[CANCER RESEARCH 64, 6652–6659, September 15, 2004]
A Unique Structure for Epidermal Growth Factor Receptor Bound to GW572016
(Lapatinib): Relationships among Protein Conformation, Inhibitor Off-Rate, and
Receptor Activity in Tumor Cells
Edgar R. Wood,2Anne T. Truesdale,2Octerloney B. McDonald,2Derek Yuan,2Anne Hassell,1Scott H. Dickerson,1
Byron Ellis,3Christopher Pennisi,3Earnest Horne,3Karen Lackey,4Krystal J. Alligood,5David W. Rusnak,5
Tona M. Gilmer,5and Lisa Shewchuk1
1Departments of Computational, Analytical and Structural Sciences,
Throughput Chemistry, and5Oncology Biology, GlaxoSmithKline, Inc., Research Triangle Park, North Carolina
2Assay Development and Compound Profiling,
3Gene Expression and Protein Biochemistry,
GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical devel-
opment for cancer that is a potent dual inhibitor of epidermal growth
factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal
structure of EGFR bound to GW572016. The compound is bound to an
inactive-like conformation of EGFR that is very different from the active-
like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva)
described previously. Surprisingly, we found that GW572016 has a very
slow off-rate from the purified intracellular domains of EGFR and
ErbB-2 compared with OSI-774 and another EGFR selective inhibitor,
ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in
down-regulation of receptor tyrosine phosphorylation. We evaluated the
duration of the drug effect after washing away free compound and found
that the rate of recovery of receptor phosphorylation in the tumor cells
reflected the inhibitor off-rate from the purified intracellular domain. The
slow off-rate of GW572016 correlates with a prolonged down-regulation
of receptor tyrosine phosphorylation in tumor cells. The differences in the
off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can
be explained by the enzyme-inhibitor structures.
Several new cancer therapies are being developed that target the
ErbB receptor tyrosine kinase family. The receptors are multidomain
proteins that contain an extracellular ligand binding domain, a single
transmembrane domain, and an intracellular tyrosine kinase domain.
The family has three catalytically active members, epidermal growth
factor receptor (EGFR; ErbB-1), ErbB-2, and ErbB-4. A fourth mem-
ber of the family, ErbB-3, does not have tyrosine kinase activity but
retains ligand-binding function and is competent for signal transduc-
Inhibition of the tyrosine kinase activity of these receptors is one
approach for therapeutic intervention (2, 3). At least four ErbB-
targeted tyrosine kinase inhibitors are currently in Phase II clinical
trials or beyond. These include ZD-1839 (Iressa), OSI-774 (Tarceva),
GW572016 (Lapatinib), and CI-1033 (3). These compounds share a
common 4-anilinoquinazoline core, but they have distinct ErbB inhi-
bition profiles and mechanisms of action. ZD-1839 and OSI-774 are
potent, selective inhibitors of EGFR (4, 5). GW572016 is a potent
inhibitor of both EGFR and ErbB-2 (6, 7). CI-1033 was designed to
covalently modify an active site cysteine from a template with high
initial binding affinity for EGFR. ErbB-2 and ErbB-4 contain the
same active site Cys, so CI-1033 inhibits these receptors as well (8).
GW572016 is a potent inhibitor of purified EGFR and ErbB-2
receptor tyrosine phosphorylation in intact cells and ErbB-driven
tumor growth in tissue culture and animal models (7). The compound
was selected for drug discovery progression because of these and
other desirable properties. During the discovery phase of the
GW572016 program, we evaluated many different substituted quin-
azoline compounds and found that potent inhibition of purified recep-
tors was not enough to guarantee potent inhibition in intact cells (7,
9–11). In this report we present a detailed evaluation of inhibitor
potency, ErbB receptor selectivity, and inhibitor-enzyme dissociation
rate in an effort to better understand the biological activity of mole-
cules in this series. We find that GW572016 has a unique mechanism
of action. It exhibits reversible, noncovalent inhibition of EGFR and
ErbB-2, but it has a very slow off-rate compared with other reversible
The crystal structures of apo-EGFR and EGFR bound to OSI-774
(OSI-774/EGFR) were described recently (12). The EGFR structure
reveals key features associated with EGFR regulation and catalytic
activity. The OSI-774/EGFR structure provides the basic binding
mode of the 4-anilinoquinazoline core and identifies key interactions
that provide potency and selectivity for EGFR inhibition. The reasons
for the slow off-rate for GW572016 relative to other 4-anilinoquina-
zoline inhibitors is not readily apparent from the compound structure
or models based on the OSI-774/EGFR protein structure. For this
reason we determined that the crystal structure of EGFR bound to
GW572016 (GW572016/EGFR). GW572016/EGFR has a signifi-
cantly different structure than OSI-774/EGFR. The potential relation-
ship between this novel EGFR structure and the off-rates for 4-anili-
noquinazoline inhibitors are discussed. Moreover, the structure may
help shed light on previously unexplained aspects of ErbB receptor
activation and selectivity of 4-anilinoquinazoline inhibitors.
