From the Inflammatory Diseases Research,
¶ Chemical Enzymology and § Chemical and Physical
Sciences, The DuPont Merck Research Laboratories,
Wilmington, Delaware 19880-0400
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ABSTRACT |
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The compound U0126
(1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) was
identified as an inhibitor of AP-1 transactivation in a cell-based
reporter assay. U0126 was also shown to inhibit endogenous promoters
containing AP-1 response elements but did not affect genes lacking an
AP-1 response element in their promoters. These effects of U0126 result
from direct inhibition of the mitogen-activated protein kinase kinase
family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and
-2, as U0126 shows little, if any, effect on the kinase activities of
protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6,
Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK
inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J.,
Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl.
Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and
PD098059 are noncompetitive inhibitors with respect to both MEK
substrates, ATP and ERK. We further demonstrate that the two compounds
bind to N3-S218E/S222D MEK in a mutually exclusive fashion,
suggesting that they may share a common or overlapping binding site(s).
Quantitative evaluation of the steady state kinetics of MEK inhibition
by these compounds reveals that U0126 has approximately 100-fold higher
affinity for
N3-S218E/S222D MEK than does PD098059. We further
tested the effects of these compounds on the activity of wild type MEK
isolated after activation from stimulated cells. Surprisingly, we
observe a significant diminution in affinity of both compounds for wild
type MEK as compared with the
N3-S218E/S222D mutant enzyme. These
results suggest that the affinity of both compounds is mediated by
subtle conformational differences between the two activated MEK forms.
The MEK affinity of U0126, its selectivity for MEK over other kinases,
and its cellular efficacy suggest that this compound will serve as a
powerful tool for in vitro and cellular investigations of
mitogen-activated protein kinase-mediated signal transduction.
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INTRODUCTION |
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Glucocorticoid hormones have been used clinically for over 40 years, and their pharmacological benefits, as well as detriments, have been extensively reviewed (1-5). Mechanistically, glucocorticoids have been shown to act by binding to intracellular glucocorticoid receptors (GR).1 The glucocorticoid·GR complex once formed migrates to the nucleus and interacts with specific target sequences known as glucocorticoid response elements (GREs) in gene promoters resulting in enhanced transcriptional activity. This feature of steroid hormone/receptor interaction is shared with a family of other hormones and effector molecules and their receptors which characterizes the steroid-thyroid receptor superfamily of ligand-activated transcription factors (6-8).
Although steroids have diverse effects on metabolism, their clinical
utility is derived from their potent anti-inflammatory and immune
modulatory properties that result from inhibition of cytokine, adhesion
molecule, and metalloproteinase gene expression (9-11). The ability of
steroid hormones to suppress transcription of key inflammatory and
immune response genes is mediated through a mechanism distinct from GRE
binding, however. Antagonism of the transcription factors AP-1 and
NF-B, which are important regulators of immune response genes, has
been demonstrated in numerous laboratories and is proposed to be the
primary mechanism for the anti-inflammatory and immune suppressive
effects of steroids (11). This functional antagonism or transrepression
of transcription factors by the GR is due to a direct protein/protein
interaction between the activated GR and the components of the AP-1 and
NF-
B complexes. These findings have led to the notion that the
anti-inflammatory and immune modulatory properties of steroids might be
due solely to protein/protein interactions between the GR and various
transcription factors, whereas the side effects of steroid hormones may
be mediated through classical GR/GRE interactions (12). Indeed, if this were the case, we envisioned that an agent that could selectively activate the GR to provide for functional antagonism of AP-1 and NF-
B without affecting GRE activation would be an ideal
anti-inflammatory agent. Therefore, we set out to identify agents that
could discriminate these GR activities using reporter gene assays in
transfected cells.
Herein, we report the results of these studies. Although we did not identify an agent that functionally antagonizes AP-1 gene transcription without affecting GRE transactivation through GR interaction, we did identify an agent that inhibited AP-1 independent of the GR. U0126 was identified as a cellular AP-1 antagonist. Mechanistically, U0126 did not prevent DNA binding by AP-1 rather it suppressed the up-regulation of c-Fos and c-Jun mRNA and protein levels in activated cells. Detailed investigation of the signaling cascades leading to c-Fos and c-Jun induction determined that U0126 was an inhibitor of the dual specificity kinase, MAP kinase kinase (MEK). This inhibition appeared to be selective as U0126 did not affect the kinase activity of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK3, MKK6, Cdk2, or Cdk4. Another compound, PD098059, has recently been reported to also function as a selective inhibitor of MEK activity in vitro and in cellular assays (13). In vitro steady state kinetic and equilibrium binding studies of both compounds with MEK reveal that their mode of inhibition is noncompetitive with respect to both substrates and that these two inhibitors share a common or overlapping binding site(s). Thus, both compounds are selective MAP kinase signaling cascade inhibitors and represent starting points for the identification and optimization of potent and pharmacologically useful MEK inhibitors.
