From the Department of Immunology, Inflammation and
Pulmonary Drug Discovery, Bristol-Myers Squibb Pharmaceutical
Research Institute, Princeton, New Jersey 08543 and the Departments of
¶ Preclinical Candidate Optimization and
Discovery
Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute,
Wallingford, Connecticut 06492
Received for publication, September 20, 2002
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ABSTRACT |
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The signal-inducible
phosphorylation of serines 32 and 36 of I The expression of many pro-inflammatory genes is regulated by the
transcriptional activator NF- A high molecular mass (500-900 kDa) multisubunit I Given the importance of NF- Materials--
GST-I Synthesis of BMS-345541
(4(2'-Aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline)--
4,5-Dihydro-1,8-dimethylimidazo(1,2-a)quinoxalin-4-one-2-carboxylic
acid, prepared by the procedure of Treiber et al. (20) was
suspended in diphenyl ether (124 mmol in 400 ml) and refluxed at
260 °C for 2 h. After the suspension cooled down to room
temperature, hexanes (C6H14; 500 ml) were added
to further precipitate the product. The solid was filtered, giving
4,5-dihydro-1,8-dimethylimidazo(1,2-a)quinoxalin-4-one as a white solid
(29.6 g). 1H NMR (500 MHz; hexadeuterio-dimethyl
sulfoxide, d6-Me2SO) Enzyme Assays--
Assays measuring the enzyme-catalyzed
phosphorylation of GST-I
Because the above-described SDS-PAGE assay is not sufficiently precise
for kinetic analyses of the IKK enzymes, assays for enzyme kinetic
studies instead measured the enzyme-catalyzed phosphorylation of a
17-amino acid peptide corresponding to amino acids 26-42 of I Protein Phosphorylation in Stimulated Cells--
To measure the
effect of the IKK inhibitor on I
In a similar manner, the stimulated phosphorylation of Ser-73 of c-Jun
was detected with immunoblotting using anti-phospho-c-Jun (Cell
Signaling) after a 15-min stimulation with 25 µg/ml anisomycin. The
levels of phosphorylated c-Jun were increased 5-fold when comparing
stimulated versus unstimulated cells. When measuring the
effect on stimulated phosphorylation of STAT3, human lung epithelial
H292 cells were resuspended at 2-5 × 106/ml
following trypsinization and plated in a 12-well plate, 1 ml/well in
serum-free medium for 18 h. Medium was removed and replenished
with 0.45 ml of fresh serum-free medium. Inhibitor was added and
incubated for 60 min at 37 °C followed by stimulation with EGF (100 ng/ml) for 5 min. Medium was extracted, and the cells were lysed and
analyzed in a similar manner as above using anti-phospho-STAT3-specific
antisera (Cell Signaling) for immunoblot analysis. The levels of
phosphorylated STAT3 were increased 200-fold when comparing stimulated
versus unstimulated cells.
MAPKAP K2 Activation in THP-1 Cells--
To measure the effect
of BMS-345541 on LPS-induced MAPKAP K2 activation in THP-1 cells, the
general procedure of Kumar et al. was followed (22).
Briefly, THP-1 cells in RPMI 1640 containing 10% fetal calf serum were
preincubated with BMS-345541 for 1 h prior to a 30-min stimulation
with LPS. The cells were then lysed and immunoprecipitated with
anti-MAPKAP K2, and the amount of enzyme activation was quantitated by
measuring the MAPKAP K2-catalyzed phosphorylation of hsp27 using
[33P]ATP as substrate. When cells were stimulated in this
manner, MAPKAP K2 activity was found to be stimulated 15-fold over
unstimulated cells.
Cytokine Production in THP-1 Cells--
THP-1 cells in 96-well
plates (2.5 × 105 cells/well in 180 µl of RPMI
1640/10% fetal bovine serum) were treated with BMS-345541 or no test
material for 1 h prior to stimulation with 100 ng/ml LPS.
Measurements of the amount of the cytokines IL-1 Pharmacokinetics in Mice--
BMS-345541 was administered either
by intravenous tail vein injection or by peroral gavage to groups of
three 18-22-g female BALB/c mice (Harlan). BMS-345541 was formulated
as a 2 mg/ml solution in 3% Tween 80, water. Mice received either a 2 mg/kg (1 ml/kg) intravenous bolus or a 10 mg/kg (5 ml/kg) peroral
gavage. Whole blood samples were taken from individual mice by orbital
bleed and cardiac puncture at 0, 0.05, 0.25, 0.5, 1.0, 3.0, 6.0, and 8.0 h after dosing. Whole blood was centrifuged at 20 × 103 × g for 5 min. Serum was stored at
Serum samples were treated with two volumes of methanol containing 1 µg/ml of an appropriate internal standard. After centrifugation to
remove precipitated proteins, a 10-µl portion of the clear supernatant was analyzed by liquid chromatography/tandem MS. The HPLC
was interfaced to a Micromass Micro Quattro liquid chromatography tandem mass spectrometer equipped with an electrospray interface. Nitrogen was used as the nebulizing and de-solvation gas at flow rates of 150 liter/h for nebulization and 900 liter/h for de-solvation. The de-solvation temperature was 300 °C, and the source temperature was 150 °C. Data acquisition was selected via reaction monitoring. Ions representing the (M+H)+ species for both the analyte
and internal standard were selected in MS1 and dissociated
collisionally with argon at a pressure of 2 × 10 LPS-induced Serum TNF Using an assay measuring the IKK-2-catalyzed phosphorylation of
GST-IB
is critical in
regulating the subsequent ubiquitination and proteolysis of I
B
,
which then releases NF-
B to promote gene transcription. The
multisubunit I
B kinase responsible for this phosphorylation contains
two catalytic subunits, termed I
B kinase (IKK)-1 and IKK-2.
