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Validation of Human Sterol 14α-Demethylase (CYP51) Druggability: Structure-Guided Design, Synthesis, and Evaluation of Stoichiometric, Functionally Irreversible Inhibitors.
Friggeri L, Hargrove TY, Wawrzak Z, Guengerich FP, Lepesheva GI
(2019) J Med Chem 62: 10391-10401
MeSH Terms: 14-alpha Demethylase Inhibitors, Animals, Catalytic Domain, Crystallography, X-Ray, Drug Design, Humans, Molecular Structure, Mutagenesis, Site-Directed, Protozoan Proteins, Sterol 14-Demethylase
Show Abstract · Added March 3, 2020
Sterol 14α-demethylases (CYP51) are the cytochrome P450 enzymes required for biosynthesis of sterols in eukaryotes, the major targets for antifungal agents and prospective targets for treatment of protozoan infections. Human CYP51 could be and, for a while, was considered as a potential target for cholesterol-lowering drugs (the role that is now played by statins, which are also in clinical trials for cancer) but revealed high intrinsic resistance to inhibition. While microbial CYP51 enzymes are often inhibited stoichiometrically and functionally irreversibly, no strong inhibitors have been identified for human CYP51. In this study, we used comparative structure/functional analysis of CYP51 orthologs from different biological kingdoms and employed site-directed mutagenesis to elucidate the molecular basis for the resistance of the human enzyme to inhibition and also designed, synthesized, and characterized new compounds. Two of them inhibit human CYP51 functionally irreversibly with their potency approaching the potencies of azole drugs currently used to inhibit microbial CYP51.
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10 MeSH Terms
Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design.
Hargrove TY, Friggeri L, Wawrzak Z, Sivakumaran S, Yazlovitskaya EM, Hiebert SW, Guengerich FP, Waterman MR, Lepesheva GI
(2016) J Lipid Res 57: 1552-63
MeSH Terms: 14-alpha Demethylase Inhibitors, Antifungal Agents, Antineoplastic Agents, Antiprotozoal Agents, Catalytic Domain, Cell Line, Tumor, Cholestadienols, Crystallography, X-Ray, Drug Design, Drug Screening Assays, Antitumor, Humans, Hydrogen Bonding, Lanosterol, Models, Molecular, Protein Binding, Protein Conformation, alpha-Helical, Sterol 14-Demethylase
Show Abstract · Added April 6, 2017
Rapidly multiplying cancer cells synthesize greater amounts of cholesterol to build their membranes. Cholesterol-lowering drugs (statins) are currently in clinical trials for anticancer chemotherapy. However, given at higher doses, statins cause serious side effects by inhibiting the formation of other biologically important molecules derived from mevalonate. Sterol 14α-demethylase (CYP51), which acts 10 steps downstream, is potentially a more specific drug target because this portion of the pathway is fully committed to cholesterol production. However, screening a variety of commercial and experimental inhibitors of microbial CYP51 orthologs revealed that most of them (including all clinical antifungals) weakly inhibit human CYP51 activity, even if they display high apparent spectral binding affinity. Only one relatively potent compound, (R)-N-(1-(3,4'-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VFV), was identified. VFV has been further tested in cellular experiments and found to decrease proliferation of different cancer cell types. The crystal structures of human CYP51-VFV complexes (2.0 and 2.5 Å) both display a 2:1 inhibitor/enzyme stoichiometry, provide molecular insights regarding a broader substrate profile, faster catalysis, and weaker susceptibility of human CYP51 to inhibition, and outline directions for the development of more potent inhibitors.
Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.
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Structural basis for rational design of inhibitors targeting Trypanosoma cruzi sterol 14α-demethylase: two regions of the enzyme molecule potentiate its inhibition.
Friggeri L, Hargrove TY, Rachakonda G, Williams AD, Wawrzak Z, Di Santo R, De Vita D, Waterman MR, Tortorella S, Villalta F, Lepesheva GI
(2014) J Med Chem 57: 6704-17
MeSH Terms: 14-alpha Demethylase Inhibitors, Carbamates, Crystallography, X-Ray, Drug Design, Imidazoles, Models, Molecular, Protein Binding, Protein Conformation, Stereoisomerism, Sterol 14-Demethylase, Trypanocidal Agents, Trypanosoma cruzi
Show Abstract · Added February 12, 2015
Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50=1.2 nM, vs (S)-2/(S)-3, EC50=1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development of more effective antiparasitic drugs.
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12 MeSH Terms
Complexes of Trypanosoma cruzi sterol 14α-demethylase (CYP51) with two pyridine-based drug candidates for Chagas disease: structural basis for pathogen selectivity.
Hargrove TY, Wawrzak Z, Alexander PW, Chaplin JH, Keenan M, Charman SA, Perez CJ, Waterman MR, Chatelain E, Lepesheva GI
(2013) J Biol Chem 288: 31602-15
MeSH Terms: 14-alpha Demethylase Inhibitors, Antiprotozoal Agents, Chagas Disease, Crystallography, X-Ray, Humans, Protozoan Proteins, Sterol 14-Demethylase, Thiazoles, Triazoles, Trypanosoma cruzi
Show Abstract · Added March 7, 2014
Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.
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In vitro and in vivo studies of the antiparasitic activity of sterol 14α-demethylase (CYP51) inhibitor VNI against drug-resistant strains of Trypanosoma cruzi.