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EXPERIMENTAL PROCEDURES |
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Methods
AP-1 Assay--
COS-7 cells were cultured at 5% CO2
at 37 °C in DMEM:F12 medium (Life Technologies, Inc.) plus 10%
fetal bovine serum (HyClone Labs, Inc.), 0.1 mM minimum
Eagle's medium non-essential amino acids (Life Technologies, Inc.), 1 mM minimum Eagle's medium sodium pyruvate solution (Life
Technologies, Inc.), and L-glutamine 500 mg/liter. The
cells were trypsinized and washed twice with Dulbecco's phosphate-buffered saline without CaCl2 and
MgCl2 and resuspended in Opti-MEM 1 medium (Life
Technologies, Inc.). COS-7 cells (8 × 106 cells) were
transiently transfected by the electroporation method (150 V, 500 microfarads, Bio-Rad electroporator) with either the AP-1 response
element (2× TRE-luciferase) or the glucocorticoid response element
(Pmam-neo-luciferase) plus human glucocorticoid receptor and
RSV--galactosidase (provided by M. Karin, University of California,
San Diego). Twenty-four hours later, cells were treated with 10 ng/ml
PMA (phorbol 12-myristate 13-acetate, Life Technologies, Inc.) with and
without 10 µM compound (in triplicate) in 1% final
concentration dimethyl sulfoxide (Me2SO) (Sigma). The cells
were incubated for an additional 24 h before harvesting for
luciferase activity and
-galactosidase activity. The cells were
lysed (Lysis Buffer, 25 mM Tris-P04, pH 7.6, 8 mM MgCl2, 1 mM EDTA, 1% Triton
X-100, 1% bovine serum albumin, 15% glycerol) for 10 min at room
temperature. Cell lysate (50 µl/well) was transferred to a 96-well
luminometer plate (Microlite 2, Dynatech Laboratories, Inc.) to which
100 µl of luciferase substrate reagent was added (20 mM
Tricine, 2.7 mM MgSO4, 0.1 mM EDTA,
1.1 mM MgCO3, 0.5 mM ATP, 0.27 mM coenzyme A, 0.47 mM luciferin, 33.3 mM dithiothreitol (DTT), pH 7.8). Luciferase activity was
immediately determined using a Dynatech ML3000 luminometer.
Chlorophenol red-
-D-galactopyranoside (Boehringer
Mannheim) 22 µl/well was added to the remaining cell lysate, and
-galactosidase activity was determined after approximately 1 h
at room temperature using a Molecular Devices UVmax
microplate kinetic reader at 570 nm. All AP-1 suppression data are
expressed relative to the PMA control. All GRE activation is compared
relative to dexamethasone.
RP5'-Luciferase Assay--
The effect of U0126 on a promoter
containing multiple inducible elements was examined in transient
transfection assays using a human renin promoter-luciferase reporter
construct, RP5'-luc. RP5'-luc was constructed by subcloning a
1.2-kilobase pair fragment (1245 to +36) of the human renin promoter
5'-untranslated region that contains both cAMP response elements and
AP-1 response elements (TRE) (14) into the NheI and
BglII sites of pGL2-basic (Promega, Madison, WI) and was
kindly provided by Dr. Gwen Wise (DuPont Merck). COS-7 cells were
transiently cotransfected with 20 µg of RP5'-luciferase and 2 µg of
human GR as described above. The transfected cells were plated in
96-well plates and incubated for 24 h at 5%
CO2, 37 °C before being treated with 10 ng/ml PMA or 1 mM N-6 2'-O-dibutyryladenosine 3',5' cyclic
monophosphate (Bucladesine; dibutyryl cyclic AMP, sodium salt) (Sigma) ± compound. After an additional 24-h incubation, the luciferase
activity was measured as described above.
Immunoprecipitations-- Jurkat cells were grown in RPMI 1640 medium (Life Technologies, Inc.) plus 10% fetal bovine serum (HyClone Labs, Inc.). Cells (1 × 107) were stimulated with 50 ng/ml PMA and 2 µg/ml PHA (phytohemaglutinin, Murex Biotech Ltd.) for 15 min at 37 °C. Drug-treated cells received U0126 at a final concentration of 10 µM in 0.1% Me2SO immediately prior to stimulation. Control cells received an equal amount of Me2SO. Cells were centrifuged at 1000 rpm, washed once with cold phosphate-buffered saline (PBS), resuspended in 1 ml of ice-cold RIPA buffer (1× PBS, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF)), and disrupted through a 21-gauge needle. Cellular debris was pelleted at 14,000 × g, and supernatant was incubated with 1 µg of the appropriate antibody (c-Raf-1(c-12), MEK-1(c-18), ERK-2(c-14) from Santa Cruz Biotechnology, Inc.) on a rocking platform at 4 °C. After 1 h, 40 µl of a protein-A agarose slurry was added, and tubes were rocked for another 2 h. Agarose beads were pelleted by centrifugation at 1500 rpm for 5 min, washed 3 times with RIPA buffer, and then once with 20 mM Hepes, pH 7.0, buffer.
Immune Complex Kinase Assays--
Immunoprecipitates were
resuspended in 25 µl of kinase assay buffer (20 mM Hepes,
pH 7.0, 5 mM 2-mercaptoethanol, 10 mM
MgCl2, 0.1 mg/ml bovine serum albumin), containing 1 µg
of His-MEK-1 (Santa Cruz Biotechnology), 5 µg of glutathione
S-transferase (GST)-(K71A)ERK-1 (Upstate Biotechnology,
Inc., eluted from agarose beads with 10 mM glutathione), or
3 µg of myelin basic protein (Upstate Biotechnology) for Raf, MEK,
and ERK kinase assays, respectively. Kinase reactions were initiated by
the addition of 10 µM ATP plus 10 µCi of
[-33P]ATP (NEN Life Science Products) and incubated at
25 °C for 30 min. Reactions were terminated by the addition of
Laemmli SDS sample buffer, boiled for 5 min, electrophoresed on a 10%
Tris glycine gel (Novex), dried, and analyzed using a Molecular
Dynamics PhosphorImager.
