Use of Phenoxyaniline Analogues To Generate Biochemical Insights

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Use of phenoxyaniline analogs to generate biochemical insights into polybrominated diphenyl ether interaction with CYP2B enzymes Chao Chen, Jingbao Liu, James R. Halpert, and P. Ross Wilderman Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01024 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 17, 2017

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Biochemistry

Use of phenoxyaniline analogs to generate biochemical insights into polybrominated diphenyl ether interaction with CYP2B enzymes

Chao Chen, † Jingbao Liu,† James R. Halpert,† P. Ross Wilderman†,* †

University of Connecticut School of Pharmacy, Storrs, Connecticut 06269, United States

*To whom correspondence should be addressed. Email: [email protected].

KEYWORDS Cytochrome P450 2B enzymes; halogenated diphenyl ethers; CYP-ligand interaction; cytochrome P450 oxidoreductase; cytochrome b5; human CYP2B6; conformational change; spectral titrations; enzyme inhibition assays; NADPH consumption ABBREVIATIONS CYP – cytochrome P450; POR – cytochrome P450 oxidoreductase; Cyt b5 – cytochrome b5; PCB – polychlorinated biphenyl; PBDE – polybrominated diphenyl ether; 4-CPI, 4-(4chlorophenyl)imidazole;

CuOOH

–

cumene

hydroperoxide;

7-EFC

–

7-ethoxy-4-

(trifluromethyl)coumarin; 7-HFC – 7-hydroxy-4-(trifluromethyl)coumarin; RNase – ribonuclease A; DNase – deoxyribonuclease I; IPTG – isopropyl β-D-1-thiogalactopyranoside; CHAPS – [(3-Cholamidopropyl)dimethylammonia]-1-propanesulfonate; Ni2+-NTA – nickel-nitrilotriacetic acid; TB – Terrific Broth; BME – 2-mercaptoethanol; PMSF – phenylmethanesulfonyl fluoride;

1 ACS Paragon Plus Environment

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EDTA – ethylenediaminetetraacetic acid; DTT – dithiothreitol CCD – charged-coupled device; SVD – singular value decomposition; E. coli – Escherichia coli;


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Biochemistry

ABSTRACT Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogs of PBDEs. For most congeners, there was less than 3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the most sensitive enzyme to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6.

This substitution partially altered the protein-ligand

interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2’4’5’TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 (cyt b5) to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2’4’5’TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2’4’5’TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of diphenyl ether binding to human CYP2B6 and changes in CYP2B6 mediated metabolism dependent on POA congener and redox partner identity.


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INTRODUCTION Polybrominated diphenyl ethers (PBDEs) have long presented major environmental challenges due to their inherent persistence and toxicity. The primary route for human exposure to such organohalogen chemicals is from ingestion of contaminated dust in an indoor environment because of the prior common use of PBDEs as flame retardants in various household products.(1) Another route of exposure is through consumption of food such as fish, as PBDEs naturally occur in all marine living resources.(2) Recent studies on PBDE metabolism by human liver microsomes and purified cytochrome P450 (CYP) dependent monooxygenases demonstrated that the main catalyst of oxidation of the major PBDE congeners is CYP2B6.(3-5) Other CYP2B6 substrates include pesticides, polychlorinated biphenyls (PCBs) and exogenous toxins.(3, 4, 612) Additionally, highly polymorphic CYP2B6 also participates in the metabolism of approximately 10 percent all drugs including artemisinin, bupropion, efavirenz and ketamine.(13) CYP2B enzymes, including human CYP2B6, were the among first mammalian CYP enzymes isolated and characterized in detail,(14) and they demonstrate remarkable species-dependent substrate stereo- and regiospecific oxidation of common substrates.(15-17) Recent advances in the understanding of how CYP2B enzymes selectively catalyze and/or interact with a broad range of structurally diverse chemicals were facilitated by a combination of biophysical, biochemical, and structural techniques. Deuterium exchange mass spectrometry (DXMS) provided solution structural information, NMR combined with molecular dynamics simulations revealed ligand binding orientations, and isothermal titration calorimetry (ITC) yielded thermodynamic signatures of ligand interactions with CYP2B enzymes.(18-21)


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Biochemistry

In addition to solution studies, X-ray crystallographic studies have demonstrated that CYP2B enzymes undergo substantial structural rearrangement upon ligand binding, while the overall CYP enzyme scaffold remains.(10, 22-25) Intriguingly, mounting structural evidence suggests that novel CYP-ligand interactions such as halogen-π bonding may play an important role in the selective metabolism and/or binding of a diverse array of chemicals including brominated monoterpenes to CYP2B6,(25, 26) and 4-(4-chlorophenyl)imidazole (4-CPI) to CYP2B37.(27) Structural characterization of CYP3A4, the major human metabolizing CYP enzyme, illustrated formation of halogen-π bonding in its complexes with bromoergocriptine(28) and ketoconazole(29).

Additionally,

non-CYP

metabolizing

enzymes

such

as

estrogen

sulfotransferase (SULT1E1) also exhibit halogen bonds in complexes with hydroxylated metabolites of PCB80 or BDE47.(30, 31) Consistent with this structural information, halogenated compounds are in fact widely preferred in drug development(32, 33) and halogenation often confers higher specificity for the proteins of interest with improvement of several orders of magnitude. Despite the growing body of evidence for halogen bonding interactions in protein-ligand complexes, how the degree and position of halogenation of hydrophobic substances affects their interactions with mammalian CYP enzymes remains to be elucidated.