The dissociation rate of the inhibitor-receptor complex or overall
enzyme receptor structure may impact the potency or duration of
receptor inhibition in biological models. Down-regulation of receptor
autophosphorylation in tumor cell lines is a reliable measure of ErbB
inhibitor action. We evaluated the recovery of receptor tyrosine phos-
phorylation in tumor cells after washing away free compound. Re-
ceptor phosphorylation after treatment with GW572016 recovers very
slowly in a manner that reflects the dissociation rate of the inhibitor
from the purified enzyme.
MATERIALS AND METHODS
Compound Synthesis. Compounds were synthesized and purified accord-
ing to the published procedures, including GW572016 (6), OSI-774 (13),
ZD-1839 (14), and CI-1033 (8).
Enzyme Assays and Data Analysis. We have described previously the
purification, general assay methods, and substrate kinetic constants for EGFR,
ErbB-2, and ErbB-4 intracellular domains (15). In the experiments presented
herein, we measured phosphorylation of the ErbB substrate peptide [Biotin-
(amino hexanoic acid)–RAHEEIYHFFFAKKK- CONH2] using the homoge-
neous time-resolved fluorescence procedure. The concentration of peptide was
Received 4/3/04; revised 7/6/04; accepted 7/13/04.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Edgar R. Wood, Department of Assay Development and
Compound Profiling, GlaxoSmithKline, Inc., Research Triangle Park, NC 27709-13398.
Phone: 919-483-3910; Fax: 919-483-3895; E-mail: firstname.lastname@example.org.
©2004 American Association for Cancer Research.
0.8 ?mol/L, and the concentration of ATP was 10 ?mol/L unless otherwise
To measure the IC50for enzyme inhibition, the indicated compound was
serially diluted 1 to 3 in dimethyl sulfoxide and added to the reactions to
produce an 11-point dose-response curve ranging from 0.00005 to 5 ?mol/L.
The IC50values were estimated from data fit to Equation A:
y ? Vmax?1 ? ?x/?K ? x??? ? y2
Vmaxis the curve fit parameter that represents the upper asymptote of the dose
response. This value is typically equivalent to the level of product formed in
the absence of inhibitor. x is the variable representing the concentration of
inhibitor. K is the curve fit parameter for the IC50value. This occurs at the
inflection point of the dose response. y2is the curve fit parameter that
represents the lower asymptote of the dose response. For a potent inhibitor, y2
is equivalent to the assay baseline (signal in the absence of enzyme activity).
eight different concentrations of ATP in the enzyme assay, 10, 40, 70, 100,
130, 160, 190, and 220 ?mol/L. Within an experiment each IC50was deter-
mined in triplicate. Three independent experiments were performed, and the
average IC50was plotted as a function of the concentration ATP. The inhibitors
that we have evaluated have been determined previously to be competitive
with ATP. Therefore, the Kiappvalues were obtained from a least squares fit of
the data to Equation B (16):
appdeterminations, IC50s were obtained for each compound using
app?1 ? ?ATP?/KmATP? ? E/2(B)
E is the curve fit parameter that represents the concentration of enzyme in the
reaction. This parameter was constrained to a value ?2 nM. Two nM is the
maximum concentration of enzyme based on the estimate of total protein in
the preparation. The parameter value for E was allowed to float, because the
precise concentration of enzyme capable of binding each compound is not
known. For all of the Ki
be at or very near 1 nM. The value of Km
and 30 ?mol/L for ErbB-2 based on our experimental values determined
previously (15). Calculated Ki
pounds with IC50s ?1 ?m. These values were obtained by determining IC50at
a single concentration of ATP (20 ?mol/L) and calculated using Equation B.
appestimates described in Table 1, E was estimated to
ATPwas fixed to 5 ?mol/L for EGFR
app) were determined for com-
We evaluated the recovery of enzyme activity from a preformed enzyme–
inhibitor complex to estimate inhibitor off-rate. Enzyme and inhibitor, 500 nM
each, were incubated together in 50 mmol/L 4-morpholinepropanesulfonic acid
(pH 7.5) and 0.01% Tween for 30 minutes at ambient temperature. This
complex was diluted 1:1,000 into standard enzyme assay mixtures containing
200 ?mol/L ATP. The reaction was allowed to proceed for the indicated time,
and the phosphorylated peptide product was measured. The data were fit to
Equation C (17):
P ? vst ? ??vr? vs?/kobs? ?1 ? exp ??kobst??