Western Blots-- COS-7 cells were seeded in 60-mm dishes at 30,000 cells/cm2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) plus 10% fetal bovine serum (HyClone Labs, Inc.). Cells were pretreated with compound in Me2SO (1% final concentration) for 15 min and then stimulated with 10 ng/ml PMA for 15 additional min. Cells were washed once with cold PBS, lysed with cold lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 50 µg/ml aprotinin, and 50 µg/ml leupeptin), scraped from the plate, and centrifuged. Supernatants were assayed for protein using Bio-Rad DC-protein assay kit. Protein samples (20 µg) were analyzed by SDS-polyacrylamide gel electrophoresis on 10% Tris-Tricine gels (Novex). Protein was electrotransferred to polyvinylidene difluoride membrane and probed with a polyclonal phosphospecific antibody against ERK (New England Biolabs). Detection of bands was carried out according to the manufacturer's protocol and analyzed on Molecular Dynamics Personal Densitometer.
For the experiments to determine the effects of U0126 on Fos and Jun protein expression, 3T3-type fibroblasts were plated in 100-mm dishes in DMEM, 10% FCS, and when they reached confluency were serum-starved for 72 h in DMEM, 0.5% FCS. The cultures were then stimulated with 50 ng/ml TPA, 10% FCS in the presence or absence of compound for various lengths of time. Compounds were added to give a final concentration of 0.1% Me2SO. At the end of the incubation period, nuclear extracts were prepared as follows. The cells were trypsinized, washed twice in cold PBS, and resuspended in 100 µl of Buffer 1 (10 mM Hepes, pH 7.9, 60 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, and 1 mM PMSF). After 5 min on ice, the samples were centrifuged at 4,000 rpm for 5 min, and the pellet was resuspended in 100 µl of Buffer 2 (10 mM Hepes, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF). The samples were centrifuged at 4,000 rpm for 5 min, and the pellet was resuspended in 100 µl of Buffer 3 (250 mM Tris, pH 7.8, 60 mM KCl, 1 mM DTT, 1 mM PMSF). After three freeze/thaw cycles, the samples were centrifuged at 9,000 rpm for 15 min, and the supernatant was saved as the nuclear extract. Ten µg of this extract was analyzed by Western blot as described above using antibodies specific for c-Jun, c-Fos, and SP-1 (Santa Cruz Biotechnology).Cloning and Expression of Kinases--
cDNA encoding a
constitutively active form of MEK-1 (N3-S218E/S222D) (N. Ahn,
University of Colorado, Boulder) was recloned into pGEX-2T to express
it as a GST fusion protein. Wild type forms of ERK1, p38, JNK1, and
MKK-3 were cloned from a mixture of mRNA from human Jurkat, HeLa,
and Raji cell lines by reverse transcriptase-polymerase chain reaction
(PCR). Wild type MEK-2 was cloned by PCR from a cDNA clone (K. Guan, University of Michigan, Ann Arbor). Wild type SEK-1 was cloned
from a mixture of mRNA made by reverse transcriptase-PCR from EL4,
7023, and WEHI265 mouse cell lines. Wild type MKK-6 was cloned from
human skeletal cDNA (CLONTECH). The catalytic
domain of MEKK, consisting of amino acids 353-673, was cloned from
mouse spleen cDNA (CLONTECH). A truncated form
of c-Jun consisting of amino acids 1-108 was cloned from the
full-length c-Jun cDNA. The wild type form of ATF-2 was cloned from
fetal brain cDNA (CLONTECH). The primers used
for the cloning described above were as follows: 5'MEK1,
CGATGGATCCCCCAAGAAGAAGCCGACG; 3'MEK1, CGATCTCGAGTTAGACGCCAGCAGCATG;
5'MEK2, CGATGGATCCAACCTGGTGGACCTGCAG; 3'MEK2,
CGATGAATTCTCACACGGCGGTGCGCGT; 5'ERK1, TTATGGATCCGCGGCGGCGGCGGCTCAG; 3'ERK1, CGATCTCGAGCTAGGGGGCCTCCAGCAC; 5'P38,
CGATGGATCCTCTCAGGAGAGGCCCACGTTC; 3'P38,
CGATCTCGAGTCAGGACTCCATCTCTTCTTG; 5'JNK1, CGATGGATCCAGCAGAAGCAAGCGTGAC; 3'JNK1, CGATCTCGAGTCACTGCTGCACCTGTGC; 5'MKK3,
GCATCTCGAGTCCAAGCCACCCGCACCCAAC; 3'MKK3,
GCATGAATTCCTATGAGTCTTCTCCCAGGATC; 5'SEK,
TAATGGATCCGCGGCTCCGAGCCCGAGC; 3'SEK,
CGATCTCGAGTCAGTCGACATACATGGG; 5'MKK6, GCATGGATCCTCTCAGTCGAAAGGCAAG; 3'MKK6, GCATCTCGAGTTAGTCTCCAAGAATCAG; 5'MEKK,
CGATGGATCCATGGCGATGTCAGCGTCTCAG; 3'MEKK,
CGATCTCGAGCTACCACGTGGTACGGAAGAC; 5'JUN,
CGATGGATCCACTGCAAAGATGGAAACG; 3'JUN,
CGATGAATTCTCACTCCTGCTCATCTGTCACGTTC; 5'ATF,
CGATGGATCCAAATTCAAGTTACATGTGAATTCTGCC; 3'ATF,CGATCTCGAGTCAAAGAGGGGATAAATCTAGAGG. The following mutants were then generated by site-directed mutagenesis using PCR: kinase inactive ERK-1(K71A), constitutively active MEK-2(S222E/S226D), MKK-3(S189E/T193D), and MKK-6(S207E/T211E). All of the above cDNAs were cloned into pGEX-2T (Amersham Pharmacia Biotech), and expressed as
GST fusion proteins in Escherichia coli BL-21 cells and
purified on glutathione beads according to the manufacturer's
directions.