Thus, we synthesized a

series of halogenated phenoxyanilines (POAs) as analogs of PBDEs to probe the structureactivity relationships of CYP2B enzymes (Figure 1, Table 1). These model ligands circumvent the challenges that the parent PBDEs would present including the highly hydrophobic nature of the compounds and the strong likelihood that the compounds bind in multiple orientations, as evidenced by their complex metabolism profiles.(4, 5) The nitrogenous congeners may also anchor themselves favorably in the active site via iron-nitrogen interaction in a limited number of


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orientations, while the freely rotating bonds still allow for an influence of halogen bonds. Using these congeners, CYP2B interactions with halogenated ligands were interrogated by spectral titrations and enzyme inhibition assays, and the role of redox partner proteins was investigated by addition or removal of these enzymes from the respective assays. Halogenation of phenoxyaniline affected ligand interaction with CYP2B enzymes for most POA congeners but not 3CPOA or POA itself. Our results demonstrate that redox partner proteins such as cytochrome b5 (cyt b5) and NADPH-cytochrome P450 reductase (POR) exerted profound roles in CYP2B-ligand interactions.

Figure 1. Chemical structures of POA congeners

Table 1. List of POA analogues and their properties Substitution at Identified Carbona POA analogs

IUPAC nomenclature 3

2’

4’

5’

POA

–H

–H

–H

–H

4-(phenoxy)aniline

3CPOA

–Cl

–H

–H

–H

3-chloro-4-(phenoxy)aniline

2’4’DCPOA

–H

–Cl

–Cl

–H

4-(2’,4’-dichlorophenoxy)aniline

2’4’DBPOA

–H

–Br

–Br

–H

4-(2’,4’-dibromophenoxy)aniline

2’4’DCBA

–H

–Cl

–Cl

–H

4-[(2’,4’-dichlorophenyl)methyl]aniline

2’4’5’TCPOA

–H

–Cl

–Cl

–Cl

4-(2’,4’,5’-trichlorophenoxy)aniline

3-2’4’TCPOA

–Cl

–Cl

–Cl

–H

3-chloro-4-(2’,4’-dichlorophenoxy)aniline


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Biochemistry

a

Carbon numbering from Figure 1.

MATERIALS AND METHODS Materials. 7-Ethoxy-4-(trifluromethyl)coumarin (7-EFC) was obtained from Invitrogen (Carsbad, CA). 7-Hydroxy-4-(trifluromethyl)coumarin (7-HFC) was purchased from Alfa Aesar. Cumene

hydroperoxide

(CuOOH),

4-(4-chlorophenyl)imidazole

(4-CPI),

β-NADPH,

ribonuclease A (RNase), deoxyribonuclease I (DNase), and high performance liquid chromatography plus-grade acetone were purchased from Sigma-Aldrich (St. Louis, MO). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was received from Anatrace (Maumee, OH). 3[(3-Cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) was obtained from EMD Millipore (San Diego, CA). Nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin was from Thermo Scientific (Rockford, IL). Macro-Prep CM cation exchange resin was received from Bio-Rad Laboratories (Hercules, CA). Amicon ultrafiltration devices were purchased from EMD Millipore (Billerica, MA). The pGro7 plasmid harboring genes for the GroEL and GroES chaperones was obtained from Takara Bio (Shiba, Japan). The QuikChange XL site-directed mutagenesis kit and Escherichia coli TOPP3 and JM109 cells were obtained Agilent (Santa Clara, CA). Oligonucleotide primers for mutagenesis and sequencing were synthesized by Integrated DNA Technologies (Coralville, IA). 4-(2’,4’-Dichlorophenoxy)aniline (CAS #: 14861-17-7) was purchased from BOC Sciences (Shirley, NY). Recombinant rat liver POR and rat cyt b5 were prepared as described previously.(34, 35) Unless described in the section regarding chemical synthesis, all other chemicals and supplies were purchased from standard sources and are of the highest quality available.


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Chemical synthesis. All halogenated POAs were synthesized in a similar manner to published methods,(36, 37) and the purity of the compounds exceeds 95% by 1H NMR. The experimental details are summarized in the Supporting Information. Protein mutagenesis. CYP2B mutants were generated using the QuikChange XL kit according to the instructions provided by the manufacturer. To create CYP2B4 I363V and CYP2B6 L363V, the pKK2B4dH (H226Y)(38) and pKK2B6dH (Y226H/K262R)(22) plasmids, respectively, were employed as templates that contain N-terminal truncation and modification and a C-terminal tetrahistidine tag. Primers were listed in Table S1. The mutant constructs were verified by DNA sequencing at the Institute for Systems Genomics at the University of Connecticut. Plasmids were then transformed into E. coli JM109 or TOPP3 competent cells and expressed as described below. Protein expression and purification. CYP2B1 was expressed in E. coli TOPP3 cells.(38) All other CYP2B wild-type and mutant proteins were expressed in E. coli JM109 cells that contained the pGro7 plasmid expressing the GroEL and GroES chaperones.(39) Protein expression and purification followed the previously described protocol with minor modification(22, 38). Briefly, pre-cultures were prepared by inoculating CYP2B glycerol stocks stored at –80 °C into Luria broth (LB) supplemented with 100 μg/mL ampicillin and 25 µg/mL tetracycline (CYP2B1) or 25 µg/mL chloramphenicol (other CYP2B enzymes), and were grown overnight at 37 °C with shaking at 220 rpm. These cultures were then inoculated into fresh Terrific Broth (TB) supplemented with the same antibiotics and L-arabinose (20 mg/mL) and were grown under the conditions described above. When the OD600 reached ~0.7, IPTG and δ-aminolevulinic acid were added to a final concentration of 1 and 0.5 mM, respectively. The cells were grown for additional 65-72 hours at 30 °C and 190 rpm, and were subsequently harvested by centrifugation for 10 min