The value kobsrepresents the rate of change from vr(velocity at t ? 0) to vs
(velocity upon equilibrium). Using these reaction conditions, essentially 100%
of the enzyme is bound by inhibitor in the preformed complex such that vr? 0.
Upon reaching equilibrium, the total inhibitor concentration is 0.5 nM. The
concentration of ATP is 40 ? Km
enzyme is essentially occupied by ATP, and vs? 100% of control reactions
run in the absence of inhibitor. To be sure that these conditions hold true,
control reactions with inhibitor added to the reaction mix (0.5 nM) without
being preincubated with enzyme were conducted, and the rate was the same as
the rate in the absence of inhibitor.
For all of the data analysis described above, nonlinear regressions were
conducted using the program Sigma Plot (Jandel Scientific, San Rafael, CA).
Cell Culture and Treatment. The source and culture conditions of the
LICR-LON-HN5 cell line (HN5) have been described previously (7). Cells
were subcultured in 150-cm2flasks before use. Cells were plated at a density
of 250,000 cells per 100 ? 20-mm dish on day 1. On day 4, cells were exposed
to growth medium containing 0.1% DMSO (vehicle) or to medium containing
vehicle and GW572016, OSI-774, or ZD-1839 at 1 ?mol/L for 4 hours. Cells
were rinsed twice with growth medium and were maintained in fresh growth
medium for 0 to 96 hours. After 0, 24, 48, 72, and 96 hours after removal of
compounds, cells were rinsed with cold PBS and were lysed on ice with 0.5
mL of cold radioimmunoprecipitation assay buffer ? [150 mmol/L NaCl, 50
mmol/L Tris-HCl (pH 7.5), 0.25% deoxycholate, 1% NP40, protease inhibitor
mixture, and 1 mmol/L sodium orthovanadate].
SDS-PAGE and Western Blot Analysis. EGFR was immunoprecipitated
from 0.1 mg of lysate using 1 ?g of anti-EGFR Ab-13 (Lab Vision, Fremont,
CA). Immune complexes were precipitated with 50 ?L of Protein G Plus/
Protein A agarose (Calbiochem, San Diego, CA). Bead pellets were resus-
pended in 50 ?L of 1? Novex Tris-Glycine SDS sample buffer (Invitrogen,
Carlsbad, CA) containing 0.25% ?-mercaptoethanol. Samples were boiled, and
20 ?L were loaded on duplicate 6% Novex Tris-Glycine gels (Invitrogen).
Gels were transferred to nitrocellulose, and membranes were blocked in
Tris-buffered saline-Tween [150 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5),
and 0.1% Tween 20] containing 4% bovine serum albumin. Phosphotyrosine
was detected using clone PT66 (Sigma, St. Louis, MO) diluted 1:1,000 in
blocking buffer. EGFR was detected using anti-EGFR Ab-12 (Lab Vision)
diluted 1:2,500 in blocking buffer. Horseradish peroxidase-conjugated donkey
antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA)
was diluted 1:10,000 in blocking buffer, and bands were visualized using
enhanced chemiluminescence (Amersham, Piscataway, NJ). Films were
scanned using a Fluor S Multi-Imager (Bio-Rad, Hercules, CA), and bands
were quantified by densitometry. The ratio of phosphorylated receptor to total
receptor was calculated for each sample and was expressed as a percentage of
the vehicle-treated control.
Crystallization and Structure Determination. EGFR, residues 672 to
998, was expressed and purified as described previously (12). EGFR was
concentrated to 3–5 mg/mL in 50 mmol/L Tris (pH 8.0) and 100 mmol/L NaCl.
The protein was complexed with a 3-fold molar excess of GW572016 and
incubated on ice for 1 hour. Crystals were grown by the hanging drop vapor
diffusion method from 100 mmol/L 3-(cyclohexylamino)propanesulfonic acid
(pH 9.0), 200 mmol/L LiSO4, and 2 mol/L NaKPO4at 22°C. Crystals typically
appeared in 2 to 5 days and were harvested and flash frozen in paraffin oil.
Data were collected at beam line 17ID on an ADSC detector and processed
with HKL0 (18). Crystals belonged to the space group P212121with cell
dimensions a ? 45.65, b ? 67.14, c ? 102.88, ? ? ? ? ? ? 90, and 1 mol/asu.