In Vitro Kinase Assays--
The amount of immunoprecipitated
wild type MEK used in these assays was adjusted to give a similar
amount of activity units as obtained with 10 nM recombinant
MEK (see below). All other assays were performed with a recombinant,
constitutively activated mutant MEK-1 (N3-S218E/S222D) or
constitutively active MEK-2(S222E/S226D). Reaction velocities were
measured using a 96-well nitrocellulose filter apparatus (Millipore,
Bedford, MA) as described below. Unless otherwise noted, reactions were
carried out at an enzyme concentration of 10 nM, in 20 mM Hepes, 10 mM MgCl2, 5 mM
-mercaptoethanol, 0.1 mg/ml BSA, pH 7.4, at room
temperature. Reactions were initiated by the addition of
[
-33P]ATP into the premixed MEK/ERK/inhibitor reaction
mixture, and an aliquot of 100 µl was taken every 6 min and
transferred to the 96-well nitrocellulose membrane plate which had 50 mM EDTA to stop the reaction. The membrane plate was drawn
and washed 4 times with buffer (see above) under vacuum. Wells were
then filled with 30 µl of Microscint-20 (Packard, Meriden, CT)
scintillation fluid, and the radioactivity of
33P-phosphorylated ERK was counted with a Top Count
(Packard, Meriden, CT) scintillation counter. Velocities were obtained
from the slopes of radioactivity versus time plots.
Concentrations of ERK and ATP were 400 nM and 40 µM, respectively, unless otherwise indicated.
Equilibrium Binding Measurements--
To determine whether U0126
and PD098059 could displace one another from MEK, we performed
displacement experiments with 3H-labeled U0126 by
equilibrium dialysis (16). Dialysis was performed with a 10-kDa cut-off
Slide-A-Lyzer dialysis cassettes (Pierce). The cassette contained 0.5 ml of a 1 µM N3-S218E/S222D MEK solution (in reaction
buffer plus 400 µM ATP and 0.1 mg/ml BSA, see above). Mixtures were dialyzed at room temperature for 4 h against 100 ml
of 0.2 µM [3H]U0126 and varying
concentrations of PD098059, in the same buffer system. A dialysis time
of 4 h was chosen because experiments in which
[3H]U0126 was dialyzed against buffer only established
that this amount of time was sufficient to reach equilibrium. At the
end of dialysis the amount of [3H]U0126 inside and
outside the dialysis cassette was quantified by scintillation counting.
Control experiments were performed in the same fashion with BSA, but
not MEK, present in the dialysis cassette. Only low levels of compound
binding to BSA were detected, and the MEK data were corrected for this
nonspecific binding.
Northern Analysis-- An immortalized 3T3-type cell line derived from mouse embryonic fibroblasts was used for these studies (17). The cells were plated in 100-mm2 dishes in DMEM, 10% FCS and when they reached confluency were serum-starved for 48 h in DMEM, 0.5% FCS. The cultures were then stimulated with 100 ng/ml TPA, 10% FCS in the presence or absence of compound for 8 h. Compounds were added to give a final concentration of 0.1% Me2SO. Total RNA was isolated using RNA-ZolB (Tel-Test Inc., Friendswood, TX), and Northern blots were performed as described (17) using 10 µg of RNA/lane. The blots were probed with digoxigenin-labeled cDNA probes for full-length murine MMP-1, c-Fos, c-Jun, or glyceraldehyde-3-phosphate dehydrogenase according to the manufacturer's instructions (Boehringer Mannheim). For effects on IL-2 mRNA levels, Jurkat cells were stimulated with 100 ng/ml PMA and 1 µg/ml PHA in the presence or absence of various concentrations of U0126. RNA was isolated after 4 h, and IL-2 mRNA levels were determined by Northern analysis using a digoxigenin-labeled human IL-2 cDNA as a probe.
Electrophoretic Mobility Shift Analysis--
A 54-mer peptide
(BBRC) containing a consensus sequence from the DNA binding region and
leucine zipper dimerization motif of the Fos and Jun proteins was used.
This peptide has been shown to bind to the TRE consensus site (19). The
sequence of the AP-1 binding oligonucleotide used was
TTATAAAGCATGACTCAGACACCTCT, which contains the TRE
site from the collagenase promoter. Single-stranded oligonucleotides
were 5'-end-labeled with [-32P]ATP using T4 kinase,
purified over a Chromaspin-10 column
(CLONTECH) to remove unincorporated
[
-32P]ATP, and annealed. For each reaction, 1 × 104 cpm (approximately 5 nM) of radiolabeled
oligonucleotide was incubated with 15 nM BBRC peptide in a
final volume of 25 µl of 1× binding buffer for 25 min at 16 °C.
The binding buffer contained 12 mM Hepes, pH 7.9, 20 mM KCl, 1 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 12% glycerol, and 50 nM poly(dI-dC). Compounds were added to the peptide/binding
buffer mixture 10 min prior to the addition of radiolabeled
oligonucleotide in a 0.4% final Me2SO concentration. All
reactions were analyzed by polyacrylamide gel electrophoresis using 5%
gels in 0.5× TBE buffer.