Soeiro Mde N, de Souza EM, da Silva CF, Batista Dda G, Batista MM, Pavão BP, Araújo JS, Aiub CA, da Silva PB, Lionel J, Britto C, Kim K, Sulikowski G, Hargrove TY, Waterman MR, Lepesheva GI
(2013) Antimicrob Agents Chemother 57: 4151-63
MeSH Terms: 14-alpha Demethylase Inhibitors, Animals, Chagas Disease, Drug Resistance, Endoplasmic Reticulum, Golgi Apparatus, Imidazoles, Male, Mice, Microscopy, Electron, Transmission, Nitroimidazoles, Oxadiazoles, Protozoan Proteins, Sterol 14-Demethylase, Thiazoles, Triazoles, Trypanocidal Agents, Trypanosoma cruzi
Show Abstract · Added March 7, 2014
Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.
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18 MeSH Terms
Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Delta7-24, 25-dihydrolanosterol.
Hargrove TY, Wawrzak Z, Liu J, Waterman MR, Nes WD, Lepesheva GI
(2012) J Lipid Res 53: 311-20
MeSH Terms: 14-alpha Demethylase Inhibitors, Binding Sites, Crystallography, X-Ray, Dose-Response Relationship, Drug, Enzyme Activation, Lanosterol, Models, Molecular, Mutation, Protein Conformation, Sterol 14-Demethylase, Trypanocidal Agents, Trypanosoma brucei brucei
Show Abstract · Added February 12, 2015
Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.
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Targeting Trypanosoma cruzi sterol 14α-demethylase (CYP51).
Lepesheva GI, Villalta F, Waterman MR
(2011) Adv Parasitol 75: 65-87
MeSH Terms: 14-alpha Demethylase Inhibitors, Antifungal Agents, Azoles, Catalytic Domain, Chagas Disease, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, Drug Discovery, Humans, Protein Conformation, Substrate Specificity, Triazoles, Trypanocidal Agents, Trypanosoma cruzi
Show Abstract · Added February 12, 2015
There are at least two obvious features that must be considered upon targeting specific metabolic pathways/enzymes for drug development: the pathway must be essential and the enzyme must allow the design of pharmacologically useful inhibitors. Here, we describe Trypanosoma cruzi sterol 14α-demethylase as a promising target for anti-Chagasic chemotherapy. The use of anti-fungal azoles, which block sterol biosynthesis and therefore membrane formation in fungi, against the protozoan parasite has turned out to be highly successful: a broad spectrum anti-fungal drug, the triazole compound posaconazole, is now entering phase II clinical trials for treatment of Chagas disease. This review summarizes comparative information on anti-fungal azoles and novel inhibitory scaffolds selective for Trypanosomatidae sterol 14α-demethylase through the lens of recent structure/functional characterization of the target enzyme. We believe our studies open wide opportunities for rational design of novel, pathogen-specific and therefore more potent and efficient anti-trypanosomal drugs.
Copyright © 2011 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum.
Hargrove TY, Wawrzak Z, Liu J, Nes WD, Waterman MR, Lepesheva GI
(2011) J Biol Chem 286: 26838-48
MeSH Terms: 14-alpha Demethylase Inhibitors, Binding Sites, Catalysis, Leishmania infantum, Leishmaniasis, Visceral, Protein Binding, Protozoan Proteins, Species Specificity, Sterol 14-Demethylase, Substrate Specificity, Trypanosoma brucei brucei, Trypanosoma cruzi
Show Abstract · Added February 12, 2015
Leishmaniasis is a major health problem that affects populations of ∼90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V(max) of ∼10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.
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12 MeSH Terms
Comparison of the inhibitory profiles of itraconazole and cimetidine in cytochrome P450 3A4 genetic variants.
Akiyoshi T, Saito T, Murase S, Miyazaki M, Murayama N, Yamazaki H, Guengerich FP, Nakamura K, Yamamoto K, Ohtani H
(2011) Drug Metab Dispos 39: 724-8
MeSH Terms: 14-alpha Demethylase Inhibitors, Androgens, Cimetidine, Cytochrome P-450 CYP3A, Cytochrome P-450 CYP3A Inhibitors, Drug Interactions, Enzyme Inhibitors, Genetic Variation, Heme, Humans, Hydroxytestosterones, Itraconazole, Recombinant Proteins, Testosterone
Show Abstract · Added March 26, 2014
CYP3A4, an important drug-metabolizing enzyme, is known to have genetic variants. We have previously reported that CYP3A4 variants such as CYP3A4.2, 7, 16, and 18 show different enzymatic kinetics from CYP3A4.1 (wild type). In this study, we quantitatively investigated the inhibition kinetics of two typical inhibitors, itraconazole (ITCZ) and cimetidine (CMD), on CYP3A4 variants and evaluated whether the genetic variation leads to interindividual differences in the extent of CYP3A4-mediated drug interactions. The inhibitory profiles of ITCZ and CMD on the metabolism of testosterone (TST) were analyzed by using recombinant CYP3A4 variants. The genetic variation of CYP3A4 significantly affected the inhibition profiles of the two inhibitors. In CYP3A4.7, the K(i) value for ITCZ was 2.4-fold higher than that for the wild-type enzyme, whereas the K(i) value for CMD was 0.64-fold lower. In CYP3A4.16, the K(i) value for ITCZ was 0.54-fold lower than that for wild-type CYP3A4, whereas the K(i) value for CMD was 3.2-fold higher. The influence of other genetic variations also differed between the two inhibitors. Docking simulations could explain the changes in the K(i) values, based on the accessibility of TST and inhibitors to the heme moiety of the CYP3A4 molecule. In conclusion, the inhibitory effects of an inhibitor differ among CYP3A4 variants, suggesting that the genetic variation of CYP3A4 may contribute, at least in part, to interindividual differences in drug interactions mediated by CYP3A4 inhibition, and the pattern of the influences of genetic variation differs among inhibitors as well as substrates.
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14 MeSH Terms