The structure was solved by molecular replacement using CNX (19) and
PDB-1M14 as the starting coordinates. The structure was rebuilt using
QUANTA (Accelrys, San Diego, CA) and refined to an Rfactor of 20% at 2.4Å
ATPso that at equilibrium, all of the free
Table 1 ErbB enzyme inhibition by compounds in clinical development
app,b? IC50,c? cKi
3.0 ? 0.2a
13 ? 1a
347 ? 16c
0.40 ? 0.1a
870 ? 90a
1130 ? 370c
0.7 ? 0.1a
1000 ? 100a
1530 ? 270c
30 ? 20b
127 ? 42b
388 ? 174b
NOTE. The method for enzyme assays, experimental design for potency estimation,
and data analysis procedures were conducted as described in Materials and Methods. The
estimates of inhibitor affinity differ according to the mode of inhibition and potency of the
particular result. Ki
(IC50? 500 nM). Calculated Ki
initial IC50? 1,000 nM. CI-1033 is a covalent modifier, so Ki
appropriate measures of potency. For this compound the nonprocessed assay IC50values
are given. For Ki
For IC50and cKi
experiments is shown.
appvalues were determined to assess tight-binding inhibition
app) are given for inhibition that yielded an
appvalues are not
appvalues, the standard error of the least squares fit of the data is shown.
appvalues, the SD of the mean determined from three independent
EGFR BOUND TO GW572016
using CNX. The Rmergeand completeness of the data were 6.7% and 95%,
respectively. The root mean squared deviation in the bonds and angles of the
final model were 0.007Å and 1.30°, respectively.
Potency and Selectivity of 4-Anilinoquinazoline ErbB Inhibi-
tors. Different laboratories have used different methods to evaluate
the potency and selectivity of the 4-anilinoquinazoline ErbB inhibitors
in clinical development. Potency and selectivity measurements can be
affected by experimental conditions, especially enzyme and substrate
concentrations and incubation times. To provide directly comparable
estimates of potency and selectivity, we evaluated enzyme inhibition
using a purified enzyme assay system consisting of the recombinant
human intracellular domain of each catalytically active ErbB family
The dissociation constant for an inhibitor, Ki, is the best standard of
comparison for potency and selectivity. We estimated Ki
termined at a single fixed concentration of peptide) by measuring the
initial rate of product formation at varied inhibitor concentrations
using eight fixed concentrations of ATP. All of the 4-anilinoquinazo-
line inhibitors being evaluated are potent inhibitors of EGFR with
reported IC50values close to the concentration of enzyme normally
required to achieve an adequate assay signal (4–8). Therefore, the
data were fit to Equation B, which takes into account the concentra-
tion of active enzyme in the assay. This allowed us to estimate Ki
values below the enzyme concentration limit (16). We determined the
value for each compound increased with increasing ATP concentra-
tion as expected for a competitive inhibitor. The Ki
estimated from the slopes of the lines formed by fitting the data to
Equation B. Similar experiments were conducted with ZD-1839,
OSI-774, and GW572016 using EGFR, ErbB-2, and ErbB-4 (Table
1). The Ki
OSI-774 were 0.4 and 0.7 nM, respectively. These values are lower
than the previously reported Kivalues of 2.1 ? 0.2 nM for ZD-1839
(4) and 2.7 nM for OSI-774 (5). The inclusion of the enzyme concen-
tration term in our analysis may contribute to these observed differ-
ences. ZD-1839 and OSI-774 are both ?1,000-fold selective for
EGFR over ErbB-2 with Ki
CI-1033 is an irreversible inhibitor that inactivates ErbB family
appfor EGFR using ZD-1839 and GW572016 (Fig. 1). The IC50
appvalues for EGFR that we obtained using ZD-1839 and
appvalues near 1 ?mol/L for ErbB-2.
enzymes by covalently modifying an active site cysteine (8). For
compounds of this sort, Ki
are not appropriate evaluations of binding potency. Nevertheless, we
measured the IC50of CI-1033 for EGFR, ErbB-2, and ErbB-4 after 20
minutes of incubation time to estimate the affinity of this compound
for the different ErbB enzymes. CI-1033 is a potent pan-ErbB inhib-
itor that inhibits EGFR, ErbB-2, and ErbB-4 with IC50values of 30,
127, and 388 nM, respectively.
GW572016 is unique among the molecules studied, because it is a
reversible inhibitor that is potent against both EGFR and ErbB-2 with
Inhibitor Dissociation Rates. Preliminary evaluation of IC50as a
function of time suggested that GW572016 exhibited time-dependent
behavior. To characterize this behavior further, we evaluated the
inhibitor off-rate using an enzyme reactivation procedure. EGFR and
inhibitor were incubated for 30 minutes to allow formation of an
enzyme-inhibitor complex. This complex was diluted extensively into
a reaction mixture containing a high concentration of ATP (40 ? Km),
and phosphorylated product was evaluated as a function of time (Fig.
2). Under these conditions, the change in the rate of product formation
reflects the dissociation of the enzyme–inhibitor complex (17). Dis-
sociation of ZD-1839, OSI-774, and GW572016 were compared (Fig.