Assay for Tyrosine Aminotransferase--
FU5 BDS.1 rat hepatoma
cells (20) kindly provided by Gary Firestone (University of California,
Berkley) were seeded at 50,000 cells/cm2 in 96-well
microtiter plates and incubated overnight at 5% CO2, 37 °C in Ham's F12:DMEM nutrient mix, 10% charcoal:dextran-treated FCS (HyClone Laboratories), 10 mM Hepes, 1 mM
sodium pyruvate, 1× nonessential amino acids, and 50 µg/ml
gentamycin (Life Technologies, Inc.). After 24 h, compounds were
added in Me2SO:media and incubated for an additional
24 h. The media were aspirated from the wells, and the cells were
washed with PBS (without calcium and magnesium) before lysis with 25 µl/well 20 mM CHAPS in 0.1 M
KHPO4 for 15 min. The cell lysates were centrifuged at
3,000 rpm for 15 min. Tyrosine aminotransferase activity was performed
on the cell lysates as described (21) with the following modifications
to accommodate analysis in a 96-well plate format. Lysates (15 µl)
were transferred to a 96-well plate to which 220 µl/well tyrosine
aminotransferase reagent was added (150 mM
L-tyrosine in 0.2 M
K2PO4, 5 mg/ml bovine serum albumin, 1.2 mM pyridoxal-5'-phosphate, pH 7.3). -Ketoglutarate (0.5 M in 0.2 M K2PO4, pH
7.3, 8 µl/well) was added to all wells except blanks and gently
mixed. The plates were covered with foil and incubated at room
temperature for 5 h. NaOH (10 N) was added to all
wells (15 µl/well) except blanks and mixed thoroughly.
-Ketoglutarate (0.5 M in 0.2 M
K2PO4, pH 7.3) was added to the blank wells
(8.0 µl/well) and mixed. The tyrosine aminotransferase activity was
measured at 340 nm using a Molecular Devices UVmax kinetic
plate reader.
Materials
Synthesis of U0126 (1,4-Diamino-2,3-dicyano-1,4-bis-(2-aminophenylthio)butadiene, U0125, and U0124-- U0124, U0125, and U0126 (22) were prepared by the addition of a thiol to tetracyanoethane as shown in Scheme 1. Tetracyanoethane (23) (6.5 g, 50 mmol) was dissolved in 20 ml of reagent grade acetone. In a separate flask ortho-aminobenzenethiol (25 g, 20 mmol) was dissolved in 50 ml of degassed 10% sodium hydroxide. The tetracyanoethane solution was added in a single portion to the sodium hydroxide solution, and the reaction mixture was vigorously stirred for 2 h, during which time an oil separated. The reaction mixture was allowed to stand for 1 h, and the solid which formed was filtered. The crude product was triturated with ethanol (2 × 50 ml) and recrystallized twice from ethanol (300 ml) and dried under vacuum, m.p. 163-165 °C. 1H NMR (CD3OD) d 7.48, d, J = 7 Hz, 2H, 7.35, t, J = 7 Hz, 2H, 6.94, d, J = 7 Hz, 2H, 6.79, t, J = 7 Hz, 2H, 3.70, q, J = 7 Hz, 2H (EtOH), 1.28, t, J = 7 Hz, 2H (EtOH). The proton NMRs for each compound were indicative of a symmetric structure. The configuration of the butadiene in U0126 was confirmed by single crystal x-ray analysis (see Scheme 1).
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RESULTS |
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Identification of a Novel Inhibitor of AP-1-driven Gene
Activation--
We screened a total of 40,000 compounds for their
ability to functionally antagonize AP-1-driven gene activation without
activating a GRE-driven gene. Our efforts identified U0126 whose
structure is shown in Fig. 1. U0126
suppressed AP-1-mediated gene activation in transient transfection
assays (Fig. 2) as described under
"Methods" with an IC50 = 0.96 ± 0.16 µM (n = 12). In these same assays, U0126
was without effect on a constitutively expressed cotransfected gene,
RSV-Gal, or constitutively expressed luciferase driven off the
cdc2 kinase promoter (data not shown). A further test of
U0126 specificity was performed in a number of other cell types using
either transfected or endogenous promoters. The results are summarized
in Table I. U0126 had no effect on
cAMP-dependent response element-driven luciferase
expression from the RP5' promoter and was also without effect on the
GRE-driven tyrosine aminotransferase gene. In contrast, U0126 did
inhibit TPA-dependent TRE-driven luciferase expression in
the RP5' reporter construct along with MMP-1, a gene known
to be highly dependent upon AP-1 for its inducible activity by multiple
stimuli (24).
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U0126 Inhibits c-Fos and c-Jun Induction in TPA-stimulated Cells-- To determine the mechanism of U0126 inhibition of AP-1-mediated gene activation, we initially studied the ability of U0126 to inhibit directly AP-1 DNA binding. U0126 did not inhibit binding of an AP-1 peptide (18) to a TRE-containing oligonucleotide probe by electrophoretic mobility shift analysis (data not shown). Based upon these findings, our next series of studies focused on the ability of U0126 to affect components that make up the AP-1 transcription factor complex. In fibroblasts treated with TPA/serum, U0126 suppressed the up-regulation of c-Fos and c-Jun proteins by 50-80%, as detected by Western analysis (Fig. 3A). This effect was mirrored in Northern blot analysis which showed that U0126 blocked c-Fos and c-Jun mRNA up-regulation (Fig. 3B). Similar effects were observed in Jurkat cells stimulated with PHA/PMA (data not shown). In contrast, dexamethasone and U0124, an inactive U0126 analog, had no effect on c-Fos and c-Jun protein or mRNA levels. Induction of the Fos family member, FosB, and the zinc finger-containing transcription factor, Egr-1, was also inhibited by U0126 (data not shown). Treatment with 10 µM U0126 did not affect the protein levels of the constitutively expressed transcription factors SP-1 (Fig. 3A) or JunD and Fra-1 (data not shown). These studies suggested that the decreased AP-1 activity observed in cells treated with U0126 resulted from a decrease in Fos and Jun protein levels.