BMS-345541
(4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of
IKK (IKK-2 IC50 = 0.3 µM, IKK-1
IC50 = 4 µM). The compound failed to inhibit
a panel of 15 other kinases and selectively inhibited the stimulated
phosphorylation of I
B
in cells (IC50 = 4 µM) while failing to affect c-Jun and STAT3
phosphorylation, as well as mitogen-activated protein kinase-activated
protein kinase 2 activation in cells. Consistent with the role
of IKK/NF-
B in the regulation of cytokine transcription, BMS-345541
inhibited lipopolysaccharide-stimulated tumor necrosis factor
,
interleukin-1
, interleukin-8, and interleukin-6 in THP-1
cells with IC50 values in the 1- to 5-µM
range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541
showed a non-linear relationship indicating non-Michaelis-Menten
kinetic binding, the use of multiple inhibition analyses indicated that
BMS-345541 binds in a mutually exclusive manner with respect to a
peptide inhibitor corresponding to amino acids 26-42 of I
B
with
Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive
manner with respect to ADP. The opposite results were obtained when
studying the binding to IKK-1. A binding model is proposed in which
BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which
then affects the active sites of the subunits differently. BMS-345541
was also shown to have excellent pharmacokinetics in mice, and peroral
administration showed the compound to dose-dependently
inhibit the production of serum tumor necrosis factor
following
intraperitoneal challenge with lipopolysaccharide. Thus, the compound
is effective against NF-
B activation in mice and represents an
important tool for investigating the role of IKK in disease models.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B. Genes dependent on activation of
NF-
B include the cytokines
TNF
,1 IL-6, IL-8, and
IL-1
; the adhesion molecules E-selectin, ICAM-1, and VCAM-1; and the
enzymes nitric-oxide synthase and COX-2 (for reviews see Refs. 1 and
2). NF-
B normally resides in the cytoplasm of unstimulated cells as
an inactive complex with a member of the I
B inhibitory protein
family. This class of protein includes I
B
, I
B
, and
I
B
, which all contain ankyrin repeats necessary to form a complex
with NF-
B (for a review see Ref. 3). In the case of I
B
, the
most carefully studied member of this class, stimulation of cells with
agents that activate NF-
B-dependent gene transcription
results in the phosphorylation of I
B
at Ser-32 and Ser-36 (4).
This is critical for subsequent ubiquitination and proteolysis of
I
B
, which then leaves NF-
B free to translocate to the nucleus
and promote gene transcription (5-7). Indeed, a mutant in which both
Ser-32 and Ser-36 have been changed to alanine prevents signal-induced
activation of NF-
B and results in an I
B
that is not
phosphorylated, ubiquitinated, or digested proteolytically (7).
Analogous serines have been identified in both I
B
and I
B
,
and phosphorylation at these residues appears to regulate the
proteolytic degradation of these proteins by a mechanism similar to
that of I
B
(8, 9).
B kinase
(termed IKK) that phosphorylates at Ser-32 and Ser-36 of I
B
has
been isolated from HeLa cells (10-12). Two catalytic subunits (termed
IKK-1 and IKK-2) of IKK have recently been identified, cloned, and
shown to be widely expressed in human tissues (12-17). The use of
gene-targeting experiments have clearly shown that all known
proinflammatory stimuli, including cytokines, viruses, and
lipopolysaccharide (LPS) require the IKK-2 subunit for NF-
B activation (for a review see Ref. 18). Although the role of IKK-1 in
NF-
B activation is still unclear, recent evidence suggests that
IKK-1 may only play a role in response to certain stimuli (e.g. RANK-ligand and Blys/BAFF) and in select cells such as
mammary epithelial cells and B lymphocytes (19).