2A). The rates of product formation in the presence of ZD-1839 and
OSI-774 were virtually indistinguishable from the rate of product
formation in the absence of inhibitor. This indicates a rapid off-rate
(half-life ? 10 min). In contrast, enzyme activity after preincubation
with GW572016 recovered very slowly suggesting that the compound
had a much slower off-rate.
To estimate this slow off-rate, we compared GW572016 to the
covalent modifier CI-1033 and ran the reactions for a longer period of
time (Fig. 2B). No recovery of enzyme activity was seen after prein-
cubation with CI-1033. In contrast, phosphorylated product was de-
tected 50 minutes after dilution of the EGFR-GW572016 complex.
The off-rate estimated from the fit of data to Equation C is
0.0023 ? 0.0002 per minute. This translates to a half-life of 300
minutes for the GW572016-EGFR complex. GW572016 is a potent
inhibitor of EGFR and ErbB-2, so we compared the dissociation of the
compound from both enzymes (Fig. 2C). GW572016 also has a very
slow off-rate for ErbB-2.
The slow recovery of enzyme activity indicates that GW572016
binds in a reversible fashion. Because the recovery was slow, how-
ever, we evaluated whether or not GW572016 forms a stable covalent
bond with EGFR using liquid chromatography-mass spectrometry.
EGFR that was incubated with CI-1033 had a molecular weight
consistent with the formation of a single modified cysteine residue as
reported previously for a closely related compound (20). EGFR incu-
bated with GW572016 had a molecular weight identical to that of
untreated EGFR, which indicated that a stable covalent bond was not
formed between the protein and compound (data not shown).
Duration of Inhibition in Tumor Cells. Exposure to kinase in-
hibitors causes the down-regulation of tyrosine phosphorylation of
ErbB receptors in tumor cells (4, 5, 7). We examined the recovery of
receptor phosphorylation after inhibitor washout to determine whether
there is a correlation between inhibitor dissociation rate and recovery
of autophosphorylation (Fig. 3). Logarithmically growing HN5 cells
that naturally overexpress EGFR (21) were treated for 4 hours with
GW572016, OSI-774, and ZD-1839 (1 ?mol/L). After treatment, the
inhibitor containing media was removed, cells were rinsed exten-
sively, and fresh media without inhibitor was added. The cells were
lysed at various times after inhibitor washout, and EGFR was captured
by immunoprecipitation. The relative receptor content was determined
by immunoblot with a separate antireceptor antibody, and the state of
phosphorylation was analyzed by immunoblot with an antiphospho-
appestimates as determined in these studies
appvalues of 3 nM and 13 nM, respectively.
Fig. 1. Determination of Kiappfor ZD-1839 and GW572016. The IC50for EGFR with
the indicated compound was determined at eight fixed concentrations of ATP. EGFR IC50
with ZD-1839 (F). EGFR IC50with GW572016 (E). Lines represents the least squares fit
of the data to Equation B. The parameters for Kiappfrom this and other experiments are
summarized in Table 1.
EGFR BOUND TO GW572016
tyrosine antibody. Treatment with all three of the inhibitors resulted in
?85% reduction in receptor tyrosine phosphorylation without any
reduction in total receptor content. In OSI-774-treated cells, receptor
phosphorylation recovered to control levels 24 hours after washout. In
ZD-1839-treated cells, recovery of receptor phosphorylation was
slower but had reached 60% of control levels after 72 hours. Receptor
phosphorylation in GW572016-treated cells had only recovered to
15% of control levels 96 hours after washout. These results suggest
that the very slow inhibitor dissociation rate of GW572016 may
increase the duration of suppression of ErbB receptor activity in tumor
Crystal Structure of GW572016/EGFR. The dramatic differ-
ences between the off-rates observed for GW572016 and OSI-774
suggested that the two compounds were binding to EGFR in different
ways. To test this hypothesis, we evaluated the binding mode of
GW572016 by determining the crystal structure of EGFR bound to the
inhibitor. The structure of GW572016/EGFR was solved to 2.4 Å
resolution and contains the kinase domain, as well as 40 residues of
the COOH-terminal tail. The overall fold of the protein is similar to
that observed in apo-EGFR and OSI-774/EGFR structures (12). How-
ever, there are significant differences in the orientation of the NH2-
and COOH-terminal lobes, the COOH-terminal tail, and the C helix,
as described below. Six residues at the NH2terminus, 8 residues at the
COOH-terminus, and five short loop regions (residues 710 to 713, 726
to 730, 844 to 851, 964 to 970, and 980 to 983) are not included in the
final model due to poor electron density.