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Biochemistry

at 7500g using a TA-10-250 rotor in a Beckman Coulter Allegra 25R centrifuge. The cell pellets were collected and stored at −80 °C. All purification steps were performed in potassium phosphate pH 7.4 at 4 °C. The cell pellet was resuspended in 10% the culture volume of buffer containing 20 mM potassium phosphate, 20% (v/v) glycerol, 10 mM 2-mercaptoethanol (BME), and 0.5 mM phenylmethanesulfonyl fluoride (PMSF), and treated with lysozyme (0.3 mg/mL) with stirring for 30 min, followed by centrifugation for 30 min at 7500g. After the supernatant was decanted, spheroplasts were resuspended in 5% of the original culture volume in the buffer described above containing 500 mM potassium phosphate as well as RNase A and DNase I at 10 µg/mL and were sonicated for 4 x 1 min on ice. CHAPS was added to the sample at a final concentration of 0.8% (w/v), and the solution was allowed to stir for 90 min at 4 °C prior to ultracentrifugation for 1 hour at 245,000g using a Ti 50.2 rotor in a Beckman L7 Ultracentrifuge. The supernatant was collected, and the CYP enzyme concentration was measured using the reduced CO difference spectrum and an absorption coefficient ∆450-490nm of 91 mM−1 cm−1.(40, 41) Following protein quantitation, the collected supernatant was applied to an equilibrated Ni2+NTA column, and the column was washed with buffer containing 100 mM potassium phosphate, 100 mM NaCl, 20% (v/v) glycerol, 10 mM BME, 0.5 mM PMSF, 0.5% CHAPS, and 5 mM histidine. The protein was eluted using the same buffer containing 50 mM histidine. The fractions with the highest purity as indicated by the A417/A280 ratio were pooled, and the CYP enzyme concentration was measured using the reduced CO difference spectra. Pooled fractions were diluted 5 times to reduce ionic strength and loaded onto an equilibrated Macro-Prep CM cation exchange column. The column was washed using 8-10 volumes of buffer containing 5 mM potassium phosphate, 20 mM NaCl, 20% (v/v) glycerol, 1 mM EDTA, and 0.2 mM DTT,


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and the protein was eluted with 500 mM NaCl in the same buffer. The total CYP enzyme concentration was determined from the reduced carbon monoxide difference spectrum. Spectral binding titrations. Spectral titrations were performed by adding ligand into a sample containing a single CYP enzyme or an enzyme complex consisting of CYP enzyme and POR. In a typical titration experiment, a POA congener in acetone was titrated into a reaction mixture of 1 mL sucrose buffer (50 mM potassium phosphate, pH 7.4, 500 mM sucrose, 500 mM NaCl, 1 mM EDTA and 0.2 mM DTT) or HEPES buffer (50 mM HEPES, pH 7.4 and 15 mM MgCl2) containing 1 µM CYP enzyme alone or 1 µM CYP enzyme with 4 µM POR, and was equilibrated at 25 °C for 1 min. Following temperature equilibration, an absorbance spectrum from 340 to 700 nm was acquired using an S2000 single-channel charged-coupled device (CCD) rapid scanning spectrometer (Ocean Optics, Inc., Dunedin, FL, USA) equipped with DH-mini light source (Ocean Optics, Inc.) and a temperature-controlled cell chamber with a magnetic stirrer. A semi-micro quartz cell with a stirring compartment (10 x 4 mm light path) from Hellma GmbH (Mulheim, Germany) was used for titration experiments. For single CYP enzyme titrations, spectra were blanked using the buffer, while for the enzyme complex system, the spectrum of the buffer containing POR was used as the blank. Acetone at concentrations up to 1% (v/v) was used as the ligand solvent, since it had negligible effects on CYP2B spectra (data not shown). All spectra were recorded using the custom data acquisition program SPECTRALAB,(42) and analysis was performed using Igor Pro version 6.37 (WaveMetrics, Inc., OR, USA). To interpret changes in the absorbance spectra in a titration experiment, singular value decomposition (SVD) was applied together with least-squares approximation of the spectra principle components with a linear combination of prototypical spectra standards, which included the spectra of ferric high-


