0090-9556/97/2511-1242–1248$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 11
Printed in U.S.A.
INACTIVATION OF CYTOCHROME P450S 2B1, 2B4, 2B6, AND 2B11 BY ARYLALKYNES
ELIZABETH S. ROBERTS, NANCY EDDY HOPKINS, MARYAM FOROOZESH,1 WILLIAM L. ALWORTH, JAMES R. HALPERT,
AND PAUL F. HOLLENBERG
Department of Pharmacology, University of Michigan (E.S.R., P.F.H.), Department of Chemistry, Tulane University (N.E.H., M.F., W.L.A.), and
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona (J.R.H.)
(Received March 20, 1997; accepted June 10, 1997)
ABSTRACT:
treated with various inducing agents was determined by measuring lidocaine, testosterone, p-nitrophenol, or erythromycin metabolism. The greatest effect was observed with the 2B-specific products from lidocaine and testosterone, whereas no effect was seen
with p-nitrophenol or erythromycin. When the covalent binding of
[3H]2EN to microsomal protein was analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and autoradiography, a
radiolabeled protein band that corresponds to 2B1 was observed
in the lanes containing microsomes from rats treated with phenobarbital and, to a lesser extent, pyridine and isosafrole after incubation with NADPH. When these microsomes were incubated with
[3H]9EPh or [3H]1EP, two NADPH-dependent bands were radiolabeled. One corresponded to 2B1/2 and the other to a protein of
approximately 59 kDa, which was observed in the lanes from phenobarbital-treated male and female rats and pyridine-treated male
rats. No radiolabeled bands were observed with [3H,14C]4PbP with
any of the microsomes.
The P4502 superfamily of enzymes is a group of heme proteins that
oxidize an extensive series of compounds including drugs, carcinogens, steroids, and fatty acids (1). Many of the P450 isozymes have
been investigated for their ability to oxidize acetylenic compounds,
resulting in a time-dependent loss of the heme chromophore after an
intermediate becomes covalently bound to a heme nitrogen (2). More
recently, several aromatic acetylenes, including 2EN and 9EPh, have
been shown to act as reversible inhibitors or mechanism-based inactivators that alkylate the protein moiety of cytochrome P450 in microsomes or reconstituted systems from rats or rabbits (3–7). In
addition, the structure-activity relationships for the inhibition and
inactivation of 1A1-, 1A2-, and 2B1-dependent reactions in rat liver
microsomes by a series of aryl acetylenes have been investigated (6,
8). It was determined that the size and the shape of the polycyclic
aromatic ring system and the placement of the alkyne function on the
ring system were critical for suicide inhibition (6, 8). In addition, there
was selectivity for different P450 isozymes when the compound
contained either an ethynyl or propynyl group. In microsomes, the
substitution of a propyne group for the ethyne moiety enhanced the
inhibition of P450 1A enzymes (6). However, ethynes were more
effective suicide inhibitors of P450 2B-dependent reactions in microsomes than the corresponding propynes (6). In addition, 9EPh and
2EN were also found to be mechanism-based inactivators of mouse
2b-10 in liver and lung microsomes (7).
The present study describes the structure-activity relationships for
the inactivation of P450s 2B1, 2B4, or 2B11 in a reconstituted system
by a series of arylalkynes (fig. 1). The most effective inactivator of
these 2B enzymes, 9EPh, was investigated for its potential as an
inactivator of human 2B6 expressed in a human lymphoblastoid cell
line. The ability of 9EPh to inactivate 2B2 was investigated using
lidocaine as a substrate monitoring the formation of the 2B2-specific
product (9). To further investigate the specificity of 9EPh for rat liver
This research was supported by NIH Grants CA16954 (P.F.H.), CA38192
(W.L.A.), and ES03619 (J.R.H.).
1
Present address: Chemistry Department, Xavier University of Louisiana.
2
Abbreviations used are: P450, cytochrome P450 (the system of P450 nomenclature as described in Nelson et al. (1) is used); reductase, NADPH-cytochrome P450 reductase; DLPC, L-a-phosphatidylcholine, dilauroyl; EFC, 7ethoxy-4-trifluoromethylcoumarin; HFC, 7-hydroxy-4-trifluoromethylcoumarin;
1EN, 1-ethynylnaphthalene; 2EN, 2-ethynylnaphthalene; 2PN, 2-(1-propynyl)
naphthalene; 1EA, 1-ethynylanthracene; 2EA, 2-ethynylanthracene; 2EPh, 2-ethynylphenanthrene; 3EPh, 3-ethynylphenanthrene; 9EPh, 9-ethynylphenanthrene,
9PPh, 9-(1-propynyl)phenanthrene;1EP, 1-ethynylpyrene; 4EP, 4-ethynylpyrene;
4PbP, 4-(1-propynyl)biphenyl; PB, phenobarbital; bNF, b-naphthoflavone; PYR,
pyridine; PCN, pregnenolone 16a-carbonitrile; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; MEGX,
monoethylglycinexylidide; Me-OH lidocaine, methylhydroxylidocaine; MALDIMS, matrix-assisted laser desorption ionization mass spectrometry; THF, tetrahydrofuran.