Inhibitor Binding Site. GW572016 binds in the ATP-binding
cleft in a fashion similar to that observed in other kinase-quinazoline
Fig. 3. Recovery of EGFR autophosphorylation after treatment with GW572016,
ZD-1839, and OSI-774. Logarithmically growing HN5 cells were treated with 1 ?mol/L
inhibitor in culture media for 4 hours. The media was removed, cells were washed twice,
and fresh compound-free media was added. The cells were lysed at the indicated time after
inhibitor washout, and EGFR was isolated by immunoprecipitation. A, tyrosine phospho-
rylated EGFR (pEGFR) was determined by Western blot using antiphosphotyrosine
antibody. Lysates used for immunoprecipitation were prepared from cells treated with the
following agent: Lane 1, DMSO control; Lane 2, GW572016; Lane 3, ZD-1839; and Lane
4, OSI-774. Total EGFR was measured from the same immunoprecipitate samples by
Western blot using anti-EGFR antibody. B, The level of Tyrosine-phosphorylated EGFR
was quantified for each condition and expressed as the percentage of vehicle-treated
sample. The results represent the mean value of three independent experiments; bars,
Fig. 2. Dissociation of 4-Anilinoquinazoline Compounds from EGFR and ErbB-2.
Phosphorylation of peptide substrate as a function of time is shown. The reaction was
initiated by diluting a preformed enzyme-inhibitor complex into reaction buffer. Under the
conditions of the reaction, the recovery of activity reflects the dissociation of inhibitor to
generate the active enzyme species. Lines represent the least squares fit of the data to
equation 3. A, Phosphorylation of peptide by EGFR after preincubation with no inhibitor
(F), GW572016 (E), OSI-774 (?), and ZD-1839 (ƒ). B, phosphorylation of peptide by
EGFR after preincubation with no inhibitor (F), GW572016 (E), and CI-1033 (?). C,
phosphorylation of peptide by EGFR or ErbB-2 after preincubation with GW572016.
ErbB-2 with no inhibitor (F), ErbB-2 with GW5720106 (E), EGFR with no inhibitor (?),
and EGFR with GW572016 (ƒ).
EGFR BOUND TO GW572016
crystal structures (refs. 12, 22; Fig. 4). The quinazoline ring is hy-
drogen bonded to the hinge region between the NH2- and COOH-
terminal lobes of the kinase. N1 of the quinazoline is hydrogen
bonded to the main chain NH of Met769, whereas N3 makes a
water-mediated hydrogen bond to the side chain of Thr830. The
quinazoline ring is sandwiched from the top and bottom by the side
chains of Ala719 and Leu820, respectively. The 3?-chloro-4?-[(3-
fluorobenzyl)oxy]aniline group is oriented deep in the back of the
ATP binding site and makes predominantly hydrophobic interactions
with the protein. The 3?-chloro-aniline group is positioned in a pocket
formed by the side chains of Val702, Lys721, Leu764, Thr766,
Thr830, and Asp831. The 3-fluorobenzyloxy group occupies a pocket
formed by the side chains of Met742, Leu753, Thr766, Thr830,
Phe832, and Leu834. The aniline nitrogen and the ether oxygen are
not involved in any direct hydrogen bonding interactions with the
protein. The methylsulfonylethylaminomethylfuryl group, off the C6
position of the quinazoline, is positioned at the solvent interface and
does not make any significant interactions with the protein. The
methylsulfonylethylamino group is bound near Asp776 but is poorly
Comparison with OSI-774/EGFR. The structure of GW572016/
EGFR is very different from the structure of OSI-774/EGFR. These
differences include the shape of the ATP binding site, the position of
the C helix, the conformations of the COOH-terminal tail and activa-
tion loop, and the hydrogen bonding pattern with the quinazoline ring
of the inhibitors.
The NH2- and COOH-terminal lobes of kinases are connected by a
flexible hinge region. The relative orientation of the two lobes with
respect to one another influences the shape of the ATP binding cleft
and is dependent on the activation state of the kinase and the presence
of ligands. The ATP binding cleft of GW572016/EGFR is in a
relatively closed conformation. This conformation is similar to the
ATP binding clefts in inactive Src and Hck (23–25). The ATP binding
cleft in OSI-774/EGFR is in a more open conformation. The NH2-
terminal lobe in EGFR/GW572016 is rotated ?12° relative to its
position in EGFR/OSI-774 (Fig. 5).
The ATP binding site of GW572016/EGFR has a larger back
pocket than apo-EGFR or OSI-774/EGFR. The larger pocket is cre-
ated by an ?9 Å shift in one end of the C helix to accommodate the
3-fluorobenzyl-oxy group of GW572016 (Fig. 6). The 3-fluorobenzyl-
oxy group occupies roughly the same space as Met742 in the C helix
of EGFR/OSI-774. The shift in the C helix results in the loss of a
highly conserved Glu-Lys salt bridge (Glu738 and Lys721) that
ligates the phosphate groups of ATP. In GW572016/EGFR, Lys721 is
hydrogen bonded to the side chain of Asp831, located in the COOH-
terminal lobe, and Glu738 points out toward solvent.