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Ras Signaling and the MAP Kinase Cascade Provide the Target for U0126 Action-- The ability to inhibit TPA up-regulation of c-Fos and c-Jun protein suggested that U0126 blocked signaling events generated at the cell surface leading to AP-1 induction. One signaling pathway known to be involved in TPA-mediated AP-1 up-regulation is the Ras pathway. Activation of the Ras pathway by TPA in fibroblasts or PHA/PMA in Jurkat cells was also consistent with our data on U0126 blockade of AP-1 up-regulation in these two cell types.
To address the effects of U0126 on members of the Ras signaling cascade, immune complex kinase assays were performed on cell lysates from Jurkat cells treated with PMA/PHA as described under "Methods." The results of these studies showed that ERK activity was decreased in cells treated with U0126, whereas Raf and MEK activities were unaffected by the compound (data not shown). The effect was reversible as pretreatment of cells with compound followed by washout prior to activation showed no effect on ERK activity (data not shown). These results suggested that U0126 was either directly affecting ERK catalytic activity or was affecting the ability of MEK to activate ERK as part of the MAP kinase signaling cascade. Therefore, we evaluated the ability of U0126 to inhibit each member of the MAP kinase cascade directly in in vitro kinase assays using enzymes isolated by immunoprecipitation from PHA/PMA-stimulated Jurkat cells. As seen in Fig. 4, U0126 did not inhibit ERK or Raf kinase activity when added directly to kinase assays in vitro but did inhibit MEK enzymic activity in a concentration-dependent manner. These data demonstrate that U0126 is an inhibitor of MEK. Similar MEK inhibition was observed with U0125 although the potency was approximately 10-fold less than U0126. U0124 did not inhibit MEK at concentrations up to 100 µM. As expected, U0126 was able to inhibit MEK-dependent intracellular ERK phosphorylation in a concentration-dependent manner (Fig. 5), indicating that the in vitro effect seen with U0126 translated into cells treated with the compound. Furthermore, the data in Table II show that inhibition of MEK is selective, as only MEK-1 and MEK-2 are sensitive to U0126 from a panel of kinases tested.
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In Vitro MEK Assay-- To characterize inhibitor interactions with MEK most efficiently, we have optimized a high throughput assay based on MEK-mediated phosphorylation of a kinase-inactive mutant (K71A) form of ERK1 and subsequent capture of ERK on nitrocellulose filters. The experimental details of this assay are described above under "Experimental Procedures." Fig. 6 illustrates typical progress curves for ERK phosphorylation by MEK-1 at varying ERK concentrations and a fixed ATP concentration of 40 µM. As illustrated in this figure, the assay provides linear enzyme kinetics over a convenient time window, with minimal background signal. For all of the data reported here we have determined the reaction velocity from the slope of the linear least squares fit of the progress curve data, as exemplified in Fig. 6.
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Steady State Evaluation of MEK-1 Inhibition by U0126 and
PD098059--
The demonstration that U0126 was a MEK inhibitor
prompted us to characterize the inhibition kinetics. For comparative
purposes we also evaluated the kinetics of the only other known MEK
inhibitor, PD098059 (13). For initial evaluation of inhibitor potency, the reaction velocity was measured over a range of inhibitor
concentrations at a single, fixed set of substrate concentrations (400 nM ERK, 40 µM ATP), and the data were fit to
a Langmuir isotherm to determine the IC50 value for each
inhibitor (data not shown). From this analysis we found that the
apparent IC50 values of U0126 and PD098059 were ~0.07 and
10 µM, respectively, for the constitutively active recombinant MEK-1. Similar studies were performed to evaluate the
inhibition of wild type activated MEK that was obtained by immunoprecipitation from stimulated Jurkat cells. In this case the
IC50 of both compounds was significantly increased to 0.53 µM and >100 µM for U0126 and PD098059,
respectively. This effect was not due to some antibody-mediated
interference with inhibitor binding, since the IC50 of
U0126 for N3-S218E/S222D MEK that was similarly immunoprecipitated
from solution was not significantly affected (IC50 = 0.12 µM). Thus, there appears to be a large difference in
affinity of both compounds for these two activated forms of MEK.
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(Eq. 1) |
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(Eq. 2) |
Equilibrium Binding Studies-- The affinity differences notwithstanding, the similarity in inhibition modality for U0126 and PD098059 begs the question of whether these inhibitors share a common binding site on the enzyme. To address this issue we have had U0126 synthesized with tritium incorporation at the 5-position (see Fig. 1) and used this radiolabeled form of the inhibitor to perform equilibrium binding displacement studies. For these studies, the preformed MEK·ATP complex was used, based on the data in Table III, to provide maximal affinity of both compounds for the enzyme. [3H]U0126 was incubated with the MEK·ATP complex, and the ability of varying concentrations of PD098059 to displace the radiolabeled inhibitor was determined by equilibrium dialysis. The results of a typical displacement curve are illustrated in Fig. 9. The data clearly indicate that PD098059 is capable of displacing U0126 from the enzyme in a concentration-dependent fashion. Hence, the two inhibitors bind to MEK in a mutually exclusive manner. Similar displacement of [3H]U0126 by PD098059 was observed using free MEK in place of the MEK·ATP complex, but in this case a slightly higher concentration of PD098059 was required to effect half-maximal displacement (data not shown).