Send reprint requests to: Dr. Paul F. Hollenberg, Department of Pharmacology, Medical Science Research Building III, 1150 West Medical Center Drive, Ann
Arbor, MI 48109-0632.
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The time-dependent loss of the 7-ethoxy-4-trifluoromethylcoumarin (EFC) O-deethylase activity of rat P450 2B1, rabbit P450 2B4, or
dog P450 2B11 by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 2-(1-propynyl)naphthalene (2PN), 1-ethynylanthracene
(1EA), 2-ethynylanthracene, 2-ethynylphenanthrene, 3-ethynylphenanthrene, 9-ethynylphenanthrene (9EPh), 9-(1-propynyl)phenanthrene (9PPh), 4-ethynylpyrene (4EP), and 4-(1-propynyl)biphenyl (4PbP) was investigated. The rate constants for inactivation
by the arylalkynes in descending order of effectiveness for the top
five compounds were 9EPh>9PPh>1EN, 2EN, 2PN for 2B1,
9EPh>2EN>4EP>1EN, 1EA for 2B4, and 9EPh>1EA>4EP,
9PPh>2EN for 2B11. The size and the shape of the aromatic ring
system and the placement of the alkyne functional group were
important for inactivation. The most effective inactivator with all
the isozymes was 9EPh. This compound also inactivated the EFC
activity in microsomes from human lymphoblastoid cells expressing human P450 2B6. The specificity of 9EPh for the inhibition or
inactivation of different P450 activities in microsomes from rats
INACTIVATION OF P450s BY ARYLALKYNES
FIG. 1. Structures of the arylhydrocarbons studied as inhibitors of P450catalyzed activities with the numbers identifying the placement of the
acetylene or propynyl groups.
Materials and Methods
Materials. EFC was from Molecular Probes, Inc. (Eugene, OR), and HFC
was from Enzyme Systems Products (Dublin, CA). 7-Ethoxycoumarin, 7hydroxycoumarin, and testosterone were from Aldrich Chemical Co. (Milwaukee, WI), and the metabolites of testosterone, including the internal standard
14a-hydroxytestosterone, were from Steraloids, Inc. (Wilton, NH) or the
Alfred-Bader Collection from Aldrich Chemical Co. (Milwaukee, WI). Catalase purified from bovine liver, lidocaine, procaine, and DLPC were from
Sigma Chemical Co. (St. Louis, MO).
Enzymes. The microsomes were prepared from the livers of either male or
female 10-week-old Fisher 344 rats (Charles River Labs, Portage, MI) given
0.25 g of clofibrate/100 g of lab chow for 17 days, 50 mg of PCN/kg ip for 4
days, 0.1% PB in the drinking water for 12 days, or 150 mg of isosafrole/kg
ip for 4 days or male 150 –175-g Fisher 344 rats (Harlan Sprague Dawley, Inc.,
Indianapolis, IN) given 0.1% PB in the drinking water for 12 days, 100 mg of
PYR/kg ip for 3 days, or 80 mg bNF/kg ip for 3 days. P450 2B1 and reductase
were purified as described by Saito and Strobel (10) and Strobel and Dignam
(11), respectively, from microsomes prepared from the livers of fasted Long
Evans rats (150 –175 g; Harlan Sprague Dawley; Indianapolis, IN) given 0.1%
PB in the drinking water for 12 days. P450 2B4 was purified from the livers
of 2.5-kg male New Zealand White rabbits (Langshaw, Augusta, MI) given
0.1% PB in the drinking water for 7 days (12). P450 2B11 was purified as
previously described by Duignan et al. (13). Microsomes from human lymphoblastoid cells expressing P450 2B6 were from Gentest Corp. (Woburn, MA).
Arylalkynes. 1EN, 2EN, 1EA, 2EA, 2EPh, 3EPh, and 9EPh were synthesized as described (8). 2PN, 9PPh, 4EP, and 4PbP were synthesized and
characterized as recently described (6).
2-([2-3H]ethynyl)Naphthalene ([3H]2EN, labeled on the acetylenic hydrogen, 170 Ci/mole) was synthesized as described (3). [2-3H]9EPh ([3H]9EPh,
labeled on the terminal ethynyl carbon with tritium, 135 Ci/mole) was synthesized as described in Roberts et al. (14).