B in regulating inflammatory processes,
the identification of selective IKK inhibitors has received considerable interest. We report here the identification of BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) as a
highly selective inhibitor of IKK that inhibits
NF-
B-dependent transcription of pro-inflammatory
cytokines both in vitro and in vivo. The compound
appears to bind to an unidentified allosteric binding site of the IKK
catalytic subunits.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
was purchased from Santa Cruz
Biotechnology, and [33P]ATP (1000 Ci/mmol) was purchased
from Amersham Biosciences, and ADP, EGF, and staurosporine were
obtained from Sigma-Aldrich. A peptide substrate corresponding to amino
acids 26-42 of I
B
(LDDRHDSGLDSMKDEEY; N-terminal acetylated and
C-terminal amidated), along with a peptide inhibitor that corresponds
to the same amino acids except with Ser-32 and Ser-36 changed to
aspartates (LDDRHDDGLDDMKDEEY; N-terminal acetylated and C-terminal
amidated; see Ref. 20), was synthesized by Research Genetics
(Huntsville, AL). GST-tagged IKK-1, IKK-2, and IKK-
were expressed
in High Five cells and purified as reported previously (20). LPS
(Salmonella typhosa) was obtained from Sigma. Proteosome
inhibitor I and anisomycin were obtained from Calbiochem.
11.7 (s, 1H),
7.91 (s, 1H), 7.31 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 2.81 (s, 3H), 2.41 (s, 3H); MS
(electrospray ionization), m/z 214.13 ((M+H)+; calculated for
C12H11N3O, 213.09). A mixture of
4,5-dihydro-1,8-dimethylimidazo(1,2-a)quinoxalin-4-one (29.6 g, 139 mmol) and N,N-diethylaniline (PhNEt2;
45 ml) was refluxed in phosphorus oxychloride (POCl3; 250 ml) for 1 h. The solvent was evaporated under vacuum; the residue
was diluted with CHCl3 (1000 ml), followed by careful
neutralization with cold saturated Na2CO3
solution. The aqueous layer was further extracted with
CHCl3. The combined organic layer was dried over
MgSO4, filtered, and concentrated. Flash chromatography
using ethyl acetate and hexanes (EtOAc/hexanes, 60/40) provided
4-chloro-1,8-dimethylimidazo(1,2-a)quinoxaline as a white solid (25 g).
1H NMR (500 MHz; CDCl3)
8.07 (s, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 0.7 Hz, 1H), 7.41 (dd, J = 8.4, 1.2 Hz, 1 H), 2.97 (s, 3H),
2.59 (s, 3H); MS (electrospray ionization), m/z 232.04 ((M+H)+; calculated for
C12H10CIN3, 231.06). A solution of
4-chloro-1,8-dimethylimidazo(1,2-a) quinoxaline (4.8 g, 21 mmol) in
ethylenediamine (300 ml) was heated at 60 °C under nitrogen gas
(N2) for 16 h. The solvent was evaporated under
vacuum. The residue was diluted with EtOAc (200 ml), washed with
saturated Na2CO3 and brine, dried over
MgSO4, filtered, and concentrated. Flash chromatography
using methanol (MeOH) provided a solid, which was then dissolved with
CHCl3 and filtered to remove any dissolved silica gel. The
filtrate was concentrated under vacuum, providing
4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline as a white
solid (4.67 g). 1H NMR (500 MHz, CDCl3)
7.88 (s, 1H), 7.63 (d, J = 8.3 Hz, 1 H), 7.26 (s, 1 H),
7.22 (d, J = 8.3 Hz, 1H), 6.31 (s, 1H), 3.63 (apparent
q, J = 5.9 Hz, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.88 (s, 3H), 1.38 (br, 2H); MS (electrospray ionization),
m/z 256.15 ((M+H)+; calculated for
C14H17N5, 255.15).
4(2'-Aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline (4.67 g,
18.3 mmol) was dissolved with aqueous hydrochloric acid, HCl (1.0 N,
18.3 ml), and water, H2O (50 ml), at room temperature. Subsequent removal of water using a lyophilizer provided the
hydrochloride salt as a white solid (5.22 g).
B
were performed by adding enzyme (IKK-2,
IKK-1, or IKK-
, typically to a final concentration of 0.5 µg/ml)
at 30 °C to solutions of 100 µg/ml GST-I
B
and 5 µM [33P]ATP in 40 mM
Tris·HCl, pH 7.5, containing 4 mM MgCl2, 34 mM sodium phosphate, 3 mM NaCl, 0.6 mM potassium phosphate, 1 mM KCl, 1 mM dithiothreitol, 3% (w/v) glycerol, and 250 µg/ml
bovine serum albumin. The specific activity of [33P]ATP
used in the assay was 100 Ci/mmol. After 5 min, the kinase reactions
were stopped by the addition of 2× Laemmli sample buffer and
heat-treated at 90 °C for 1 min. The samples were then loaded on to
NuPAGE 10% BisTris gels (Novex, San Diego, CA). After completion of
SDS-PAGE, gels were dried on a slab gel dryer. The bands were then
detected using a 445Si PhosphorImager (Molecular Dynamics), and the
radioactivity was quantified using ImageQuant software. Under these
conditions, the degree of phosphorylation of GST-I
B
was linear
with time and concentration of enzyme.