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Biochemistry

spin, ferric low-spin, type-II CYP species obtained from full length CYP2B4.(43) Spectral binding constant (KS) values were generally determined by fitting the data to the quadratic velocity equation 2∆A = (∆Amax/[E0])((KD + [I0] + [E0]) – (KD + [I0] + [E0])2 – 4[E0][I0])1/2)(44) or the Michaelis-Menten equation ∆A = ∆Amax[I]/(KD + [I]) + offset where ∆Amax is the maximum change in the fraction of Type-II CYP enzyme, KD is the KS, E0 is total CYP enzyme concentration, and I0 is total inhibitor or ligand concentration. NADPH-dependent enzyme inhibition assay. NADPH-dependent 7-EFC O-deethylation assays were performed as previously described with slight modifications.(45) CYP2B proteins were reconstituted with POR and cyt b5 at a molar ratio of 1:4:2 or 1:4:0. Inhibition assays were performed at 37 °C in a reaction mixture of 100 µL HEPES buffer pH 7.4 containing a POA congener at varying concentrations, 7-EFC at a fixed concentration close to the MichaelisMenten constant (KM) and 2.5 - 5 picomol of CYP enzyme in the reconstituted system, and reactions were initiated with 1 mM NADPH after a 5 min preincubation. The reaction was quenched by addition of 50 µL ice-cold acetonitrile after 5 min. Fifty µL of the quenched reaction was then transferred to a vial containing 950 µL of 0.1 M Tris (pH 9.0). Quantitation of the product of 7-EFC O-deethylation, 7-HFC, was measured using a λex of 410 nm and a λem of 510 nm in a Cary Eclipse Fluorimeter (Agilent, Santa Clara, CA, USA). Commercial 7-HFC was prepared at concentrations from 0 to 50 μM in the reaction mixture, and a fresh standard curve was made each day when data were collected. To determine inhibition constants (KI) for selected POAs, the reaction was carried out at 37 °C in a mixture of 100 µL HEPES buffer consisting of 0 – 200 µM 7-EFC, 2.5 – 5 pmol of reconstituted CYP enzyme with POR and cyt b5 at a ratio of 1:4:2, and the POA analog at various concentrations. The quantity of 7-HFC produced was detected by fluorescence intensity as described above. Data analysis for inhibition assays was


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processed using Prism 5 (GraphPad Software, Inc., San Diego, CA). IC50 values were determined by using a four-parameter logistic function, and KI values were quantified by global fitting of the untransformed data to equations for various inhibition types. Cumene hydroperoxide (CuOOH)-driven enzyme inhibition assay. Peroxide-driven inhibition assays like NADPH-driven assays were carried out at 37 °C in a mixture of 100 µL HEPES buffer containing POA congener with varying concentrations, 7-EFC close to the KM, and 40 pmol CYP enzyme alone or with 80 pmol cyt b5. Kinetic parameters of peroxidemediated 7-EFC O-deethylation were determined by continuously measuring 7-HFC formation in 500 µL of the assay buffer described above containing 0-200 µM 7-EFC with stirring. The reactions were initiated by addition of 0.5 mM cumene hydroperoxide. Rates of 7-EFC Odeethylation were determined from the slopes of the initial phase of 7-HFC formation, and the KM and kcat values were calculated using the Michaelis-Menten equation. Inhibition experiments were performed in the same manner using varying concentrations of the selected POA and 2.5 – 5 pmol of CYP enzyme; in assays containing cyt b5, the ratio of CYP enzyme to cyt b5 was 1:2. IC50 values were derived by fitting data to a four-parameter logistic function using GraphPad Prism 5. NADPH oxidation assay. NAPDH depletion rates were assayed in a 300 µL reaction volume containing 20 - 200 µM ligand, a reconstituted enzyme mixture consisting of CYP, POR and cyt b5 with a molar ratio of 1:4:2 or 0:4:2, and 1% acetone. The reaction was preincubated at 37 °C for 5 min, and was subsequently initiated by adding 0.2 mM NADPH. The rate of NADPH oxidation was kinetically measured by monitoring the decrease in absorbance at 340 nm with an extinction coefficient of 6.22 mM-1cm-1 using SPECTRALAB, and data were analyzed using Igor Pro version 6.37.


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Biochemistry

Statistical analysis. Mean and standard deviation values were calculated Microsoft Excel. The significance of changes due to addition of inhibitor, changing buffer conditions, or the addition of cyt b5 or POR to the assay was quantified using an independent sample two-tailed t-test in Microsoft Excel; the null hypothesis for each comparison was the change in assay conditions did not lead to a difference in the analyzed quantity. Results generated using Microsoft Excel were validated using Igor Pro.

RESULTS Interaction of POA analogs with CYP2B enzymes. To understand how halogenation of lipophilic molecules influences their binding to CYP2B enzymes, rat CYP2B1, rabbit CYP2B4, and human CYP2B6 were titrated with the seven compounds described in Table 1. In all cases, a type-II spectral shift from 417 to 419 nm was observed with increasing concentrations of POA congener in the presence of the CYP enzyme, consistent with a weak direct iron–nitrogen interaction or an indirect iron–nitrogen interaction via an axial water molecule (Figure 2).(46) All tested compounds exhibited high affinity for CYP2B1 (KS = POA ≈ monochloro POA ≈ dichloro POA > 2’4’5’TCPOA; for CYP2B4, the relative affinity order was 2’4’DCBA > trichloro POA > POA > dihalo POA > monochloro POA, and the relative affinity order for CYP2B6 was 2’4’DCBA ≈ 2’4’DBPOA > 2’4’5’TCPOA > POA ≈ monochloro POA ≈ 2’4’DCPOA ≈ 3-2’4’TCPOA. In contrast, the inhibitory potency of POA 14 ACS Paragon Plus Environment

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Biochemistry

analogs in the activity-based inhibition assay generally increased with increased halogenation. There was also no correlation between the effect of halogen identity and substitution pattern on binding affinity in spectral titrations and inhibitory potency. Furthermore, while the magnitude of the type-II spectral shift was not linked with POA affinity for CYP2B enzymes, in general, binding of trichlorinated POAs produced less type-II complex than other congeners with CYP2B4 and CYP2B6. In contrast, only 3-2’4’TCPOA reduced the amount of type-II component in the spectra of CYP2B1.