[4,5,9,10-3H]1EP. [4,5,9,10-3H]Pyrene (100 mCi, 23.6 Ci/mol, Chemsyn
Science Laboratories, Lenexa, KS) and 0.5 mmol (100 mg) of unlabeled
pyrene (Aldrich Chemical Co., Milwaukee, WI) were dissolved in 10 ml of
tetrachloroethylene (Aldrich); 1.5 mmol of AlCl3 (Aldrich) were added, and
the reaction mixture cooled to 220°C under an N2 atmosphere. Three equivalents (107 ml, 118 mg) of acetyl chloride (Aldrich) was added to the reaction
mixture, which was stirred with a magnetic stir bar, and maintained at 220°C
under an N2 atmosphere for 4 hr and then allowed to warm to room temperature over a 0.5-hr period. The reaction was quenched with water, and the
product was extracted with CH2Cl2. The CH2Cl2 extract was dried with
Na2SO4, evaporated to dryness in vacuo, and the crude 1-acetylpyrene product
obtained was purified by chromatography on silica gel with CH2Cl2 elution.
The fractions containing 1-acetylpyrene were identified by thin layer chromatography on silica gel plates (CH2Cl2 elution), pooled, and evaporated to
dryness to yield 88 mg (0.36 mmol, 72%) of [4,5,9,10-3H]1-acetylpyrene.
The [4,5,9,10-3H]1-acetylpyrene was dried in vacuo overnight, dissolved in
5.0 ml of dry tetrahydrofuran (THF), freshly distilled from benzophenone
dianion under N2 atmosphere, cooled to 278°C under N2 atmosphere, and
treated with 2.5 equivalents (0.9 mmol) of base prepared by treating 152 ml of
2,2,6,6-tetramethylpiperidine (Aldrich) in 15 ml of dry THF with 0.56 ml of
1.6 M butyllithium solution in hexanes (Aldrich) for 0.5 hr. The combined
solution was stirred with a magnetic stir bar at 278°C under N2 atmosphere for
1 hr, and then 1.5 equivalents (78 ml, 93 mg) of diethyl chlorophosphate
(Aldrich) were added. After 2 hr at 278°C, the reaction mixture was allowed
to warm to room temperature and was then transferred with N2 pressure into
a second freshly prepared solution of 2.5 equivalents of 2,2,6,6tetramethylpiperidine and butyllithium in THF at 278°C. After an additional
0.5 hr at 278°C, the reaction was allowed to warm slowly to room temperature
and was then cooled again to 278°C and quenched with 5.0 ml of dilute
sodium bicarbonate solution. The product was extracted with CH2Cl2, and the
CH2Cl2 extract was washed twice with cold diluted HCl and then H2O and then
dried over Na2SO4. The CH2Cl2 was evaporated in vacuo, and the crude
[3H]1EP product was purified by flash chromatography on silica gel with
petroleum ether as the eluting solvent. The yield of [4,5,9,10-3H]1EP was 32
mg (0.14 mmol, 36%); radiochemical analysis performed with a Beckman LS
7000 liquid scintillation system with internal standardization established that
the specific activity was 61.2 Ci/mole. The use of fresh, anhydrous reagents
and carefully dried glassware is critical to the success of this synthetic
conversion of aryl methyl ketones to alkynes on the small scale.
4-(1-propynyl)Biphenyl ([3H,14C]4PbP). 4-Acetylbiphenyl was labeled by
catalytic exchange with tritium gas by Chemsyn Science Laboratories (Lenexa,
KS). The tritiated sample was diluted with nonradioactive 4-acetylbiphenyl
(Aldrich) to a total of 225 mg (1.15 mmol), dissolved in dry THF, and reacted
with diethyl chlorophosphate (Aldrich) at 278°C under an N2 atmosphere as
described for 1EP. The reaction mixture generated by adding 2.5 additional
equivalents of lithium diisopropylamide (Aldrich) at 278°C was then treated
with 1.1 equivalents of [14C]CH3I (Amersham Corp., Arlington Heights, IL),
and the reaction mixture was allowed to warm to room temperature overnight.
The reaction, still under N2 atmosphere, was then cooled to 278°C, 5.0 ml of
dilute sodium bicarbonate solution was added, and the solution was allowed to
warm to room temperature. The reaction product was extracted and purified by
flash chromatography on silica gel as described above for [3H]1EP. The yield
of [3H,14C]4PbP was 80 mg (36%). The specific activities were determined
with a Beckman LS 7000 liquid scintillation system with internal standardization using a program that counts 3H and 14C in separate channels. The specific
activities were 31 Ci/mole for 3H and 0.32 Ci/mole for 14C.