B
as described previously (21, 28). In this assay, either IKK-1 or IKK-2
was added at 30 °C to solutions containing peptide and
[33P]ATP (1000 Ci/mmol) in 50 mM Tris·HCl,
5 mM MgCl2 at pH 8. After 60 min, the kinase
reactions were quenched by addition of EDTA to a concentration of 10 mM. HPLC analysis was performed as described previously
(22), and the amount of IKK-catalyzed incorporation of 33P
into each peptide was quantitated by liquid scintillation counting. Under these conditions, the degree of phosphorylation of peptide substrate was linear with time and concentration of enzyme.
B
phosphorylation in cells, 1-ml
aliquots of THP-1 cells suspended at 2 × 106/ml in
RPMI 1640 supplemented with 10% fetal bovine serum were preincubated
for 60 min with inhibitor at 37 °C. To prevent degradation of
phosphorylated I
B
upon stimulation, proteosome inhibitor I was
also added to a final concentration of 1 µM during this preincubation. Cells were then stimulated for 5 min with TNF
(100 ng/ml) and pelleted by rapid centrifugation, and the pellets were
solubilized using 150 µl of ice-cold lysis buffer (0.5% Nonidet P-40
in 40 mM Tris·HCl, pH 7.5, containing 200 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM
-glycerophosphate, 1 mM NaF, 1 mM
Na3VO4, and 1 µM okadaic acid).
SDS-PAGE and anti-phospho-I
B
immunoblot analysis were used to
visualize the amount of phospho-I
B
produced. Quantitation of the
bands was accomplished using luminescence capture (Roche Molecular
Biochemicals Lumi-Imager) with data analysis using Lumi-Analyst
software. Under these conditions, the levels of phosphorylated I
B
were increased 17-fold upon stimulation of the cells.
, TNF
, and IL-8
in the supernatants were made after 6 h of LPS stimulation and
after 24 h for IL-6 production. Specific enzyme immunoassay kits
from Pharmingen (OptEIA) were used in quantitating each cytokine. Under
these conditions, IL-1
levels increased from 26 pg/ml in unstimulated cells to 2435 pg/ml in LPS-stimulated cells. TNF
levels
increased from 51 to 905 pg/ml, IL-8 levels increased from 12 to 92 pg/ml, and IL-6 levels increased from <5 to 272 pg/ml.
20 °C until analysis.
3
torr to form specific product ions, which were subsequently
monitored by MS2. The transition monitored for BMS-345541 was
m/z 256
239 and m/z 326
270 for internal standard. Cone voltage and collision energy were
optimized at 40 V and 20 eV, respectively. The retention time for
BMS-345541 was 2.04 min. The 6-point standard curve ranged from 1 to
4000 ng/ml and was fitted with a quadratic regression weighted by
reciprocal concentration (1/X). Standards were analyzed in
duplicate. Quality control samples, prepared in blank plasma, at three
concentrations within the range of the calibration curve were also
analyzed in duplicate with each plasma analytical set. For this
compound, the predicted concentrations of at least two thirds of the
plasma quality controls were generally within ±15% of nominal
concentration, indicating acceptable assay performance.
in Mice--
The general procedure of
Ghezzi et al. (23) was followed. Briefly, 0.2 ml of an
aqueous solution of BMS-345541 was administered by peroral gavage to
6-8-week-old female BALB/c mice (Harlan) 1 h prior to a challenge
with an intraperitoneal dose of 1 µg of Escherichia
coli LPS (from strain O111:B4; Sigma) in 500 µl of
phosphate-buffered saline. Blood was collected from mice 90 min after
LPS challenge, and the levels of TNF
in the serum were measured by
enzyme immunoassay (R&D Systems). Inhibition by BMS-345541 was
calculated from control animals that received LPS challenge but no
BMS-345541 (vehicle only). Mice receiving no LPS challenge gave no
detectable serum TNF
.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
, BMS-345541 was identified as an inhibitor of the enzyme.
As shown in Fig. 1, BMS-345541
dose-dependently inhibited IKK-2 with an IC50
value of ~0.3 µM. The compound was considerably less
potent against IKK-1 with an IC50 value of 4 µM and did not inhibit the related kinase IKK-
. When
tested against a panel of both serine/threonine and tyrosine kinases
such as human recombinant protein kinase C
, protein kinase C
,
protein kinase C
, protein kinase C
, protein kinase A, Her1, Her2,
p38
, MAPKAP K2, JAK3, EMT kinase, lck, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase,
extracellular signal-regulated kinase 1/2, and insulin-like growth
factor-1R, BMS-345541 (Structure TI)
failed to inhibit any kinase at concentrations as high as 100 µM (results not
shown).
View larger version (13K):
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Fig. 1.