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DISCUSSION |
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The mitogen-activated protein kinase (MAPK) signaling pathways are involved in cellular events such as growth, differentiation, and stress responses (26-32). These pathways are linear kinase cascades in that MAP kinase kinase kinase phosphorylates and activates MAP kinase kinase (MAPKK) which phosphorylates and activates MAP kinase. To date, seven MAPK kinase homologs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK6, and MKK7) and four MAPK families (ERK1/2, JNK, p38, ERK5) have been identified. The MAPK kinase family members are unique in that they are dual specificity kinases, phosphorylating MAPKs on both threonine and tyrosine. Specifically, MEK1 and MEK2 share 80% amino acid sequence identity and appear to be functionally redundant in cells (33). The same is true for ERK1 and ERK2 (34). Activation of these pathways regulates the activity of a number of substrates through phosphorylation. These substrates include transcription factors such as p62TCF (Elk-1), c-Myc, ATF2 and the AP-1 components, c-Fos and c-Jun (35).
We identified U0126 as an inhibitor of the MAPK cascade leading to ERK1 and ERK2 activation. U0126 is a potent and specific inhibitor of the dual specificity kinases MEK1 and MEK2 both in in vitro enzymic assays as well as intracellularly where U0126 blocked phosphorylation and activation of ERK. Blockade of ERK activation would prevent downstream phosphorylation of a number of factors including p62TCF (Elk-1) preventing induction of c-Fos and c-Jun, components of the AP-1 complex. Discovery of U0126 was based upon blockage of AP-1 activation which relied upon this activation pathway.
We have demonstrated that U0126 is a potent (Ki
41-109 nM), noncompetitive inhibitor of N3-S218E/S222D
MEK-1. Steady state kinetic analysis indicates that PD098059 likewise
is a noncompetitive inhibitor of this enzyme. The noncompetitive nature
of U0126 and PD098059 inhibition of MEK may be a pharmacological
advantage for compounds of this type. Although undefined at present,
the binding site for these compounds on MEK is clearly distinct from those for the two substrate molecules, ATP and ERK. Hence, such compounds may not suffer large diminution in efficacy in cellular systems due to high concentrations of substrate (i.e. ATP)
or the existence of preformed binary enzyme-substrate complexes
(i.e. MEK·ERK or MEK·ATP). The results of equilibrium
binding studies indicate that the two compounds bind to
N3-S218E/S222D MEK-1 in a mutually exclusive fashion. The simplest
interpretation of this result is that the two compounds share a common
binding site on the enzyme. Although we consider it less likely, we
cannot rule out the possibility that the two compounds bind to
different sites on the protein that are somehow in anti-cooperative
communication with one another. Attempts are underway presently to
clarify this issue further.
For both U0126 and PD098059 we observed a significant (at least 7-fold)
reduction in affinity for wild type-activated MEK-1 as compared with
the N3-S218E/S222D MEK-1 mutant. In fact, the diminution in affinity
is so great for PD098059 that we were not able to achieve more than
~25% inhibition of wild type activated MEK-1 at the highest
concentration tested for this compound (100 µM). This
result is consistent with previous data for PD098059 reported by Dudley
et al. (13) and by Alessi et al. (36). In these
papers the authors demonstrated that PD098059 was an effective
inhibitor (IC50 ~10 µM) of the
ERK-phosphorylating activity of both nonactivated wild type
(i.e. basal activity) and of a partially activated mutant
MEK-GST fusion construct. In contrast, however, these authors found
that PD098059 was incapable of inhibiting wild type MEK-1 that had been
activated through Raf-mediated phosphorylation in stimulated Swiss 3T3
cells. From these data the authors suggested that PD098059 binds only
to the inactive (i.e. nonphosphorylated) form of MEK-1,
binding being mediated by the conformational change that attends
activation of the enzyme. Our present data lead to a slightly different
interpretation. The mutant MEK-1 that we have used (
N3-S218E/S222D)
has been shown to be fully activated and to stimulate AP-1-regulated
gene transcription when expressed in mammalian cells (37). Hence any
conformational transition that attends activation of MEK-1 must clearly
be mimicked in the
N3-S218E/S222D mutant. Both PD098059 and U0126
display potent binding to this activated MEK-1 form. Yet neither
compound appears to bind as well to wild type MEK-1 that has been
activated through Raf-mediated phosphorylation. We conclude, therefore,
that there must be subtle conformational differences between
N3-S218E/S222D MEK-1 and S218/S222-phosphorylated MEK-1 that mediate
binding of U0126 and PD098059 to the former, but not the latter, enzyme form, despite the fact that both forms display high ERK-phosphorylating and subsequent AP-1-activating ability. The potency of PD098059 as an
inhibitor of cellular AP-1-regulated gene transcription cannot be
accounted for by its inhibitory potency toward wild type-activated
MEK-1 (see above). This discrepancy was rationalized by Alessi et
al. (36) by demonstrating that PD098059 blocked Raf-mediated
activation (i.e. phosphorylation) of wild type MEK in cells,
by direct binding to nonactivated MEK. Thus, despite the ability of
this compound to inhibit activated and partially activated mutant forms
of MEK-1 in vitro, these authors concluded that the cellular
effects of PD098059 were mainly due to binding of this compound to
nonactivated MEK, leading to inhibition of MEK activation by Raf. This
mechanism clearly does not apply for U0126, since MEK phosphorylation
in stimulated Jurkat cells is unaffected by U0126 at concentrations
that completely block downstream ERK phosphorylation (Fig. 4). Thus,
whereas U0126 and PD098059 appear to bind to
N3-S218E/S222D MEK-1 in
similar fashions, and in fact may bind to a common site on the enzyme,
there remain some questions about the details of their individual modes
of action in a cellular context.
MEK inhibition has been shown to differentially affect signaling events
through a variety of cell surface receptors. Growth factor-mediated
proliferation and chemotactic responses are blocked by PD098059 (24,
38). In addition, mitogenic effects of insulin on DNA synthesis and
pp90Rsk activation are also inhibited by PD098059, whereas
insulin-mediated PHAS-I activation is not inhibited (39, 40).