Inactivation of P450 Enzymes by Arylalkynes. For inactivation experiments of purified P450 in the reconstituted system by various aryl acetylenes
(table 1), a single time point EFC O-deethylase assay (15) was employed to
determine the enzyme activity remaining after incubation with the arylalkynes.
The primary incubations contained 0.25 mM P450, 0.5 mM reductase, 15 mg of
DLPC, 125 units of catalase, 7.5 mM inactivator, or DMSO in control incubations and 50 mM potassium phosphate buffer, pH 7.4, in a total volume of
0.12 ml. The mixtures were preincubated at 30°C for 3 min before the addition
of 0.83 mM NADPH to start the reaction. Aliquots containing 5 pmol of P450
were removed at various time points and added to 1.0-ml secondary reaction
mixtures containing 0.2 mM NADPH, 100 mM EFC, 40 mg BSA, and 50 mM
potassium phosphate buffer, pH 7.4. Secondary incubations were allowed to
proceed for 5 min before the reactions were quenched with 0.3 ml of cold
acetonitrile. For incubations with P450 2B4 or 2B11, the amount of reductase
in the primary incubation was 0.75 mM. The fluorescent product, HFC, was
measured on an SLM-Aminco model SPF-500C spectrofluorometer with excitation at 410 (slit width, 5 nm) and emission at 510 nm (slit width, 5 nm).
For the determination of the maximal rate constant of inactivation and the
concentration of inactivator required for half-maximal inactivation of the
O-deethylation of 7-ethoxycoumarin by 2B1, 2B4, and 2B11 (table 2), the
primary incubations contained 0.4 mM P450, 0.8 mM reductase, 10 mg DLPC,
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P450s, we monitored the effect on testosterone, p-nitrophenol, and
erythromycin metabolism in liver microsomes from rats treated with
inducers of different forms of P450. Finally, the NADPH-dependent
binding of several radiolabeled arylalkynes to microsomal proteins
were determined by SDS-PAGE and autoradiography.
1243
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ROBERTS ET AL.
TABLE 1
Rate constants for inactivation of the EFC O-deethylase activity of 2B1,
2B4, or 2B11 by arylalkynes
Experiments were carried out as described under Materials and Methods.
Rate constants of inactivation were derived by linear regression analysis of
the natural logarithm of the residual activity as a function of time. Values
represent the average from two independent experiments that did not differ
by more than 5%. The time 0 deethylase activities for P450 2B1, 2B4, and
2B11 were 6.17, 1.04, and 3.82 nmol HFC formed/min/nmol P450,
respectively. Control values with no inhibitor were ,0.010 min21. ND,
values not different from control values.
P450 2B1
P450 2B4
Arylalkyne
0.16
0.14
0.12
0.07
0.03
ND
0.03
0.88
0.45
0.02
0.07
0.11
0.34
0.04
0.10
ND
ND
0.03
0.43
ND
0.18
0.08
0.06
0.11
ND
0.23
ND
0.05
ND
0.49
0.17
0.19
ND
TABLE 2
Inactivation of the 7-ethoxycoumarin O-deethylase activity of purified 2B1,
2B4, or 2B11 in a reconstituted system by 9-ethynylphenanthrene
Experiments were carried out as described under Materials and Methods.
The kinetic values were obtained as described previously (14).
P450
kinactivation(min)21a
KI(mM)b
2B1
2B4
2B11
0.33
0.25
0.24
0.12
0.56
0.44
a
Maximal rate constant of inactivation.
b
Concentration of inactivator that gives half-maximal inactivation.
125 units of catalase, 0.05, 0.1, 0.25, 1.0, or 2.0 mM 9EPh, or DMSO in control
incubations and 50 mM potassium phosphate buffer, pH 7.4, in a total volume
of 0.15 ml. The mixtures were preincubated at 20°C for 3 min before the
addition of 1.0 mM NADPH to start the reaction. Aliquots containing 10 pmol
of P450 were removed at various time points and added to 1.0-ml secondary
reaction mixtures containing 1.0 mM NADPH, 300 mM 7-ethoxycoumarin, 40
mg of BSA, and 50 mM potassium phosphate buffer, pH 7.4. Secondary
incubations were allowed to proceed for 10 min before the reactions were
quenched with 0.1 ml of 2 N HCl. The reaction mixtures were extracted with
3.0 ml of CH2Cl2, and the organic phase was back-extracted with 2.0 ml of 30
mM sodium borate. 7-Hydroxycoumarin was determined spectrofluorometrically at 454 nm with an excitation wavelength of 366 nm (16).