Dose-dependent inhibition of
IKK-2 and IKK-1 by BMS-345541. Inhibition of IKK-2 (open
circles), IKK-1 (closed circles), and IKK-
(closed squares) assayed using GST-tagged I
B
as
substrate. The data represent the average of triplicate measurements.
The error bars represent the standard deviation. See
"Experimental Procedures" for details.
View larger version (8K):
[in a new window]
Structure I.
BMS-345541.
When assayed in THP-1 monocytic cells, the compound
dose-dependently inhibited the TNF-stimulated
phosphorylation of I
B
with an IC50 value of ~4
µM (Fig. 2A).
The compound was not cytotoxic in this concentration range as
determined by trypan blue exclusion (results not shown). Consistent
with the compound being a highly selective inhibitor of IKK, BMS-345541
at concentrations as high as 100 µM failed to block both
the anisomycin-stimulated phosphorylation of c-Jun and LPS-stimulated
activation of MAPKAP K2 in THP-1 cells, as well as the EGF-stimulated
phosphorylation of STAT3 in H292 cells.
|
As shown in Fig. 2B, the compound was also effective in
THP-1 cells at inhibiting the stimulated production of a number of cytokines from THP-1 cells including TNF, IL-1
, IL-8, and IL-6. The IC50 against cytokine production in these cells fell in
the range of 1-5 µM, which matched the inhibition seen
against I
B
phosphorylation. This observation is consistent with
both the essential requirement of NF-
B in promoting the
transcription of these cytokines and the important role of IKK in
regulating NF-
B activation.
Enzyme Kinetic Analysis of Inhibition--
To investigate the
binding mechanism of BMS-345541 to IKK-2, a simple analysis of whether
the inhibitor was competitive with respect to ATP or peptide substrate
was undertaken. However, non-linear fits of the data to both
competitive and mixed non-competitive inhibition were very poor, and a
definitive answer could not be obtained (results not shown). The reason
for these poor fits is that the inhibitor shows non-linear binding
kinetics. Indeed, Fig. 3 shows that
BMS-345541 does not give a linear Dixon plot of [Inhibitor]
versus rate1. For an inhibitor that follows
simple Michaelis-Menten kinetics, a linear correlation would be
expected for competitive, non-competitive, mixed, and uncompetitive
inhibitors. This non-linearity does not appear to result from
solubility problems, because the compound is very water-soluble.
Moreover, the Dixon plot of the inhibition of IKK-1 by BMS-345541 is
more linear even at concentrations nearly 100-fold higher (results not
shown), which indicates the effect is dependent on IKK-2.
|
As an alternative way to investigate the binding mechanism, the effect
of BMS-345541 on the inhibition kinetics of ADP and a peptide inhibitor
corresponding to amino acids 26-42 of IB
with Ser-32 and Ser-36
changed to aspartates was determined. Not surprisingly, ADP has been
shown to be a competitive inhibitor with respect to the ATP substrate
(24), and the peptide inhibitor is a competitive inhibitor with respect
to peptide substrate (20). As shown in Fig.
4A, a Dixon plot of the
inhibition of IKK-2 by ADP at different fixed concentrations of peptide
inhibitor gave non-parallel relationships. At each fixed concentration
of inhibitor, the value of kinetic constant
A,2 which is
directly proportional to the slope of the Dixon plots, were calculated
by a non-linear fit of the data to be 775 ± 75, 1952 ± 229, 3286 ± 212, and 4564 ± 347 s·counts
1·M
1 at 0, 157, 314, and
471 µm peptide inhibitor, respectively. Non-parallel lines
(i.e. increasing values of A) in this type of
multiple inhibition analysis is the hallmark of non-mutually exclusive
inhibitors and means that both inhibitors can bind to the enzyme
simultaneously (25). This conclusion is expected, because the ATP and
peptide binding sites of kinases are distinct regions of the active
site, which allows both substrates (or inhibitors in the case of ADP and peptide inhibitor) to be bound simultaneously. Indeed, both ATP and
peptide substrates are required to bind simultaneously for kinases to
function as catalysts.
|
A similar relationship exists between BMS-345541 and ADP as shown in
Fig. 4B. Indeed, the values for kinetic constant
A at different fixed concentrations of BMS-345541 were found
to be 853 ± 88, 1180 ± 203, 1287 ± 144, 1617 ±
104, and 2021 ± 207 s·counts1·M
1 at 0, 0.5, 1, 1.5, and 2 µM BMS-345541, respectively. The resulting non-parallel lines indicate that BMS-345541 and ADP bind to different sites on IKK-2. Therefore, BMS-345541 does not bind to the ATP binding site.
Confirmatory evidence in support of this conclusion comes from the
third permutation of this multiple inhibition analysis: the effect of
BMS-345541 on the inhibition of peptide substrate. As shown in Fig.