Interestingly, PD098059 did not affect glucose uptake, glycogen
synthase activation, or lipogenesis in insulin-treated cells (39).
Similar differential effects of MEK inhibition on heterotrimeric
G-protein-coupled seven transmembrane receptor (7TM) signaling have
appeared. For example, PD098059 blocks angiotensin II-induced ERK
phosphorylation and thymidine incorporation into DNA in aortic smooth
muscle cells but has no effect on AII induction of phospholipase C,
phospholipase D, or pp70Rsk (41). Also, PD098059 inhibits
ERK phosphorylation in myocytes exposed to phenylephrine but fails to
block atrial natriuretic factor expression (42). In neutrophils, ERK
activation occurs in response to the agonists N-formyl
peptide, IL-8, C5a, and leukotriene B4 which is blocked
by PD098059 (43). In addition, PD098059 blocks neutrophil chemotaxis in
response to all agents but does not alter superoxide anion production.
Similarly, U0126 blocks ERK activation in N-formyl peptide-
and leukotriene B4-stimulated neutrophils but does not
impair NADPH-oxidase activity or bacterial cell
killing.2 Analogous
differential effects of PD098059 have recently been shown in T cells
stimulated with anti-CD3 monoclonal antibody in conjunction with either
PMA or anti-CD28 monoclonal antibody (44). The MEK inhibitor blocked
IL-2, tumor necrosis factor-, granulocyte-macrophage
colony-stimulating factor, interferon-
, and IL-6 production but
enhanced production of IL-4, IL-5, and IL-13. These findings obviate
the need to ensure that ERK activation is indeed coupled to the
cellular event or response attributed to MAP kinase pathway
activation.
Another intriguing differential effect observed with MEK inhibition involves thrombin-induced arachidonic acid release in endothelial cells and platelets. PD098059 inhibits ERK activation in both platelets and endothelial cells in response to thrombin treatment. In platelets, this effect does not alter arachidonic acid release nor affect aggregation (45). In contrast, PD098059 blocks prostacyclin I2 release in thrombin-stimulated endothelial cells while not affecting van Willebrand factor secretion (46). Thus, blockade of thrombin signaling events in different cell types leads to dramatically different results upon arachidonic acid metabolism. Our own data support these findings. U0126 is unable to block arachidonic acid release or thromboxane synthesis in thrombin-stimulated platelets, whereas U0126 is able to block arachidonic acid release along with prostaglandin and leukotriene synthesis in keratinocytes stimulated with a variety of agents.3 Thus, the putative effector target, cytosolic phospholipase A2, is insensitive to MEK inhibition in platelets while showing sensitivity and a blunting of response in other cell types. These effects presumably reflect cytosolic phospholipase A2 activation by non-ERK mechanisms in platelets, whereas ERK is the activating kinase in other cell types (47).
The proximal involvement of Ras in the activation of the ERK pathway
suggests that MEK inhibition might show efficacy in cancers where
oncogenic RAS is a determinant in the cancer phenotype. Indeed,
PD098059 (36) as well as U0126 are able to impede the growth of
Ras-transformed cells in soft agar, even though these compounds show
minimal effects on cell growth under normal culture conditions.
PD098059 has also been shown to reduce urokinase secretion controlled
by growth factors such as epidermal growth factor, transforming growth
factor-, and fibroblast growth factor in an autocrine fashion in the
squamous cell carcinoma cell lines UM-SCC-1 and MDA-TV-138 (48).
In vitro invasiveness of UM-SCC-1 cells through an
extracellular matrix-coated porous filter was blocked by PD098059,
although the cellular proliferation rate was not affected. These
results suggest that control of the tumor invasive phenotype by MEK
inhibition may be a possibility.
Functional antagonism of AP-1 activity without activation of GRE-mediated gene activation was the original intent of our screening effort. The notion that we could find a compound to interact with the GR and preferentially inhibit AP-1 was based upon the finding that steroids suppress gene transcription through this interaction. Although we were not successful in finding an agent with the ability to separate GR gene activation from gene suppression, identification of such compounds using other members of the steroid-thyroid receptor superfamily have been reported. By using the retinoic acid receptor as the vehicle to functionally antagonize AP-1-mediated gene activation, a number of compounds have been described that perform this function without activating retinoic acid response element-driven genes (49). U0126 and MEK inhibitors in general seem to accomplish the same result, although in a mechanistically distinct fashion.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Karin (University of California, San Diego) for providing AP-1 and glucocorticoid receptor constructs and reporter genes used in these studies. We also thank Dr. Sarah Cox and Katie Burton (DuPont Merck Pharmaceutical Co.) for performing the Cdk2 and Cdk4 kinase assays and Gwendolyn Wise for providing the RP5'-luciferase construct.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Inflammatory
Diseases Research, The DuPont Merck Research Laboratories, P. O. Box
80400, Wilmington, DE 19880-0400. Tel.: 302-695-7110; Fax: 302-695-9401; E-mail: James.M.Trzaskos{at}dupontmerck.com.
1 The abbreviations used are: GR, glucocorticoid receptors; GRE, glucocorticoid response element(s); MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; MEKK, MEK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTT, dithiothreitol; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; TRE, TPA response element; GST, glutathione S-transferase; FCS, fetal calf serum; TPA, 12-O-tetradecanoylphorbol-13-acetate; PCR, polymerase chain reaction; PHA, phytohemaglutinin; IL, interleukin; ATF, activating transcription factor; BSA, bovine serum albumin, CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2 B. Schnyder, B. Car, and J. M. Trzaskos, unpublished observations.
3 G. Fisher and J. M. Trzaskos, unpublished observations.
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REFERENCES |
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