The COOH-terminal tail of EGFR contains several sites of auto-
phosphorylation and plays a key role in signal transduction by serving
as a docking site for signaling molecules that bind to the phosphoryl-
ated tyrosines. Forty residues of the COOH-terminal tail were in-
cluded in the EGFR constructs used in these crystallographic studies.
In apo-EGFR and OSI-774/EGFR, the COOH terminus is poorly
defined and is loosely associated with two symmetry-related mole-
cules. In GW572016/EGFR, residues 971 to 980 of the COOH-
terminal tail form a short ? helix that packs along the hinge region
connecting the NH2- and COOH-terminal lobes (Fig. 5). This helix
partially blocks the front of the ATP binding cleft. A second helical
segment containing residues 983 to 990 extends along the NH2-
terminal lobe of the protein.
Protein kinases contain a large flexible loop, called the activation
loop or A-loop that regulates kinase activity (26, 27). Apo-EGFR and
OSI-774/EGFR have A-loop conformations (residues 831 to 860) that
are similar to A-loops found in active kinase structures. GW572016/
EGFR has an A-loop conformation similar to that observed in crystal
Fig. 4. GW572016 in the ATP binding site of
EGFR. GW572016 is shown in purple. Hydrogen
bonds are indicated by dashed lines. The figure was
prepared using QUANTA (Accelrys).
EGFR BOUND TO GW572016
structures of inactive Src and Hck. Eight residues in the middle of the
activation loop are disordered (844 to 851). The DFG motif of kinases
is part of the A-loop and is involved in coordinating ATP. The side
chains of Asp831 and Phe832 of the DFG motif form part of the
expanded back pocket in GW572016/EGFR. Unlike apo and OSI-
774/EGFR, residues 834 to 838 form a short helical segment that
packs against the shifted C helix.
Finally, the quinazoline rings of OSI-774 and GW572016 hydrogen
Fig. 5. Overlay of EGFR in the GW572016 and OSI-774
complexes. EGFR in the GW572016 and OSI-774 structures
is shown as red and green ribbons, respectively. GW572016
is shown as a yellow space-filling model. The two proteins
were overlaid based on residues in the COOH-terminal do-
main of the kinase. The COOH-terminal tail in both struc-
tures is CT. Disordered residues in the COOH-terminal tail of
EGFR are indicated by a dashed line. The figure was pre-
pared using QUANTA (Accelrys).
Fig. 6. Difference in C helix position in the
GW572016 and OSI-774
GW572016 and OSI-774 are shown as yellow and
blue ball and stick figures, respectively. The C
helix in the GW572016 and OSI-774 structures is
shown as red and green cylinders, respectively.
The hydrogen bonds between Lys721 and Glu738
in the OSI-774 structure and Lys721 and Asp831 in
the GW572016 structure are indicated by dashed
lines. The figure was prepared using QUANTA
EGFR BOUND TO GW572016
bond with EGFR differently. The quinazoline N1 of both compounds
accepts a hydrogen bond from the main chain NH of Met769. How-
ever, N3 of GW572016 makes a water-mediated hydrogen bond to the
side chain of Thr830 (Fig. 4). N3 of OSI-774 makes a water-mediated
hydrogen bond with the side chain of Thr766. The side chain of
Thr766 in GW572016/EGFR points away from the quinazoline and
hydrogen bonds to the backbone carbonyl of Arg752. Interestingly,
Blencke et al. (28) showed that mutation of Thr766 to methionine in
EGFR leads to resistance to the 4-anilinoquinazoline PD153035.
PD153035, like OSI-774, has a small 4-aniline substituent and is
predicted to share a similar binding mode. This mutation may not
affect inhibition by compounds that bind like GW572016.
It is important to note that the structures for GW572016/EGFR and
OSI-774/EGFR described above were derived from crystals obtained
using different methods. The published OSI-774/EGFR structure was
derived from apo-EGFR crystals that had been soaked with the
inhibitor to obtain the bound complex (12). The GW572016/EGFR
structure was derived from crystals created by cocrystallization of
EGFR and inhibitor. Unfortunately, acceptable cocrystals could not be
obtained by soaking GW572016 into the apo-EGFR crystal. This
probably results from the inability of the bulky aniline substitution of
the compound to form a complex with the small back pocket found in
the active-like conformation of apo-EGFR. Nevertheless, we believe
these technical differences do not contribute to the observed structural
differences for three reasons: (1) the magnitude of the protein con-
formation differences exceeds the degree normally caused by different
crystal packing interactions, (2) we have obtained quinazoline/EGFR
structures for compounds with small aniline substitutions similar to
OSI-774 by cocrystallization and find active-like conformations (data
not shown), and (3) we have obtained compound/EGFR structures for
several other compounds with bulky-aniline substitutions similar to
GW572016, and all of these have had similar inactive-like conforma-
tions (data not shown).
