SummaryThe CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.Keywords sterol 14α-demethylase (CYP51); sterol biosynthesis; substrate preferences; catalytic activity; inhibition Family overviewSterol 14α-demethylation as a general part of sterol biosynthetic pathways in eukaryotes [1] has been known and studied for more than 30 years [2][3][4][5][6][7]. The enzyme catalyzing this reaction was first purified from yeast in 1984 (Sacharomyces cerevisiea [8]), and following determination of its primary structure [9] the cytochrome P450 sterol 14α-demethylases were placed into the CYP51 family, a number reserved for fungal sequences [10]. In 1986 the orthologous mammalian P450 was purified from rat liver microsomes [11], in 1996 the first sterol 14α-demethylase was found in plants (Sorghum bicolor [12]), and in 2000 the orthologous nature of a CYP51-like gene [13] from Mycobacterium tuberculosis to eukaryotic CYP51s was confirmed [14].Currently the CYP51 family joins proteins found in 82 organisms from all biological kingdoms. In addition, several plants and fungi contain multiple CYP51 genes (e.g. rice (10), black oats (2), tobacco (2), Arabidopsis thaliana (2), Fuzarium graminearum (3) or Aspergillus species: A. fumigatus (2), A.nidulans (2), A. orizae (3)). As a result, the number of known CYP51 sequences exceeds 100. The reasons for the existence of homologous CYP51 genes in the same species or their precise functions remain unknown, though it was reported that only one of the two CYP51 genes from A. thaliana (CYP51A2) is functional, while the other is an expressed pseudogene [15]. Mammalian genomes contain only one CYP51 gene yet sometimes † Corresponding author: Michael R. Waterman, Tel.: 615-343-1373; Fax: 615-322-4349; E-mail: michael.waterman@vanderbilt.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The average amino acid sequence identity in the CYP51 family is about 30%, varying from relatively high between the proteins from evolutionary closely related species (e.g. 95% in mammals) to lower values within the highly diverse kingdoms of lower...

With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14α-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: ()-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against and , pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.

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Sterol 14alpha-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 that 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 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.

Sterol 14␣-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.