For the inactivation of 2B6, 1.0-ml reaction mixtures containing 100 mg of
microsomal protein from human B lymphoblastoid cells expressing human
P450 2B6, 0.75 mM 9EPh, 120 units of catalase (when included), and 50 mM
potassium phosphate buffer, pH 7.4, were preincubated for 3.0 min at 30°C
before the addition of NADPH at a final concentration of 0.2 mM to start the
reaction or water in the control reactions without NADPH. At 0 or 5 min, 100
mM EFC and NADPH, for those reactions where NADPH was not previously
included, were added to the reaction mixtures, and HFC formation was
measured spectrofluorometrically as described above. The rate of product
formation was calculated from the slope of the recorded trace.
Lidocaine Metabolism. The metabolism of lidocaine by liver microsomes
from PB-treated rats was determined as described (9). The reaction mixtures
contained 0.2 mg of protein, 20 mM 9EPh, 2EN, or DMSO in the control
reactions, and 100 mM potassium phosphate buffer, pH 7.4, in a total volume
Results
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1EN
2EN
2PN
1EA
2EA
2EPh
3EPh
9EPh
9PPh
4EP
4PbP
P450 2B11
ki(min21)
of 0.5 ml. The mixtures were preincubated for 3 min at 37°C before the
addition of NADPH to a final concentration of 0.4 mM. Lidocaine (1.0 mM)
was added either before the addition of NADPH in the time 0 reactions or after
5 min in all others. After a 15-min incubation with lidocaine, the reactions
were quenched by the addition of 1 N NaOH. Procaine was added as an
internal standard, and the mixtures were extracted with ethyl acetate. The
organic layer was dried down, and the residue was dissolved in high performance liquid chromatography solvent and injected onto a C18 reversed phase
column. The compounds were eluted with an isocratic buffer system consisting
of 5% CH3CN and 95% 0.1M potassium phosphate, pH 3.0, at a flow rate of
1.7 ml/min. Detection was at 214 nm.
Testosterone Metabolism. The metabolism of testosterone by liver microsomes from untreated or PB-treated rats was determined as described (17, 18).
The mixtures contained 1.0 mg of protein, 250 mM testosterone, 20 mM 9EPh
(when included), and 50 mM potassium phosphate buffer, pH 7.4, in a total
volume of 1.0 ml. The mixtures were preincubated at 37°C for 3 min before the
addition of 1.0 mM NADPH. After a 10-min incubation, the reactions were
quenched with 3.0 ml of CH2Cl2. An internal standard (14a-hydroxytestosterone) was added, and the mixtures were extracted. The organic layer was
dried down, and the residue was dissolved in methanol, filtered, and injected
onto a C18 reversed phase column. The solvent system consisted of mixture A
(MeOH:H20:CH3CN, 39:60:1) and mixture B (MeOH:H20:CH3CN, 80:18:2).
The metabolites were eluted with a concave gradient (curve #7 using a Waters
automated gradient controller) from 11% to 85% mixture B over 23 min with
analysis of the eluate at 254 nm.
SDS-PAGE Analysis. The incubation mixtures contained 0.25 mg of microsomal protein, 1.0 mM NADPH, 30 mM radiolabeled arylalkyne, and 50
mM potassium phosphate buffer, pH 7.4, in 0.2 ml. The mixtures were
incubated at 30°C for 15 min, and the reactions were quenched with an equal
volume of sample loading buffer. After heating for 5.0 min at 90°C, the
samples were analyzed by SDS-PAGE on a 7.5% polyacrylamide gel using the
Laemmli buffer system (19). The gels were stained with Coomassie Blue,
destained, and then treated with Entensify (NEN Research Products, Boston,
MA) before drying. The gels were exposed to Hyperfilm-MP (Amersham
Corp., Arlington Heights, IL) for 12–16 days before developing.
The ethynyl-substituted compounds 1EN, 2EN, 1EA, 2EA, 2EPh,
3EPh, 9EPh, and 4EP and the propynyl-substituted compounds 2PN,
9PPh, and 4PbP (fig. 1) at a concentration of 7.5 mM were each
investigated for their ability to cause a time-dependent loss of 2B1-,
2B4-, or 2B11-dependent EFC O-deethylase activity in a reconstituted
system containing rat reductase and lipid. Fig. 2 shows representative
graphs when 2B1, 2B4, or 2B11 was incubated with substituted
phenanthrylalkynes, and the activity remaining at various time points
was determined by removing an aliquot and assaying for the EFC
O-deethylase activity. The 50-fold dilution into the secondary reaction
mixture was necessary to minimize the reversible inhibition described
previously with rat liver microsomes (8). Incubation with 9EPh resulted in a time-dependent loss of activity with all three isozymes.
