Human sterol 14α-demethylase as a target for anticancer chemotherapy: towards structure-aided drug design
2016; Elsevier BV; Volume: 57; Issue: 8 Linguagem: Inglês
10.1194/jlr.m069229
ISSN1539-7262
AutoresTatiana Y. Hargrove, Laura Friggeri, Z. Wawrzak, Suneethi Sivakumaran, Eugenia M. Yazlovitskaya, Scott W. Hiebert, F. Peter Guengerich, Michael R. Waterman, Galina I. Lepesheva,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoRapidly 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. 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. Sterol biosynthesis is an essential metabolic pathway in most eukaryotes and in some bacteria (1Lepesheva G.I. 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Furthermore, certain types of cancer exhibit CYP51 gene amplification (http://www.cbioportal.org). It is likely that the earlier attempts to develop human CYP51 inhibitors were unsuccessful because they concentrated on substrate analogs, which had rather low inhibitory potency (10Frye L.L. Leonard D.A. Lanosterol analogs: dual-action inhibitors of cholesterol biosynthesis.Crit. Rev. Biochem. Mol. Biol. 1999; 34: 123-140Crossref PubMed Scopus (27) Google Scholar) and could not compete in efficiency with statins. Here, we performed a biochemical characterization of human CYP51, including substrate preferences, catalytic parameters, inhibition, and the structure of a complex with (R)-N-(1-(3,4′-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-l)benzamide (VFV), the most potent inhibitor that we have identified. VFV was further tested in cancer cells and found to be active against different types of cancers. VNI, VNT, VNF, and VFV were synthesized by the Chemical Synthesis Core Facility (Vanderbilt Institute of Chemical Biology) (48Hargrove T.Y. Kim K. de Nazaré Correia Soeiro M. da Silva C.F. da Gama Jaen Batista D. Batista M.M. Yazlovitskaya E.M. Waterman M.R. Sulikowski G.A. Lepesheva G.I. CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds.Int. J. Parasitol. Drugs Drug Resist. 2012; 2: 178-186Crossref PubMed Scopus (43) Google Scholar). VFV-Cl was synthesized using the same procedure. Voriconazole, ketoconazole, itraconazole, and posaconazole were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); fluconazole was from ICN Biomedicals. Hydroxypropyl-β-cyclodextrin (HPCD) was purchased from Cyclodextrin Technology Development (Gainesville, FL). DEAE- and CM-Sepharose were from GE Healthcare, and Ni2+-nitrilotriacetate (NTA) agarose was purchased from Qiagen. All cell lines were purchased from American Type Culture Collection (Manassas, VA). Trypanosoma cruzi, Aspergillus fumigatus, and full-length human CYP51 and T. brucei and rat NADPH-cytochrome P450 reductase were expressed in Escherichia coli and purified as described previously (49Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. CYP51 from Trypanosoma brucei is obtusifoliol-specific.Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (66) Google Scholar, 50Hargrove T.Y. Wawrzak Z. Lamb D.C. Guengerich F.P. Lepesheva G.I. Structure-functional characterization of cytochrome P450 sterol 14α-demethylase (CYP51B) from Aspergillus fumigatus and molecular basis for the development of antifungal drugs.J. Biol. Chem. 2015; 290: 23916-23934Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The full-length proteins were used for functional studies (ligand binding, enzymatic activity, and inhibition). For crystallization, human CYP51 [UniProt protein accession number Q16850, 503 amino acid residues plus the (His)4-tag at the C terminus, 57,300 Da] was truncated (458 amino acid residues, 53,400 Da) as described previously (51Lepesheva G.I. Park H.W. Hargrove T.Y. Vanhollebeke B. Wawrzak Z. Harp J.M. Sundaramoorthy M. Nes W.D. Pays E. Chaudhuri M. et al.Crystal structures of Trypanosoma brucei sterol 14 alpha-demethylase and implications for selective treatment of human infections.J. Biol. Chem. 2010; 285: 1773-1780Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Briefly, the 50-amino acid membrane anchor sequence at the N terminus (up to the conserved CYP51 proline, Pro61 in human CYP51) was replaced with the MAKKTSSKGKL-fragment (52von Wachenfeldt C. Richardson T.H. Cosme J. Johnson E.F. Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modifications of its N-terminus.Arch. Biochem. Biophys. 1997; 339: 107-114Crossref PubMed Scopus (106) Google Scholar) and subcloned into the pCW expression vector using the NdeI (5′) and HindIII (3′) sites. The truncated human CYP51 was purified in two steps, including affinity chromatography on Ni2+-NTA agarose and cation exchange chromatography on CM-Sepharose. The Ni2+-NTA bound protein was washed with 10 bed volumes of 50 mM potassium phosphate buffer (pH 7.2) containing 100 mM NaCl, 10% glycerol (v/v), 1 mM imidazole, and 0.1% Triton X-100 (v/v) and then with 20 mM potassium phosphate buffer (pH 7.2) containing 500 mM NaCl, 10% glycerol (v/v), and 5 mM imidazole until the Triton X-100 was eliminated (as judged by A280 measurements). The P450 was eluted with a linear gradient of imidazole (10–80 mM), and the fractions with a spectrophotometric index (A425/A280) ≥1 were pooled and concentrated using an Amicon Ultra 50 K (Millipore) concentration device to a volume of 1–2 ml. The concentrated protein was then diluted 10-fold with 20 mM potassium phosphate buffer (pH 7.2) containing 10% glycerol (v/v) and 0.1 mM EDTA (CM-buffer) and applied to a CM-Sepharose column (5 ml bed volume) equilibrated with CM-buffer containing 50 mM NaCl. The column was washed with five bed volumes of equilibration buffer and then 40 bed volumes of CM-buffer with an increasing