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Sterol 14α-demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane-bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane-bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen-bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes., (Copyright © 2015 John Wiley & Sons, Ltd.)

Several studies have shown that differences in lipid composition and in the lipid biosynthetic pathway affect the aluminium (Al) tolerance of plants, but little is known about the molecular mechanisms underlying these differences. Phospholipids create a negative charge at the surface of the plasma membrane and enhance Al sensitivity as a result of the accumulation of positively charged Al(3+) ions. The phospholipids will be balanced by other electrically neutral lipids, such as sterols. In the present research, Al tolerance was compared among pea (Pisum sativum) genotypes. Compared with Al-tolerant genotypes, the Al-sensitive genotype accumulated more Al in the root tip, had a less intact plasma membrane, and showed a lower expression level of PsCYP51, which encodes obtusifoliol-14α-demethylase (OBT 14DM), a key sterol biosynthetic enzyme. The ratio of phospholipids to sterols was higher in the sensitive genotype than in the tolerant genotypes, suggesting that the sterol biosynthetic pathway plays an important role in Al tolerance. Consistent with this idea, a transgenic Arabidopsis thaliana line with knocked-down AtCYP51 expression showed an Al-sensitive phenotype. Uniconazole-P, an inhibitor of OBT 14DM, suppressed the Al tolerance of Al-tolerant genotypes of maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa), wheat (Triticum aestivum), and triticale (×Triticosecale Wittmark cv. Currency). These results suggest that increased sterol content, regulated by CYP51, with concomitant lower phospholipid content in the root tip, results in lower negativity of the plasma membrane. This appears to be a common strategy for Al tolerance among several plant species., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Recent clinical trials investigating treatment of chronic indeterminate Chagas disease with two re-purposed azole anti-fungal drugs, posaconazole and ravuconazole, revealed their inferiority to the current standard-of-care benznidazole and highlighted the inadequacy of the existing pre-clinical testing paradigm for this disease. A very limited number of controlled clinical trials for Chagas disease have been conducted to date. The selection of these compounds for clinical evaluation relied heavily on pre-clinical data obtained from in vitro screens and animal studies. This chapter reviews the evolution of CYP51 as a target for Trypanosoma cruzi growth inhibition and also explores the impact of clinical trial data on contemporary Chagas disease drug discovery. Advances in pre-clinical profiling assays, the current compound landscape and progress towards the identification of new drug targets to re-invigorate research are reviewed., (© 2015 Elsevier B.V. All rights reserved.)

The cholesterol biosynthesis regulation is the important part of the hypercholesterolemia diseases therapy. The inhibition of the post-squalene cholesterol biosynthesis steps provide the alternative to classic statin therapy. Sterol-14a-demethylase (CYP51) is one of the hypothetical targets for it. In this work the screening of the ability to interact with human CYP51 (CYP51A1) for the nature low-weight compounds with steroid-like scaffold were performed by integration of the surface plasmon resonance biosensor and spectral titration methods. The results of the selection were 4 compounds (betulafolientriol, holothurin A, teasaponin, capsicoside A) witch had high affinity to the CYP51A1 active site. These data extend the range of compounds which may be used as specific inhibitors of CYP51 and give the permission to suggest the dynamic of the enzyme.

Fungal diseases are an increasing global burden. Fungi are now recognised to kill more people annually than malaria, whilst in agriculture, fungi threaten crop yields and food security. Azole resistance, mediated by several mechanisms including point mutations in the target enzyme (CYP51), is increasing through selection pressure as a result of widespread use of triazole fungicides in agriculture and triazole antifungal drugs in the clinic. Mutations similar to those seen in clinical isolates as long ago as the 1990s in Candida albicans and later in Aspergillus fumigatus have been identified in agriculturally important fungal species and also wider combinations of point mutations. Recently, evidence that mutations originate in the field and now appear in clinical infections has been suggested. This situation is likely to increase in prevalence as triazole fungicide use continues to rise. Here, we review the progress made in understanding azole resistance found amongst clinically and agriculturally important fungal species focussing on resistance mechanisms associated with CYP51. Biochemical characterisation of wild-type and mutant CYP51 enzymes through ligand binding studies and azole IC50 determinations is an important tool for understanding azole susceptibility and can be used in conjunction with microbiological methods (MIC50 values), molecular biological studies (site-directed mutagenesis) and protein modelling studies to inform future antifungal development with increased specificity for the target enzyme over the host homologue.