Rate constants of inactivation were determined from the slopes of the
lines when the natural logarithm of the per cent activity remaining was
plotted vs. time. Each of the isozymes was incubated with each of the
11 compounds, and the rate constants of inactivation were determined
(table 1). With every isozyme, the most effective inactivator at a
concentration of 7.5 mM was 9EPh. With P450 2B1, the order of
effectiveness for the top five compounds was 9EPh.9PPh.1EN,
2EN, 2PN. The order of effectiveness with the rabbit isozyme, P450
2B4, was 9EPh.2EN.4EP.1EN, 1EA. With 2B11, the rate constants of inactivation in descending order were 9EPh.1EA.4EP,
9PPh.2EN. Similar experiments were performed to obtain the maximal rate constant of inactivation (kinactivation) and the 9EPh concentration required for half-maximal inactivation (KI) of the 7-ethoxycoumarin O-deethylase activity. There are two differences between
the two sets of experiments described in tables 1 and 2. One is the
1245
INACTIVATION OF P450s BY ARYLALKYNES
TABLE 3
Inactivation of P450 2B6-dependent deethylation of EFC by 9EPh
Reaction mixtures (1.0 ml) containing 0.1 mg of microsomal protein
from human B lymphoblastoid cells expressing human 2B6, 0.75 mM
9EPh, 120 units of catalase (when included), and 50 mM potassium
phosphate buffer, pH 7.4, were preincubated for 3 min at 30°C before
the addition of NADPH to a final concentration of 0.2 mM or water to
the mixtures without NADPH. The deethylase activity was measured
spectrofluorometrically as described under Materials and Methods after
the addition of 100 mM EFC and NADPH in the reaction mixtures
where NADPH was not previously included. The activities presented are
the average of duplicate determinations, and the values in parentheses
are the percent activities remaining as compared with the incubation
with no 9EPh at time 0.
Activity
(pmol HFC/min/mg protein)
(%)
9EPh NADPH
FIG. 2. Effect of phenanthrylalkynes on P450 2B1- (panel A), 2B4- (panel
B), or 2B11- (panel C) dependent EFC activity.
Incubations were performed with DMSO (M) or 7.5 mM 2EPh (L), 3EPh
(E), 9EPh (‚), or 9PPh (Œ) and P450 that had been reconstituted with
reductase and lipid. Aliquots were removed at various time points to assay for
EFC activity as described under Materials and Methods. Data are representative of two individual experiments.
enzyme activity used to monitor the 2B activity remaining at various
time points and the other is the temperature of the reactions. Although
the assay used to measure the product of EFC metabolism in table 1
is convenient for screening a large number of compounds, the enzyme
activity associated with several 2B isozymes is low. Therefore, 7EC
was used to more accurately examine the characteristics of these
mechanism-based inhibitors. The experiments described in table 1
were performed at 30°C, while those described in table 2 were
performed at 20°C. It was easier to monitor the linear portion of the
very rapid inactivation by 9EPh when the reaction was slowed by
lowering the temperature. Various concentrations of 9EPh were added
to primary incubations containing 2B1, 2B4, or 2B11 in reconstituted
systems, and 7-ethoxycoumarin O-deethylase activity was used as a
marker enzyme in the secondary reaction mixture to monitor the loss
of activity at various time points. First order inactivation constants
were determined by linear regression analysis of the slopes of the
lines. From the plot of the reciprocal of the initial rate constant of
inactivation as a function of the reciprocal of the 9EPh concentration,
the maximal rate constant of inactivation and the inactivator concentration required for half-maximal inactivation were determined (table
2) as previously described (14).
9EPh was further investigated for its ability to inactivate human
2B6 expressed in human lymphoblastoid cells. The 2B6-dependent
activity was followed by measuring the O-deethylation of EFC. Microsomes from cells that did not express 2B6 showed no measurable
EFC deethylase activity. When microsomes from the cells expressing
2B6 were incubated with 0.75 mM 9EPh and NADPH, only 23% of
the activity measured at time 0 remained after 5 min (table 3). The
addition of catalase to the reaction mixtures during the incubation did
not protect against the loss of activity.