Figure 3. KS and IC50 values for POAs with CYP2B wild-type enzymes and 363V mutants

Contributions of residue 363 to ligand interactions. The identity of residue 363 contributes to differences in substrate metabolism, coupling efficiency, and time-dependent inhibition among CYP2B enzymes.(17, 47-49) Since all tested POAs had high affinity for CYP2B1, which has a Val at residue 363, CYP2B4 I363V and CYP2B6 L363V were created. As shown in Figure 3 and Table S3, most tested POAs had lower affinity for the valine-containing mutant than for the


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wild-type enzyme. CYP2B4 I363V and CYP2B6 L363V exhibited large discrepancies between the KS and IC50 values for POA and 3CPOA, as seen in the respective wild-type enzyme. Increased halogen substitution increased inhibitory potency but decreased the formation of the type-II complex. The introduction of valine into CYP2B4 and CYP2B6 partially shifted their ligand selectivity toward that of CYP2B1. The congener with the highest affinity for the valine mutants was still 2’4’DCBA, but 2’,4’,5’TCPOA affinity and inhibition potency decreased significantly compared to the wild-type enzymes. Interactions of dihalogenated POAs with CYP2B6 L363V were similar to those seen with CYP2B1 with similar values for KS and IC50. For trichlorinated POAs, the relative interaction of 2’4’5’TCPOA and 3-2’4’TCPOA with CYP2B1, CYP2B4, and CYP2B6 was unique to each enzyme, and introduction of valine at residue 363 in CYP2B4 and CYP2B6 shifted these ratios toward those of CYP2B1. Mechanism of inhibition of CYP2B enzymes by POAs. The KS and IC50 values for di- and trihalogenated POA congeners were similar. However, large discrepancies between the two measurements were observed with CYP2B4 and CYP2B6 with POA and 3CPOA. As differences between IC50 values, representing inhibition potency or functional strength of the inhibitor, and inhibition constants (Ki) can vary depending on the mode of inhibition,(50) a comprehensive analysis of CYP2B enzyme inhibition by POA and 2’4’5’TCPOA was performed for CYP2B4, CYP2B6, and CYP2B6 L363V (Table 2). To limit effort, further analysis was focused on the two congeners, since POA revealed a large difference between KS and IC50 values while 2’4’5’TCPOA did not. The KI values of these POA congeners were generally consistent with the respective IC50 values and not necessarily related to the KS values. Non-linear regression analysis suggested that both POA and 2’4’5’TCPOA were competitive inhibitors of CYP2B4 and


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Biochemistry

CYP2B6, and POA was a non-competitive inhibitor of CYP2B6 L363V. Determination of the mechanism of inhibition of CYP2B1 and CYP2B4 I363V by these POA congeners was not performed, since the Michaelis constants of these enzymes for 7-EFC (~120 μM) were too high for accurate determination of the KI. Thus, the IC50 value is a good indicator of the potency of POA inhibition of CYP2B enzymes. Table 2. Inhibition constants of POAs in CYP2B enzymesa KI (µM)

POA

2’4’5’TCPOA

CYP2B1

NDb

ND

CYP2B4

1.3±0.65

0.37±0.04

CYP2B4 I363V

ND

ND

CYP2B6

13±1.9

0.64±0.08

CYP2B6 L363V

67±10

0.75±0.10

a

Results are the mean ± standard deviation of three independent experiments. bND means not determined due to high 7-EFC Michaelis constants for these enzymes. Table 3. NADPH oxidation rate of CYP2B6 in the presence of POAs and 4-CPIa CYP2B4 CYP2B6 35±1 (20 µM) POA

33±2 35±2 (200 µM)

2’4’5’TCPOA

20±1

19±1

4-CPI

10±1

13±1

Ctrl (CYP: POR:cyt b5=1:4:2)

38±1

36±1

Ctrl (POR:cyt b5=4:2)

11±1

Ctrl (POR:cyt b5=4:2; 4-CPI)

12±1

Ctrl (CYP:POR:cyt b5=1:4:2), 30 °C 26±2


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a

Results are the mean ± standard deviation of three independent experiments. Reactions were performed at 37 °C in a mixture containing 20 µM inhibitor unless otherwise specified. The rate unit is nmol/min/nmolP450. POA effect on NADPH oxidase activity of CYP2B enzymes. NADPH consumption rates were determined for reconstituted CYP2B enzymes in the presence of POA, 2’4’5’TCPOA or 4CPI as a control. The former two substances are representative of different classes of ligands based on their IC50 and KS values, and the latter is a potent inhibitor of CYP2B enzymes.6, 57 As expected, inclusion of 4-CPI led to the lowest rate of NADPH oxidation, similar to the rate seen when only redox partners were present (Table 3). The NADPH depletion rate in the presence of POA was the highest, approximating that in the absence of inhibitor, and the rate of NADPH oxidation in the presence of 2’4’5’TCPOA was intermediate between those in the presence of 4CPI and POA. Increasing POA concentration 10-fold did not change the NADPH depletion rate for CYP2B6, indicating that POA was saturated based on its KS value, so ligand concentration is not a limiting factor in the consumption of reducing equivalents. Notably, NADPH consumption includes basal NADPH oxidase activity, and possible metabolism of the “inhibitor”. To rule out that the relatively high NADPH oxidation rate by POA is due to oxidation of the ligand, POA was also incubated with CYP2B6, but no potential CYP-mediated oxidative metabolites, such as p-aminophenol (the ether cleavage product, [M+H]+: m/z = 110.053 ± 0.01) or a hydroxy-POA (monohydroxylation product, [M+H]+: m/z = 202.079 ± 0.01; dihydroxylation product, [M+H]+: m/z = 218.074 ± 0.01) were identified within mass spectrometry detection limits as analyzed by UPLC-MS (Supplementary Figures S1-S4). However, in both the presence and absence of NADPH, a peak corresponding to POA ([M+H]+: m/z = 186.084 ± 0.01) is readily identifiable (Supplementary Figures S1-S2). Comparing the UPLC-MS results from the incubations with paminophenol, the ion for POA (expected [M+H]+: 186.084, found: 186.094) is detected in the incubations by not the p-aminophenol standard (Supplementary Figure S3), and the ion for p18 ACS Paragon Plus Environment