4C, the Dixon plots gave essentially parallel relationships,
which reflects the relatively unchanging values of kinetic constant
A at different fixed concentrations of BMS-345541: 103 ± 7, 101 ± 2, 99 ± 4, 109 ± 2, and 119 ± 6 s·counts1·M
1 at 0, 0.3, 0.7, 1.3, and 2 µM BMS-345541, respectively. Parallel relationships in this type of Dixon plot are indicative of mutually exclusive inhibitors in which both BMS-345541 and peptide inhibitor cannot bind to IKK-2 simultaneously (26). Although this may suggest
that BMS-345541 and peptide inhibitor/substrate bind at the same site,
mutually exclusive inhibition also can result from inhibitors that bind
at different sites but in a mutually exclusive manner (e.g.
occupation at one site induces a protein conformational change that
affects and prevents binding at the other site).
With IKK-1, ADP and peptide inhibitor showed the same relationship as
seen with IKK-2. That is, non-parallel Dixon plots of the inhibition of
ADP at different fixed concentrations of peptide inhibitor (see Fig.
5A) with the values for
kinetic constant A determined to be 345 ± 13, 1300 ± 74, 2200 ± 81, and 2771 ± 114 s·counts1·M
1 at 0, 423, 471, and 518 µM peptide inhibitor,
respectively.3 As with IKK-2,
non-parallel relationships indicates that ADP and peptide inhibitor
bind at different sites to IKK-1 and can bind simultaneously.
|
Unlike the result with IKK-2, however, Fig. 5B indicates
that BMS-345541 and peptide inhibitor bind to IKK-1 in a non-mutually exclusive manner (i.e. can bind to the enzyme at the same
time). Indeed, the Dixon plot showed non-parallel relationships with values for kinetic constant A determined to be 112 ± 5, 233 ± 37, 284 ± 24, and 446 ± 57 at
s·counts1·M
1 at 0, 471, 518, and 565 µM peptide inhibitor, respectively. A similar dichotomy between IKK-2 and IKK-1 was also observed using BMS-345541 and ADP in a multiple inhibition analysis as shown in Fig.
5C. In this case, the Dixon plots gave essentially parallel relationships that reflect the relatively unchanging values of kinetic
constant A for the inhibition by ADP at different fixed concentrations of BMS-345541: 439 ± 14, 439 ± 29, 477 ± 42, 487 ± 57, and 486 ± 78 s·counts
1·M
1 at 0, 35, 70, 105, and 140 µM BMS-345541, respectively. This indicates
that BMS-345541 and ADP are mutually exclusive inhibitors in which both
cannot bind to IKK-1 simultaneously. This is in contrast to IKK-2 where
ADP and BMS-345541 were shown to be non-mutually exclusive inhibitors.
Characterization of BMS-345541 in Vivo--
Because BMS-345541 is
both highly selective for IKK and potent against stimulated activation
of NF-B-dependent gene transcription in cells, the
biological activity in vivo was investigated. As shown in
Fig. 6, peroral administration of
BMS-345541 at a concentration of 10 mg/kg to mice resulted in prolonged
serum drug levels, with concentrations sustained at or above 1 µM for many hours. By comparing the area under the curves
for the peroral administration and intravenous legs, an oral
bioavailability of ~100% was determined. The results from this study
were used to obtain the pharmacokinetic parameters shown in
Table I. Additional studies showed
that the drug levels and total exposure after peroral administration of
BMS-345541 were proportional at doses up to 100 mg/kg (results not
shown).
|
|
Because of the excellent pharmacokinetics displayed by BMS-345541, its
biological activity in mice was investigated. As shown in Fig.
7, BMS-345541
dose-dependently inhibited the production of TNF
measured in the serum of animals challenged with an intraperitoneal administration of LPS. Approximately 50% inhibition was observed at 10 mg/kg, consistent with the micromolar exposure shown in Fig. 6 and the
cell potency shown in Fig. 2. Near complete inhibition of serum TNF
was observed at a dose of 100 mg/kg BMS-345541, with six of eight
animals in the group showing undetectable serum TNF
levels.
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DISCUSSION |
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We have shown that BMS-345541 is highly selective for the IKK
catalytic subunits versus numerous other kinases. This
selectivity is also evident in cells; only the stimulus-induced
phosphorylation of IB
was inhibited by BMS-345541 whereas other
signal transduction cascades were unaffected. This is especially
important, because there is a cascade of kinases working sequentially
in many of these signaling pathways such that blocking any of the
upstream kinases would have resulted in inhibition of the measured end point (e.g. inhibition of c-Jun NH2-terminal
kinase, SEK, or mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase kinase, etc. would have inhibited c-Jun
phosphorylation). Lack of effects on these signal transduction pathways
in cells, therefore, provides additional evidence of the selectivity of
BMS-345541 for the catalytic subunits of IKK.
Between IKK-1 and IKK-2, BMS-345541 showed approximately an
order-of-magnitude selectivity for the latter. Having selectivity for
IKK-2 over IKK-1 may be advantageous from the standpoint of the
toxicity profile of a therapeutic agent, because IKK-2 has been shown
to play the critical role in IB
phosphorylation and NF-
B
activation in response to pro-inflammatory stimuli whereas IKK-1 may
play only a minor role (for a review, see Ref. 18). Indeed, IKK-1 is
involved in keratinocyte differentiation, although this appears not to
be dependent on its kinase activity or role in NF-
B activation
(27).