While the orally-active azoles such as voriconazole and itraconazole are effective antifungal agents, they potently inhibit a broad range of off-target human cytochrome P450 enzymes (CYPs) leading to various safety issues (e.g., drug-drug interactions, liver toxicity). Herein, we describe rationally-designed, broad-spectrum antifungal agents that are more selective for the target fungal enzyme, CYP51, than related human CYP enzymes such as CYP3A4. Using proprietary methodology, the triazole metal-binding group found in current clinical agents was replaced with novel, less avid metal-binding groups in concert with potency-enhancing molecular scaffold modifications. This process produced a unique series of fungal CYP51-selective inhibitors that included the oral antifungal 7d (VT-1161), now in Phase 2 clinical trials. This series exhibits excellent potency against key yeast and dermatophyte strains. The chemical methodology described is potentially applicable to the design of new and more effective metalloenzyme inhibitor treatments for a broad array of diseases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The acidic residues of the "acid-alcohol pair" in CYP51 enzymes are uniformly replaced with histidine. Herein, we adopt the Mycobacterium tuberculosis (mt) enzyme as a model system to investigate these residues' roles in finely tuning the heme conformation, iron spin state, and formation and decay of the oxyferrous enzyme. Properties of the mtCYP51 and the T260A, T260V, and H259A mutants were interrogated using UV-Vis and resonance Raman spectroscopies. Evidence supports that these mutations induce comprehensive changes in the heme environment. The heme iron spin states are differentially sensitive to the binding of the substrate, dihydrolanosterol (DHL). DHL and clotrimazole perturb the local environments of the heme vinyl and propionate substituents. Molecular dynamics (MD) simulations of the DHL-enzyme complexes support that the observed perturbations are attributable to changes in the DHL binding mode. Furthermore, the rates of the oxyferrous formation were measured using stopped-flow methods. These studies demonstrate that both HT mutations and DHL modulate the rates of oxyferrous formation. Paradoxically, the binding rate to the H259A mutant-DHL complex was approximately four-fold that of mtCYP51, a phenomenon that is predicted to result from the creation of an additional diffusion channel from loss of the H259-E173 ion pair in the mutant. Oxyferrous enzyme auto-oxidation rates were relatively constant, with the exception of the T260V-DHL complex. MD simulations lead us to speculate that this behavior may be attributed to the distortion of the heme macrocycle by the substrate., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes., Methods: PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS., Results: Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26μM for ketoconazole and 0.64μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s., Conclusions: Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s., General Significance: The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4-aminopyridyl-based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub-nanomolar compound 3, have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å X-ray structure, TcCYP51 is in a conformation that has been influenced by compound 3 and is distinct from the previously characterized ground-state conformation of CYP51 drug-target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of triazole antifungal agents with piperidine side chains was designed and synthesized. The results of antifungal tests against eight human pathogenic fungi in vitro showed that all the compounds exhibited moderate-to-excellent activities. Molecular docking between 8d and the active site of Candida albicans CYP51 was provided based on the computational docking results. The triazole interacts with the iron of the heme group. The difluorophenyl group is located in the S3 subsite and its fluorine atom (2-F) can form H-bonds with Gly307. The side chain is oriented into the S4 subsite and formed hydrophobic and van der Waals interactions with the amino residues. Moreover, the phenyl group in the side chain interacts with the phenol group of Phe380 through the formation of π-π face-to-edge interactions.