linear gradient of NaCl (50–200 mM). The protein was eluted with CM-buffer containing 300 mM NaCl, the fractions containing the enzyme were pooled, and VFV was added from a 10 mM DMSO stock solution (molar ratio VFV/CYP51 = 2.5:1). Then the complex (complex 1) was concentrated to about 500 μM P450 using an Amicon Ultra 50 K concentration device, aliquoted, frozen in liquid nitrogen, and stored at −80°C until use. The yield was between 300 and 400 nmol/liter culture. The purity was verified by SDS-PAGE. Alternatively, when human CYP51 was copurified with the inhibitor (complex 2), all of the buffer solutions used for CM-chromatography contained 10 μM VFV. The sample was concentrated to about 500 μM P450 and used for crystallization immediately. The crystals were obtained by the hanging drop vapor diffusion technique. Crystals of complex 1 were grown at 20°C. Equal volumes of the P450-VFV complex solution preincubated with 0.18 mM Thesit (Hampton Research) and 5.5 mM tris(carboxyethyl)phosphine were mixed with mother liquor [0.2 M trisodium citrate dehydrate, 0.10 mM HEPES (pH 7.5), and 12% PEG 8,000 (w/v)] and equilibrated against the reservoir solution. Crystals of complex 2 were grown at 18°C by mixing equal volumes of the complex solution preincubated with 5.8 mM CYMAL-5 (Anagrade) and 5.5 mM tris(carboxyethyl)phosphine with 0.20 M potassium fluoride (pH 7.2) and 15% PEG 6,000 (w/v). In both cases crystals appeared after several days and were cryoprotected by soaking them in mother liquor with 30% glycerol (v/v) and flash-cooled in liquid nitrogen. UV-visible absorption spectra were recorded using a dual-beam Shimadzu UV-2401PC spectrophotometer. P450 concentrations were determined from the Soret band intensity using ε417 117 mM−1 cm−1 for the low-spin ferric form of the protein or Δε450-490 91 mM−1 cm−1 for the reduced carbon monoxide difference spectra; the spin state of the P450 samples was estimated from the absolute absorbance spectra (53Hargrove T.Y. Wawrzak Z. Liu J. Nes W.D. Waterman M.R. Lepesheva G.I. Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14α-demethylase (CYP51) from Leishmania infantum.J. Biol. Chem. 2011; 286: 26838-26848Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Substrate binding was monitored as a "type I" spectral response (blue shift in the Soret band maximum from 417 to 393 nm) (54Schenkman J.B. Remmer H. Estabrook R.W. Spectral studies of drug interaction with hepatic microsomal cytochrome.Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar) reflecting low-to-high spin transition of the ferric P450 heme iron as a result of displacement of the heme-coordinated water molecule (50Hargrove T.Y. Wawrzak Z. Lamb D.C. Guengerich F.P. Lepesheva G.I. Structure-functional characterization of cytochrome P450 sterol 14α-demethylase (CYP51B) from Aspergillus fumigatus and molecular basis for the development of antifungal drugs.J. Biol. Chem. 2015; 290: 23916-23934Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Various aliquots of sterols (dissolved in 45% HPCD, w/v) (49Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. CYP51 from Trypanosoma brucei is obtusifoliol-specific.Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (66) Google Scholar) were added to the sample cuvette (1 cm optical path length), and the same volume of HPCD was added to the reference cuvette. The apparent dissociation constants of the enzyme-substrate complex (Kd) were calculated in Prism 6 software (GraphPad, La Jolla, CA) by fitting the data for the substrate-induced absorbance changes in the difference spectra Δ(A390-A420) versus substrate concentration to a one site-total binding equation (binding-saturation). Inhibitor binding was monitored as a "type II" spectral response (red shift in the Soret band maximum from 417 to 421–427 nm) (54Schenkman J.B. Remmer H. Estabrook R.W. Spectral studies of drug interaction with hepatic microsomal cytochrome.Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar) reflecting coordination of the basic heterocyclic nitrogen to the P450 heme iron (55Hargrove T.Y. Wawrzak Z. Alexander P.W. Chaplin J.H. Keenan M. Charman S.A. Waterman M.R. Chatelain E. Lepesheva G.I. Complexes of Trypanosoma cruzi sterol 14α-demethylase (CYP51) with two pyridine-based drug candidates for Chagas disease: structural basis for pathogen selectivity.J. Biol. Chem. 2013; 288: 31602-31615Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Difference spectra were generated by recording the P450 absorbance in a sample cuvette (5 cm optical path length) versus the absorbance in a reference cuvette, both containing the same amount of the protein. Aliquots of azoles (dissolved in DMSO) were added to the sample cuvette in the concentration range of 0.1–20 μM (5–200 μM for fluconazole). At each step, the corresponding volume of DMSO was added to the reference cuvette. The apparent dissociation constants of the enzyme-ligand complex (Kd) were calculated in GraphPad Prism 6 software by fitting the data for the ligand-induced absorbance changes in the difference spectra Δ(Amax-Amin) versus ligand concentration to the quadratic equation 1 (for tight-binding ligands), ΔA=(ΔAmax/2E)((L+E+Kd)−((L+E+Kd)2−4LE)0.5)(Eq. 1) where (L) and (E) are the total concentrations of ligand and enzyme used for the titration, respectively. The standard reaction mixture (49Lepesheva G.I. Nes W.D. Zhou W. Hill G.C. Waterman M.R. CYP51 from Trypanosoma brucei is obtusifoliol-specific.Biochemistry. 2004; 43: 10789-10799Crossref PubMed Scopus (66) Google Scholar) contained 0.5 μM P450 and 1.0 μM cytochr
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