neoformans, the SREBP ortholog Sre1 is important for adaptation and growth in nutrient-limiting host tissues. In this study, we characterize the C. neoformans serotype A Sre1 and its activating protease, Stp1. We demonstrate that Stp1 is a functionally conserved ortholog of the mammalian Site-2 protease and that Stp1 cleaves Sre1 within its predicted first transmembrane segment. Gene expression analysis revealed that Stp1 is required for both Sre1-dependent and Sre1-independent gene transcription, indicating that other substrates of Stp1 may exist. Using gas chromatography, we showed that Sre1 and Stp1 are required for both normoxic and hypoxic ergosterol biosynthesis, and therefore cells lacking SRE1 or STP1 are defective for growth in the presence of low levels of the ergosterol biosynthesis inhibitors, itraconazole and 25-thialanosterol. Importantly, our studies demonstrated fungicidal effects of itraconazole and 25-thialanosterol toward sre1Δ and stp1Δ cells, demonstrating that the Sre1 pathway is required for both growth and survival in the presence of sterol biosynthesis-inhibiting antifungal drugs. Given the need for fungicidal drugs, we propose that inhibitors of Stp1, Sre1, or other regulators of Sre1 function administered in combination with a sterol synthesis inhibitor could prove an effective anti-cryptococcal therapy.
PMCID: PMC2917040  PMID: 19818023
Cryptococcus neoformans; SREBP; hypoxia; sterol; ergosterol; azole; Sre1
7.  Indomethacin Amides as a Novel Molecular Scaffold for Targeting Trypanosoma cruzi Sterol 14α-Demethylase 
Journal of medicinal chemistry  2009;52(9):2846-2853.
Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14α-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51 inhibitors has been identified using optical high throughput screening followed by web-database search for similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-amides were found to bind to TCCYP51, inhibit its activity in vitro and produce strong antiparasitic effects in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds is by inhibition of sterol biosynthesis in the parasite.
PMCID: PMC2744100  PMID: 19354253
8.  CYP51: A Major Drug Target in the Cytochrome P450 Superfamily 
Lipids  2008;43(12):1117-1125.
The cytochrome P540 (CYP) superfamily currently includes about 9,000 proteins forming more than 800 families. The enzymes catalyze monooxygenation of a vast array of compounds and play essentially two roles. They provide biodefense (detoxification of xenobiotics, antibiotic production) and participate in biosynthesis of important endogenous molecules, particularly steroids. Based on these two roles, sterol 14|*alpha*|-demethylases (CYP51) belong to the second group of P450s. The CYP51 family, however, is very special as its members preserve strict functional conservation in enzyme activity in all biological kingdoms. At amino acid identity across the kingdoms as low as 25–30%, they all catalyze essentially the same three-step reaction of oxidative removal of the 14|*alpha*|-methyl group from the lanostane frame. This reaction is the required step in sterol biosynthesis of pathogenic microbes. We have shown that specific inhibition of protozoan CYP51 can potentially provide treatment for human trypanosomiases. Three sets of CYP51 inhibitors tested in vitro and in trypanosomal cells in this study include azoles [best results being 50% cell growth inhibition at <1 and at 1.3 µM for Trypanosoma cruzi (TC) and Trypanosoma brucei (TB), respectively], non-azole compounds (50% TC cell growth inhibition at 5 µM) and substrate analogs of the 14|*alpha*|-demethylase reaction. 32-Methylene cyclopropyl lanost-7-enol exhibited selectivity toward TC with 50% cell growth inhibition at 3 µM.
PMCID: PMC2715142  PMID: 18769951
Cytochrome P450 (CYP51); Sterol biosynthesis; Sterol 14α-demethylase; Enzymatic activity; Inhibition; Sleeping sickness; Chagas disease; Antifungal agents; Antitrypanosomal drugs
9.  Sterol 14α-demethylase as a potential target for antitrypanosomal therapy: enzyme inhibition and parasite cell growth 
Chemistry & biology  2007;14(11):1283-1293.
Sterol 14α-demethylases (CYP51) serve as primary targets for antifungal drugs and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands which demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a novel lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.
PMCID: PMC2324070  PMID: 18022567
10.  Biosynthesis of Cholesterol and Other Sterols 
Chemical Reviews  2011;111(10):6423-6451.
PMCID: PMC3191736  PMID: 21902244

Results 1-10 (10)