A series of 2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl dithiocarbamates as new analogs of fluconazole were synthesized and their antifungal activities were evaluated. Among these compounds, 2a-f and 3a-q exhibited higher activities than fluconazole against nearly all fungi tested except Aspergillus fumigatus. Noticeably, the in vitro biological activities of 2b, 3a, 3c, 3h-k, and 3o-q against Candida species were much better than those of fluconazole and ketoconazole. Also, 2a-d, 3a-d, 3e-f, 3h-k, 3p and 3q showed higher activities against A. fumi than fluconazole. Computational docking experiments indicated that the inhibition of CYP51 involved a coordination bond with iron of the heme group, the hydrophilic H-bonding region, the hydrophobic region, and the narrow hydrophobic cleft., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Since the invasion of Phakopsora pachyrhizi (Syd. & P. Syd.) in Brazil, there have been detrimental yield losses of soybeans [Glycine max (L.) Merr.]. Disease management is mainly based on fungicide treatment. The sensitivity of single P. pachyhrizi isolates towards different demethylation-inhibitors (DMIs) and quinone-outside-inhibitors (QoI) was surveyed and the corresponding resistance mechanisms were analysed., Results: The QoI-response remained stable, while a loss of sensitivity towards DMIs occurred. Molecular analyses of cytochrome b showed an intron after codon 143 which is reported to prevent the development of a G143A mutation. Analysis of cyp51 revealed that point mutations and overexpression are involved in the sensitivity reduction towards DMIs. Of the detected mutations, Y131F and Y131H, respectively, and K142R are likely homologous to mutations found in other pathogens. As suggested by modelling studies, these three mutations as well as additional mutations F120L, I145F and I475T correlate to increased effective doses of 50%, ED50 -values, towards all tested DMIs. Furthermore, a constitutive up-regulation of the cyp51-gene up to ten-fold was noticed in some of the DMI-adapted isolates, while all sensitive isolates responded as the wild type., Conclusion: The G143A mutation is thought to result in significant as well as stable resistance factors towards QoIs, while other mutations play only a minor role. Since G143A development is prevented in Phakopsora pachyhrizi, a stable control of soybean rust with QoIs in future is rather likely. In contrast, a shifting in sensitivity towards DMIs has been observed, which is due to multiple independent mechanisms., (© 2013 Society of Chemical Industry.)

Sterol 14α-demethylase (CYP51) is an important therapeutic target for fungal and parasitic infections due to its key role in the biosynthesis of ergosterol, an essential component of the cell membranes of these pathogenic organisms. We report the development of potent and selective d-tryptophan-derived inhibitors of T. cruzi CYP51. Structural information obtained from the cocrystal structure of CYP51 and (R)-2, which is >1000-fold more potent than its enantiomer (S)-1, was used to guide design of additional analogues. The in vitro efficacy data presented here for (R)-2-(R)-8, together with preliminary in vitro pharmacokinetic data suggest that this new CYP51 inhibitor scaffold series has potential to deliver drug candidates for treatment of T. cruzi infections.

Background: The fungus Sclerotinia homoeocarpa causes dollar spot, the most important turfgrass disease worldwide. Demethylation inhibitor (DMI) fungicides have been relied upon heavily to manage this disease. Presently, populations of S. homoeocarpa with reduced sensitivity or resistance to DMIs are widespread in the United States., Results: Cytochrome P450 sterol 14α-demethylase (ShCYP51) and its flanking regions were identified and sequenced in 29 isolates of S. homoeocarpa with a range of DMI sensitivities. No modifications were found in the gene coding and upstream regions that were consistently related to DMI sensitivity. In the absence of propiconazole, ShCYP51 was expressed at a similar low level among DMI baseline and resistant isolates. In the presence of propiconazole, DMI-resistant isolates were induced to express ShCYP51 at significantly higher levels than baseline isolates by propiconazole at 5 mg L(-1) for 5 h or at 0.5 mg L(-1) for 72 h. The ShCYP51 expression level after 72 h exposure to 0.5 mg L(-1) of propiconazole was linearly related to EC50 values and ΔRG (the change in relative growth rate over time), with R(2) values equal to 83.7 and 90.0% respectively., Conclusion: Induced overexpression of ShCYP51 in resistant isolates following DMI exposure is an important factor determining DMI sensitivity in S. homoeocarpa., (© 2013 Society of Chemical Industry.)