To investigate the specificity of the inhibition of several rat P450
isozymes by 9EPh, lidocaine, and testosterone metabolism in microsomes from untreated and PB-treated rats was determined in the
presence or absence of 9EPh (tables 4 and 5). Many forms of P450
have the ability to N-deethylate lidocaine to form monoethylglycin-
2
2
1
1
1
1
1
1
2
1
1
1
2
2
2
2
2
1
0
5
5
0
5
5
145 6 13 (100)
127 6 4 (88)
123 6 7 (83)
136 6 7 (94)
33 6 3 (23)
29 6 4 (20)
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Catalase
Incubation time
(min)
Additions
exylidide (MEGX), although 2B1 does have one of the highest activities. Methylhydroxylidocaine (Me-OH lidocaine) is formed exclusively by 2B2 (9). In microsomes from PB-treated rats, there was both
competitive inhibition and time-dependent loss in the formation of the
two lidocaine products, MEGX and Me-OH lidocaine, in the presence
of either 2EN or 9EPh. 9EPh resulted in a dramatic time-dependent
loss in MeOH-lidocaine, the 2B2-specific product. The metabolism of
testosterone by microsomes from uninduced and PB-treated rats in the
presence or absence of 9EPh is described in table 5. The formation of
2a- and 16a-hydroxytestosterone and androstenedione (17 oxidation)
by untreated rat liver microsomes was unchanged in the presence of
9EPh. There was a 60% decrease in the 6b/7a-hydroxytestosterone
peak. In microsomes from PB-treated rats, there was no change in the
6b/7a-hydroxytestosterone metabolites or androstenedione formation, and there was an increase in the 2a- (2C11-dependent) and
2b-hydroxytestosterone metabolites. There was a significant decrease
in the formation of 16a- and 16b-hydroxytestosterone as well as
16-ketotestosterone, primarily reflecting an inhibition of 2B1 activity.
This inhibition of activity probably reflects both the competitive
inhibition and time-dependent inactivation.
To further investigate the effect of 9EPh on other rat P450-dependent activities, we added 9EPh into the reaction mixtures with the
substrates p-nitrophenol or erythromycin. The activity at the end of
the incubation would reflect a combination of the inhibition and
inactivation of the P450 by 9EPh. If we saw an effect, we could then
determine whether this was due to inhibition or inactivation. The
p-nitrophenol hydroxylase (P450 2E1-dependent) activity of microsomes from untreated, PB-, or PYR-treated rats was determined in the
presence and absence of 10 mM 9EPh. There was no difference in the
activity when 9EPh was included in the reaction mixture (data not
shown). In addition, there was no effect of 10 mM 9EPh on the
erythromycin N-demethylase (P450 3A-dependent) activity of liver
microsomes from uninduced and PB-treated rats (data not shown). At
this concentration and under these conditions, 9EPh did not inhibit or
inactivate the 2E1- or 3A-dependent activities.
Finally, the covalent binding of radiolabeled arylalkynes to microsomal proteins was investigated using SDS-PAGE followed by autoradiography after incubation in the presence of NADPH. As shown in
fig. 3A, when [3H]2EN was incubated with microsomes from rats
1246
ROBERTS ET AL.
TABLE 4
Effect of 2EN or 9EPh on lidocaine metabolism by microsomes from PB-treated rats
Incubation conditions and analysis of the products were as described under Materials and Methods. The values represent the average 6 SD from three
determinations. The numbers in parentheses represent the percentage of activity relative to the control incubation at time 0.
Me-OH lidocaine formationa
nmol/min/mg
Addition
None
2EN
9EPh
MEGX formationb
nmol/min/mg
Time 0c
Time 5
Time 0
Time 5
8.1 6 0.3 (100)
4.5 6 0.2 (56)
7.1 6 0.3 (88)
7.6 6 0.7 (94)
3.8 6 0.3 (47)
2.3 6 0.1 (28)
61.4 6 2.7 (100)
38.7 6 0.3 (63)
39.1 6 0.6 (64)
59.5 6 1.9 (97)
26.1 6 2.6 (43)
24.4 6 0.62 (40)
a
2B2-specific product.
2B1 and other isozymes contribute to this product.
c
Time in the presence of 2EN or 9EPh before addition of lidocaine.
b
TABLE 5
Effect of 9EPh on testosterone metabolism in microsomes from untreated and PB-treated rats
Testosterone metabolism (nmol/min/mg protein)
Inducer
9EPh
None
None
2
1
PB
PB
2
1
6b/7a
2.11 6 0.13
1.32 6 0.10
(63)*
3.72 6 0.09
3.87 6 0.36
(104)
16-keto
b
ND
ND
2.51 6 0.13
0.22 6 0.1
(8.8)**
16a
16b
2a
2b
17a
2.61 6 0.19
2.44 6 0.06
(93)
4.84 6 0.12
1.88 6 0.05
(39)***
,0.0025
ND
1.22 6 0.08
1.16 6 0.05
(95)
0.1c
0.24 6 0.06
(240)
,0.0025
ND
0.85 6 0.06
0.88 6 0.09
(104)
1.05 6 0.15
1.12 6 0.18
(107)
3.46 6 0.33
1.00 6 0.18
(29)**
0.27 6 0.04
0.33 6 0.03
(122)
a
17-Oxidation, androstenedione formation.
ND, not detected.
c
Only one determination.
b
FIG. 3. SDS-PAGE and autographic analysis of rat liver microsomes after
incubation with radiolabeled arylacetylenes and NADPH.