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Biochemistry

aminophenol (expected [M+H]+: 110.053, found: 110.064) is detected in the standard but not in the incubations (Supplementary Figure S4). Ligand effects on NADPH depletion rate in CYP2B4 and CYP2B6 were in the rank order of POA < 2’4’5’TCPOA < 4-CPI. This trend mirrors the rank order of the IC50 and KI values for these ligands, suggesting that the probe substrate 7-EFC in those assays does not alter interaction between CYP2B enzymes and POA congeners. Effects of buffer composition and the presence of POR on CYP2B6 POA and 2’4’5’TCPOA affinity. To better understand the large discrepancy between the affinity of POA for CYP2B6 as measured in spectral titrations and in NADPH-driven assays, the actual conditions for the two sets of experiments were carefully compared. Spectral titrations had been performed in a sucrose-containing buffer used for preparing CYP2B X-ray diffraction-quality crystals, while a HEPES buffer was used for CYP enzyme assays. The redox partners POR and cyt b5 are also included in the NADPH-driven enzyme assay. Thus, spectral titrations with CYP2B6 were carried out considering 1) buffer identity and 2) the presence of POR in the titration. Buffer composition had minimal influence on the affinity of POA and 2’4’5’TCPOA for CYP2B6. As shown in Table 4, the congeners had comparable affinities for CYP2B6 alone in HEPES buffer and the sucrose buffer within the limits of experimental error. Including POR in spectral titrations of CYP2B6 increased the KS by 4.2-fold for POA and by 5.8-fold for 2’4’5’TCPOA. Table 4. Effects of buffer type and reductase on spectral binding of POA congeners with CYP2B6a POA POR

2’4’5’TCPOA

buffer KS (µM)

Type-II

KS (µM)

Type-II


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Page 20 of 34

(%)

a

26±1

(%)

-

sucrose

1.2±0.2

-

HEPES

0.67±0.36 16±1

1.4±0.4

19±1

+

HEPES

2.8±1.4

8.1±1.7*

17±3

9.2±3.3

0.70±0.30 9±1

Results are the mean ± standard deviation of three independent experiments.

*

Statistically different from 2’4’5’TCPOA-POR in sucrose buffer or in HEPES buffer at p≤0.05. The effect of cyt b5 on inhibition of NADPH-driven CYP2B6 7-EFC dealkylation. To gain mechanistic insight into the role of cyt b5 in protein-ligand interaction, inhibition assays were carried out in the absence of cyt b5. Removing cyt b5 had no observed effect on 7-EFC metabolism by CYP2B6, as the KM and kcat remained unchanged. In sharp contrast, cyt b5 displayed pronounced effects on inhibition of CYP2B6 7-EFC O-dealkylation activity by the POA congeners (Table 5). While cyt b5 continued to increase the IC50 of POA to CYP2B6 by 1.8-fold, a large decrease in the IC50 of 2’4’5’TCPOA for CYP2B6 with the inclusion of cyt b5 was seen in the enzyme inhibition assay. The effect of reductase alone on the protein-ligand interaction in turnover conditions cannot be determined, because POR generally delivers two electrons to mammalian CYP enzymes in the CYP catalytic cycle and is the only enzyme capable of fulfilling the first electron transfer in this cycle.(51) At the single substrate concentration, the difference in apparent inhibitor affinity (Ki,app) can be calculated from the IC50 values

using

the

Cheng-Prusoff

equation

for

competitive

inhibition:

, = ⁄[ ]⁄1 + [ ]⁄ .(50) For POA, the calculated values for Ki,app in the absence and presence of cyt b5 are 7.5 µM and 13.5 µM, respectively. For 2’4’5’TCPOA, the calculated values for Ki,app in the absence and presence of cyt b5 are 1.8 µM and 0.22 µM, respectively. Cumulatively, individual contributions of POR or cyt b5 interacting with CYP2B6 induced a total decrease in the affinity of POA for CYP2B6, which in large part explains the discrepancy 20 ACS Paragon Plus Environment

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Biochemistry

between the KS and IC50 values. For 2’4’5’TCPOA, the increase in affinity for CYP2B6 in the presence of cyt b5 in NADPH-driven enzyme inhibition assays and decreased affinity when POR was included in spectral titrations only slightly alter the affinity of this ligand for CYP2B6, reflected in the observed consistency of the KS and IC50 values. Table 5. NADPH- or CuOOH-based IC50 values for POA congenersa NADPH-based IC50 (µM)

CuOOH-based IC50 (µM)

CYP:POR

CYP:POR:cyt b5

CYP

CYP:cyt b5

POA

15±1

27±5*

38±7

44±14

2’4’5’TCPOA

3.7±0.3

0.43±0.04*

NDb

ND

a

Results are the mean ± standard deviation of three independent experiments.

b

IC50 values for 2’4’5’TCPOA cannot be determined due to that at its highest concentration (20 µM) in aqueous solution, over 60% activity still remained. *

Statistically significant difference at p≤0.05 between the absence or presence of cyt b5.