New dialkylimidazole based sterol 14α-demethylase inhibitors were prepared and tested as potential anti-Trypanosoma cruzi agents. Previous studies had identified compound 2 as the most potent and selective inhibitor against parasite cultures. In addition, animal studies had demonstrated that compound 2 is highly efficacious in the acute model of the disease. However, compound 2 has a high molecular weight and high hydrophobicity, issues addressed here. Systematic modifications were carried out at four positions on the scaffold and several inhibitors were identified which are highly potent (EC50 <1 nM) against T. cruzi in culture. The halogenated derivatives 36j, 36k, and 36p, display excellent activity against T. cruzi amastigotes, with reduced molecular weight and lipophilicity, and exhibit suitable physicochemical properties for an oral drug candidate., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Due to the repeated discovery of new members of the Fusarium graminearum species complex (FGSC), some of the F. graminearum sensu stricto (s.s.)-specific qPCR assays developed to date have since been shown to be non-specific. In this study, a probe-based qPCR method was developed, targeting a sterol 14-alpha demethylase (CYP51) paralogue, CYP51C unique to the genus Fusarium, for the simultaneous detection of F. asiaticum, F. ussurianum and F. vorosii. Specificity of the assay was demonstrated for a wide range of Fusarium species, including all tested FGSC members (n=6), originating from different hosts and geographic regions. Alongside a previously published assay for detection of F. graminearum, we were able to show that members of the Asian clade of the FGSC (i.e. F. asiaticum, F. ussurianum and F. vorosii) were the primary etiological agent in wheat seeds samples originating from Central-East China. The grain samples from the UK tested negative for the presence of the FGSC's Asian clade and positive for presence of F. graminearum. It is likely that only F. graminearum s.s. is present in the UK, but the presence of other FGSC members cannot be ruled out and need further investigation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The antiparasitic activity of azole and new 4-aminopyridine derivatives has been investigated. The imidazoles 1 and 3-5 showed a potent in vitro antichagasic activity with IC50 values in the low nanomolar concentration range. The (S)-1, (S)-3, and (S)-5 enantiomers showed (up to) a thousand-fold higher activity than the reference drug benznidazole and furthermore low cytotoxicity on rat myogenic L6 cells.

Inhibition of the Trypanosoma cruzi cysteine protease cruzain has been proposed as a therapeutic approach for the treatment of Chagas' disease. Among the best-studied cruzain inhibitors to date is the vinylsulfone K777 (1), which has proven effective in animal models of Chagas' disease. Recent structure-activity studies aimed at addressing potential liabilities of 1 have now produced analogues such as N-[(2S)-1-[[(E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-3-(4-methylphenyl)-1-oxopropan-2-yl]pyridine-4-carboxamide (4), which is trypanocidal at ten-fold lower concentrations than for 1. We now find that the trypanocidal activity of 4 derives primarily from the inhibition of T. cruzi 14-α-demethylase (TcCYP51), a cytochrome P450 enzyme involved in the biosynthesis of ergosterol in the parasite. Compound 4 also inhibits mammalian CYP isoforms but is trypanocidal at concentrations below those required to significantly inhibit mammalian CYPs in vitro. A chemical-proteomics approach employing an activity-based probe derived from 1 was used to identify mammalian cathepsin B as a potentially important off-target of 1 and 4. Computational docking studies and the evaluation of truncated analogues of 4 reveal structural determinants for TcCYP51 binding, information that will be useful in further optimization of this new class of inhibitors.

Background: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α-demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI-resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real-time polymerase chain reaction (PCR)., Results: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC50 results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post-inoculation with the two other isolates., Conclusion: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch., (Copyright © 2011 Society of Chemical Industry.)