Lanes 1– 4 are from rats treated with no inducer, PB, PYR, or bNF,
respectively, with either [3H]2EN (panel A) or [3H]9EPh (panel B). Experimental procedures were as described under Materials and Methods.
treated with no inducer, PB, PYR, or bNF, the band corresponding to
2B1 and 2B2 in the lanes containing microsomes from PB- or PYRtreated rats showed radiolabel bound to protein. Only a portion of the
gel is shown, but there was no radioactivity in other areas of the lanes.
When [3H]9EPh was incubated with these microsomes, there were
two bands that were covalently modified by radiolabeled substrate
(fig. 3B). One corresponds to that seen with [3H]2EN and migrates
with 2B1/2. The other band migrates at approximately 59 kDa and
was seen in the lanes containing the microsomes from male PB- and
PYR-treated rats. The same doublet pattern was observed with microsomes from PB-treated female rats (data not shown). No bands
were seen in the lanes containing microsomes from the clofibrate-,
PCN-, or bNF-treated rats with either [3H]2EN or [3H]9EPh (data not
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
Testosterone metabolism by liver microsomes from untreated or PB-treated rats in the presence or absence of 20 mM 9EPh. Analysis of metabolites was
as described under Materials and Methods. The values represent the mean 6 SD from three determinations. The numbers in parentheses represent the
percentage of testosterone activity relative to the incubations without 9EPh. * p , 0.05, ** p , 0.002, *** p , 0.001.
shown). The band corresponding to 2B1/2 was very faint in the lane
containing microsomes from isosafrole-treated rats with either
[3H]2EN or [3H]9EPh. Fig. 4 shows the results for microsomes from
PB-induced male rats incubated with [3H]9EPh, [3H]2EN, [3H]1EP,
or [3H,14C]4PbP. The doublet is seen after incubation with [3H]9EPh
or [3H]1EP. No bands were observed when [3H,14C]4PbP was incubated with any of the microsomal preparations (data not shown).
[3H]1EP showed the doublet only with microsomes from female and
male PB-treated rats (data not shown). In microsomes from both male
and female rats, [3H]2EN covalently labeled only 2B1, while
[3H]9EPh covalently labeled 2B1/2 and another protein whose induction pattern followed that of 2B1.
Discussion
The results of these studies establish that the size and shape of the
aromatic ring system and the placement of the acetylene group on the
ring system are important determinants for the inactivation of P450s
2B1, 2B4, or 2B11 in a reconstituted system containing reductase and
lipid. With each of the isozymes, the most effective inactivator at a
concentration of 7.5 mM was 9EPh. The effectiveness of 9EPh as an
inactivator of 2B1, 2B4, and 2B11 is further supported by the high
kinactivation values and low KI values. When the ethynyl group was
placed at either the 2 or 3 position of the phenanthryl backbone, little
or no inactivation was seen. 9EPh was also found to inactivate human
2B6. The specific metabolism at the nine position correlates with the
regiospecific metabolism of phenanthrene where 84% or 93% of the
metabolites can be accounted for by the 9,10-dihydrodiol with 2B1 or
2B6, respectively (20). The addition of 9EPh to PB microsomes
resulted in a time-dependent loss of the 2B2-specific product of
1247
INACTIVATION OF P450s BY ARYLALKYNES
FIG. 5. Sequence alignment of residues around the highly conserved
threonine residue (T*).
FIG. 4. SDS-PAGE and autoradiographic analysis of liver microsomes from
PB-treated rats after incubation with radiolabeled arylalkynes and NADPH.
Lanes 1– 4 are from incubations with [3H]9EPh, [3H]2EN, [3H]1EP, and
[3H,14C]4PbP. Experimental procedures were as described under Materials
and Methods.
addition, there was another band at approximately 59 kDa that was
labeled by [3H]9EPh and [3H]1EP in the PB- and PYR-induced
microsomes. The presence of this band followed the induction patterns observed for 2B1, strong induction by PB and a weaker induction by PYR (26). Given the specificity of 9EPh and the results from
the induction studies, it is possible that this is an as yet uncharacterized member of the rat 2B subfamily.
In attempting to define the catalytic specificity of the cytochrome
P450 enzymes, one approach that is used is selective inhibition of
individual isozymes (29). These acetylenic compounds may prove
themselves to be relatively specific inhibitors of cytochrome P450
enzymes. 9EPh, in particular, may prove to be a selective mechanismbased inactivator of 2B6 in human liver microsomes and thus aid in
the identification of 2B6-dependent drug metabolism.
Acknowledgments. We thank David A. Putt for preparation of rat
liver microsomes and the purification of 2B4 and Hsia-lien Lin for the
purification of reductase.
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Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
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