The use of multiple inhibition analyses with IKK-2 indicated that BMS-345541 bound in a non-mutually exclusive manner with respect to ADP and a mutually exclusive manner with respect to peptide inhibitor. This suggests that BMS-345541 binds not to the ATP binding site but to either the peptide binding site or an allosteric site, which, when occupied, alters the peptide binding site and prevents peptide binding. Curiously, the exact opposite relationship was observed with multiple inhibition analyses using IKK-1. The results are consistent with one of the following two possible binding mechanisms. 1) BMS-345541 binds to the ATP binding site of IKK-1 but not of IKK-2, or 2) BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, but binding to the allosteric site on IKK-2 leads to a conformational change in the enzyme that alters the peptide binding site whereas the conformational change that occurs upon binding of BMS-345541 to the corresponding site on IKK-1 causes a perturbation of the ATP binding site. It is difficult to differentiate between these two possibilities without crystallographic evidence. However, structure-activity relationships with numerous other inhibitors synthesized in this chemical class show the same -fold selectivity for IKK-2 for IKK-1 across a wide range of potencies (results not shown). If BMS-345541 bound to a different site on IKK-2 than on IKK-1, the structure-activity relationships would not be expected to track between IKK-1 and IKK-2. Therefore, it is likely that BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2 but that the conformational change that then affects the active site is somewhat different (i.e. mechanism 2 above).
That BMS-345541 binds to an allosteric site on IKK-2 and IKK-1 is also consistent with the high degree of selectivity for the IKK catalytic subunits versus other kinases. Indeed, most kinase inhibitors bind to the ATP binding site, which, because the site is highly conserved among kinases, makes it difficult to obtain selective inhibitors. The high selectivity of BMS-345541 for IKK-2 and IKK-1 suggests that the putative allosteric site is unique to the IKKs, although the site may be present within other kinases not tested for selectivity.
It has been reported recently (27) that the catalytic subunits IKK-2 and IKK-1, which are expressed as dimers, contain non-equivalent binding sites acting in a cooperative fashion such that binding of substrate or inhibitor at one active site affects the conformation at the other active site. The non-linear kinetics observed with BMS-345541 binding to IKK-2 are also consistent with this model. From the relationship shown in Fig. 3, it would appear that either the two active sites within the IKK-2 homodimer are non-equivalent and bind BMS-345541 with different affinities, or the two active sites are equivalent when unoccupied but that binding of BMS-345541 to one active site within the dimer adversely affects binding of BMS-345541 to the second site. Although this certainly adds a layer of complexity to the analysis of inhibitor binding studies, the multiple inhibition analyses detailed in the present work demonstrate that questions about binding sites/mechanisms of the IKK catalytic subunits can be answered.
An especially important characteristic of BMS-345541 is the excellent
pharmacokinetic profile in mice. Complete absorption after peroral
administration, coupled with a very long intravenous half-life, results
in blood levels that should be sufficient to inhibit IKK in
vivo (e.g. BMS-345541 inhibits IKK in cells in the
micromolar range, and a 10 mg/kg peroral administration dose gives
micromolar levels of drug for many hours). Indeed, BMS-345541 dose-dependently inhibited LPS (intraperitoneal)-induced
serum TNF production in mice at doses in the 3 to 100 mg/kg range. To our knowledge, BMS-345541 represents the first selective IKK inhibitor reported with in vivo activity. The potency and
selectivity of BMS-345541 described herein, allied with an excellent
pharmokinetics profile, makes this compound ideally suited to
probe the role of IKK in vivo. Work is ongoing in our
laboratory to evaluate BMS-345541 in multiple murine models of human disease.
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ACKNOWLEDGEMENTS |
---|
We thank Valerie Bidwell and Carrie Xu for bioanalytical support, along with Murray McKinnon for helpful suggestions regarding the manuscript.
![]() |
FOOTNOTES |
---|
* 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: Bristol-Myers Squibb, P. O. Box 4000, Princeton, NJ 08543. Tel.: 609-252-3445; Fax: 609-252-6058; E-mail: james.burke@bms.com.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M209677200
3 Because the peptide inhibitor shows non-linear inhibition with IKK-1 (28), a narrow concentration range was used.
2
Regardless of whether two inhibitors are
mutually exclusive or non-mutually exclusive inhibitors, Dixon plots in
dual inhibition studies should give straight lines such that the
following relationship, shown in Equation 1, holds under the conditions
where the concentration of one inhibitor is varied whereas the other is
kept constant (26).