The effect of cyt b5 on inhibition of CuOOH-driven CYP2B6 7-EFC dealkylation. In parallel, the role of cyt b5 was also investigated in inhibition assays using cumene hydroperoxide as sole electron and oxygen donor via the peroxide shunt. As shown in Table 5, the addition of cyt b5 in CuOOH-driven assay had no effect on the IC50 for POA. Likewise, although actual IC50 values could not be determined, the presence of cyt b5 did not alter the inhibition by 2’4’5’TCPOA in CuOOH-based assays. Consistently, CYP2B6 had approximately two orders of magnitude lower catalytic efficiency with CuOOH than in the NADPH-based assay, which was contributed by a greater Km (56 µM) and lower kcat (0.42 min-1). The effect of cyt b5 on the stoichiometry of CYP2B6 7-EFC metabolism. To discern how cyt b5 affects stoichiometry in the CYP catalytic cycle, both NADPH consumption and 7-HFC formation were monitored (Table 6). Addition of cyt b5 to the reconstituted enzyme system


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Page 22 of 34

containing CYP2B6 and POR did not change the 7-HFC formation rate. Adding cyt b5 to the system when POA was present increased the rate of product formation by ~2-fold, while adding cyt b5 to the reaction when 2’4’5’TCPOA was present resulted in a ~4-fold decrease in rate of product formation. The NAPDH consumption rate was stimulated in all cases by the addition of cyt b5. Consequently, cyt b5 had small effects on the coupling of NADPH consumption to 7-HFC formation in the absence of inhibitor or in the presence of POA; in contrast in the presence of 2’4’5’TCPOA, cyt b5 caused a sharp decrease in the efficiency of coupling of NADPH consumption to product formation mostly due to the diminished 7-HFC formation. Table 6. NADPH oxidation and 7-HFC formation rates of CYP2B6 in the presence 7-EFC and/or POAsa CYP2B6+POR

CYP2B6+POR+cyt b5

NADP H

Product

Product/NADP H

NADPH

Product

Product/NADP H

7-EFC

41±2

2.0±0.2

0.05

53±4*

2.1±0.2

0.04

POA+7-EFC

32±2

‡

0.86±0.05

0.03

47±3**

1.5±0.1**,

2’4’5’TCPO A + 7-EFC

17±1‡

0.17±0.03‡

0.01

22±1**,‡

0.05±0.01**,‡

†,‡

†

0.03 0.002

a

Results are the mean ± standard deviation of three independent experiments. Reactions were performed at 37 °C in a mixture containing 5 µM 7-EFC and/or 20 µM ligands unless otherwise specified. The rate unit is nmol/min/nmolP450. *


**


†

Statistically significant difference at p≤0.05 between the absence or presence of inhibitor.

‡

Statistically significant difference at p≤0.005 between the absence or presence of inhibitor.

DISCUSSION


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Biochemistry

CYP2B6 is the major human CYP enzyme involved in the metabolism of the PBDE congeners that make up >95% of previously available penta-BDE technical mixtures.(52) A disproportionately high number of CYP2B substrates also contain halogen atoms, but the biochemical basis for this selectivity has not previously been elucidated. Knowledge of how highly halogenated hydrocarbons interact with CYP2B enzymes will help to reveal the relationship between the metabolism and human health effects of these compounds. Overall, differences between POA and 3CPOA affinity and inhibitory potency in 7-EFC based O– dealkylation assays can be explained in part by the presence or absence of the protein redox partners needed for CYP2B function, indicating a role of protein-protein interaction driven conformational changes in the selectivity of POA congener interaction with CYP2B enzymes. Conformational differences in CYP2B enzymes due to amino acid substitutions, ligand binding, or interaction with redox partners are implicated in the ligand and species specific interactions with POA congeners. POR decreased the binding affinity of POA and 2’4’5’TCPOA for CYP2B6 in spectral binding titrations 4-6 fold. Even more strikingly, the presence of POR decreased the affinity of (S)-flurbiprofen, a nonsteroidal anti-inflammatory drug, for CYP2C9 by a factor of 48.(53) Structures of the complex of CYP101A1 and its redox partner putidaredoxin (Pdx) have been reported in open and closed conformations based on data from X-ray crystallography, NMR and EPR spectroscopy, and molecular dynamics simulations.(54-58) Subtle differences in the CYP enzyme conformation dependent on ligand identity or buffer environment could also alter the redox partner affinity to the CYP enzyme, as seen in CYP101A1 binding to Pdx measured using ITC.(58) Interaction of Pdx with CYP101A1 is based on ligand identity and solution environment, and the equilibrium between CYP enzyme conformers in solution is likely easily perturbed by subtle changes in the CYP enzyme


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 34

environment, as seen in the change in affinity of 2’4’5’TCPOA for CYP2B6 when buffer conditions are changed (Table 5). Furthermore, ion pairs identified in CYP101A1 involving R186, K178, and D251 in the closed conformation of the enzyme are disrupted when the enzyme opens.(59) Poulos and coworkers hypothesize that conformational changes induced by redox partner binding are coupled to changes in the topology of the substrate access channel and may stabilize the CYP enzyme active site.(57, 60) The magnitude of these changes may depend on the conformational changes induced by specific ligands. When amlodipine is bound to CYP2B6, E301 interacts with T305 and R308.(23) Yet, when 4-(4-nitrobenzyl)pyridine or α-pinene is bound to CYP2B6, E301 forms an interaction with Q172 (Figure 4).(22, 24) Structural alterations upon interactions between POR and the human steroidogenic CYP17A1 were observed using solution NMR.(61) POR effects on ligand binding to CYP enzymes is also ligand or substrate dependent with no change in affinity seen in multiple enzyme-ligand pairs.(62, 63)


Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4. Overlay of CYP2B6 structures in complex with amlodipine (PDB ID 3UA5, green), 4(4-nitrobenzyl)pyridine (PDB ID 3QU8, magenta) and (+)-α-pinene (PDB ID 4I91, cyan). Protein is shown in cartoon representation. The side chains of E301, T305, R308 and Q172 and the heme are shown as sticks. As seen in multiple ligand interactions with other CYP enzymes, the effect of cyt b5 on POA interactions with CYP2B enzymes was unique for each ligand. As proposed by Waskell and colleagues, the allosteric effect on the CYP2B enzyme active site could enhance productive catalysis and be altered differentially by different inhibitors.(64) The binding sites for POR and cyt b5 are unique for each redox partner but are overlapping, so any allosteric effects of redox partner binding to CYP enzymes should also be unique to the redox partner.(65-67) The difference in IC50 values for the NADPH-driven versus CuOOH-driven assays likely reflects a combination of competition for the redox partner binding sites and POR-induced conformational changes in the CYP enzyme altering the effects of cyt b5 binding to CYP2B6. However, conformational changes due to ligand binding could also alter the affinity of cyt b5 for CYP2B6 changing the ability of POR to bind and subsequently transfer electrons to the heme. Furthermore, feedback may also occur between conformational changes caused by ligand binding and any allosteric effects mediated by redox partner binding with ligand identity governing redox partner affinity for the specific CYP enzyme. Unique effects of cyt b5 binding to CYP17A1 were observed by NMR, and the conformational changes due to cyt b5-CYP17A1 binding were hypothesized to modulate human steroidogenesis.(68) Subsequently, Waskell and coworkers demonstrated that binding of cyt b5 to CYP17A1 stimulates its 17,20-lyase activity.(69) In our experiments, binding of cyt b5 to CYP2B6 did not alter the product/NADPH stoichiometry for 7-EFC metabolism in the absence of inhibitor or the presence of POA, but the


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Page 26 of 34

presence of 2’4’5TCPOA resulted in a reduced efficiency in product production upon the addition of cyt b5 to the system. The CYP2B subfamily of CYP enzymes is known to have low preservation of catalytic activity across mammalian species.(48) In CYP2B enzymes, altering the identity of residue 363 alters substrate specificity and product profiles.(17,21,47) The identity of active site residues in CYP2B enzymes can in part explain catalytic differences among these enzymes.(47-49) However, differences in the identity of amino acid residues lining the substrate access channel and product egress routes could also lead to differences in catalytic activity.(70) Furthermore, the lipophilicity of CYP2B substrates and inhibitors is directly related to binding affinity and inhibition potency.(71) Thus the lack of differences in binding affinity or inhibition potency with changes in the halogenation pattern but increased affinity and potency with addition of one or more halogens to the POA backbone seen in Figure 3 could be due to increased hydrophobicity alone. CYP2B6

is

also

highly

polymorphic

with

38

verified

haplotypes

(www.pharmvar.org/gene/CYP2B6). Most of these polymorphisms are distal from the enzyme active site, and only a small number have been shown to produce clinically relevant changes in substrate metabolism.(72-74) Differences in metabolism of halogenated compounds by CYP2B6 polymorphisms are likely to correlate with altered metabolism of efavirenz, cyclophosphamide, or nevirapine by CYP2B6*4, *5, *6, *8, or *9. In conclusion, an initial structure-activity relationship was generated for POA interactions with CYP2B enzymes. The identity of residue 363 is also a determinant of the POA interaction profile with CYP2B enzymes, but this residue alone is not sufficient to fully describe species-specific interactions between halogenated compounds and CYP2B enzymes. The role of halogen-π


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Biochemistry

bonding has been suggested to influence ligand interactions with CYP2B enzymes.(26) However, increased hydrophobicity with increased halogenation may describe the role of CYP2B6 in human metabolism of halogenated compounds. Furthermore, conformational changes transmitted from the binding site of CYP enzyme redox partners to the substrate access channel and the substrate binding pocket likely occur in all CYP enzyme systems, and the changes in CYP enzyme conformation upon ligand binding likely affect the binding of redox partner proteins and vice versa.(60)

ASSOCIATED CONTENT Supporting information A description of what is in the supporting information. The following files are available free of charge at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed. Email: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This research was supported by the National Institutes of Health (R01-ES003619) to J.R.H. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. Dennis Hill at the University of Connecticut for assistance with the UPLC-MS analysis. 27 ACS Paragon Plus Environment

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Biochemistry

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FOR TABLE OF CONTENTS USE ONLY

Use of phenoxyaniline analogs to generate biochemical insights into polybrominated diphenyl ether metabolism by CYP2B enzymes

Chao Chen,† Jingbao Liu,† James R. Halpert,† P. Ross Wilderman*,† †

University of Connecticut School of Pharmacy, Storrs, Connecticut 06269, United States


Use of Phenoxyaniline Analogues To Generate Biochemical Insights

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