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor
;
EGF, epidermal growth factor;
IKK, I
B kinase;
LPS, lipopolysaccharide;
IL, interleukin;
GST, glutathione
S-transferase;
MS, mass spectrometry;
HPLC, high pressure
liquid chromatography;
STAT, signal transducers and activators of
transcription;
MAPKAP K2, mitogen-activated protein kinase-activated
protein kinase 2.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, 405-455[CrossRef] |
2. | Bauerle, P. A., and Baltimore, D. (1997) Cell 87, 13-20[CrossRef] |
3. | Whiteside, S. T., and Israel, A. (1997) Sem. Cancer Biol. 8, 75-82[CrossRef][Medline] [Order article via Infotrieve] |
4. | Brown, K., Gertsberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488[Medline] [Order article via Infotrieve] |
5. |
Finco, T. S.,
Beg, A. A.,
and Baldwin, A. S., Jr.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11884-11888 |
6. |
Baldi, L.,
Brown, K.,
Franzoso, G.,
and Siebenlist, U.
(1996)
J. Biol. Chem.
271,
376-379 |
7. |
Roff, M.,
Thompson, J.,
Rodriquez, M. S.,
Jacque, J.-M.,
Baleux, F.,
Arenzana-Seisdedos, F.,
and Hay, R. T.
(1996)
J. Biol. Chem.
271,
7844-7850 |
8. |
Weil, R.,
Laurent-Winter, C.,
and Israel, A.
(1997)
J. Biol. Chem.
272,
9942-9949 |
9. |
Whiteside, S. T.,
Epinat, J.-C.,
Rice, N. R.,
and Israel, A.
(1997)
EMBO J.
16,
1413-1426 |
10. | Chen, Z. J., Parent, L., and Maniatis, T. (1996) Cell 84, 853-862[Medline] [Order article via Infotrieve] |
11. | Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve] |
12. | DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve] |
13. | Zandi, E., Rothwarf, D., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve] |
14. |
Mercurio, F.,
Zhu, H.,
Murray, B. W.,
Shevchenko, A.,
Bennett, B. L., Li, J. W.,
Young, D. B.,
Barbosa, M.,
Mann, M.,
Manning, A.,
and Rao, A.
(1997)
Science
278,
860-866 |
15. |
Woronicz, J. D.,
Gao, X.,
Cao, Z.,
Rothe, M.,
and Goeddel, D. V.
(1997)
Science
278,
866-869 |
16. |
Li, J.,
Peet, G. W.,
Pullen, S. S.,
Schembri-King, J.,
Warren, T. C.,
Marcu, K. B.,
Kehry, M. R.,
Barton, R.,
and Jakes, S.
(1998)
J. Biol. Chem.
273,
30736-30741 |
17. | Régnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383[Medline] [Order article via Infotrieve] |
18. | Ghosh, S., and Karin, M. (2002) Cell 109, S81-S96[Medline] [Order article via Infotrieve] |
19. | Cao, Y., Bonizzi, G., Seagroves, T. N., Greten, F. R., Johnson, R., Schmidt, E. V., and Karin, M. (2001) Cell 107, 763-775[Medline] [Order article via Infotrieve] |
20. | Treiber, H.-J., Behl, B., and Hoffman, H. P. (1994) German patent 4,329,970 |
21. |
Burke, J. R.,
Miller, K. R.,
Wood, M. K.,
and Meyers, C. A.
(1998)
J. Biol. Chem.
273,
12041-12046 |
22. | Kumar, S., McDonnell, P. C., Gum, R. J., Hand, A. T., Lee, J. C., and Young, P. R. (1997) Biochem. Biophys. Res. Commun. 235, 533-538[CrossRef][Medline] [Order article via Infotrieve] |
23. | Ghezzi, P., Sacco, S., Agnello, D., Marullo, A., Caselli, G., and Bertini, R. (2000) Cytokine 12, 1205-1210[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Huynh, Q. K.,
Boddupalli, H.,
Rouw, S. A.,
Koboldt, C. M.,
Hall, T.,
Sommers, C.,
Hauser, S. D.,
Pierce, J. L.,
Combs, R. G.,
Reitz, B. A.,
Diaz-Collier, J. A.,
Weinberg, R. A.,
Hood, B. L.,
Kilpatrick, B. F.,
and Tripp, C. S.
(2000)
J. Biol. Chem.
275,
25883-25891 |
25. | Segel, I. H. (1975) Enzyme Kinetics , pp. 474-504, John Wiley & Sons, Inc., New York |
26. | Hu, Y., Baud, V., Oga, T., Kim, K. I., Yoshida, K., and Karin, M. (2001) Nature 410, 710-714[CrossRef][Medline] [Order article via Infotrieve] |
27. | Burke, J. R., and Strnad, J. (2002) Biochem. Biophys. Res. Commun. 293, 1508-1513[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Burke, J. R.,
Wood, M. K.,
Ryseck, R.-P.,
Walther, S.,
and Meyers, C. A.
(1999)
J. Biol. Chem.
274,
36146-36152 |