Our results begin to establish a framework for understanding how
ErbB receptor structure influences the biological activity of kinase
inhibitors in clinical development. We have identified several novel
relationships including connections between protein structure and
inhibitor off-rate, protein structure and receptor selectivity, and pro-
tein structure and mechanism of receptor activation.
ErbB Structure and Inhibitor Off-Rate. Significant differences
in the structure of EGFR bound to OSI-774 and GW572016 may
explain the differences in their observed dissociation rates. OSI-774
and ZD-1839, which have small aniline substituents off the quinazo-
line ring, have relatively rapid off-rates. OSI-774/EGFR has a con-
formation that is virtually identical to apo-EGFR. Thus, the rapid
off-rate may reflect the fact that the inhibitor binds and dissociates
from an active form of the enzyme without requiring any major
changes in protein conformation. GW572016, on the other hand, has
a bulky aniline substituent that reaches deep into an opened back
pocket. This back pocket, which is not apparent in apo or OSI-774/
EGFR, results from a shift in the position of the C-helix. The COOH-
terminal tail is also shifted to a position that partially blocks the
opening of the inhibitor-binding site. It appears that dissociation of
GW572016 may require a conformational change in EGFR. A slow
protein conformational change may explain the slow dissociation rate
of GW572016. Alternatively, the different structure of GW572016/
EGFR may provide a very tight binding affinity that results in the
slow-off rate, and a conformational change may not be involved in
Treatment of tumor cells with OSI-774, ZD-1839, and GW572016
results in down-regulation of tyrosine phosphorylation of ErbB recep-
tors (4, 5, 7). The recovery of receptor tyrosine phosphorylation after
down-regulation is a complex process that involves the regulation of
receptor kinase activity through inhibitor concentration and dissocia-
tion, receptor activation, and autophosphorylation. The rate of recov-
ery may also be affected by additional regulatory mechanisms such as
receptor recycling, degradation, and resynthesis. Nevertheless, we
have found that the rate recovery of receptor phosphorylation in tumor
cells reflects the inhibitor off-rate observed in enzyme assays. We
observed very little recovery of EGFR tyrosine phosphorylation 96
hours after removal of GW572016. Thus, the inhibitor off-rate may be
an important factor that affects the duration of drug activity in vivo.
ErbB Structure and Inhibitor Selectivity. ZD-1839 and OSI-774
are potent inhibitors of EGFR but not ErbB-2, although ErbB-2 has a
very similar catalytic domain (88% identical). GW572016 on the
other hand inhibits both enzymes with similar potency. The
GW572016/EGFR structure is very different from the OSI-774/EGFR
structure. The magnitude of the difference suggests that the two
inhibitors target different forms of the enzyme, active and inactive.
The lack of inhibition of ErbB-2 by OSI-774 may reflect the inability
of ErbB-2 to adopt an active-like conformation as seen in the apo and
OSI-774/EGFR structures. Interestingly, we and others have found
that purified ErbB-2 is not very active using ATP-Mg as a substrate.
The enzyme is 15-fold more active using ATP-Mn (15). The presence
of ATP-Mn in the active site of ErbB-2 may compensate for a
catalytically inefficient structure that differs from apo-EGFR. If apo-
ErbB-2 cannot achieve this active-like structure, then compounds
similar to OSI-774 may not be able to bind with high affinity.
Our results suggest that ErbB receptor tertiary structure may be a
key component of inhibitor selectivity. The enzyme assays that we
and others have used to describe the EGFR/ErbB2 inhibitor selectivity
used purified intracellular domains of the receptors (4, 15, 29). Thus,
it is possible that inhibitor selectivity could be different in vivo.
Experiments with tumor cell lines suggest that this is not the case. For
example, OSI-774 effectively inhibits EGFR tyrosine phosphorylation
and proliferation of EGFR overexpressing tumor lines (5). The com-
pound is not effective, however, at blocking ErbB-2 tyrosine phos-
phorylation (29) or blocking proliferation of tumor cells that overex-
press ErbB-2 (7). A selective ErbB-2 inhibitor, CP-654577, has been
described recently (29). This compound is a 4-anilino-quinazoline
with a bulky aniline substitution somewhat similar to GW572016.
CP-654577 effectively blocks ErbB-2 tyrosine phosphorylation in
intact cells but not epidermal growth factor-stimulated EGFR tyrosine
phosphorylation. The dual inhibitor, GW572016, effectively blocks
EGFR and ErbB-2 autophosphorylation in cells and inhibits the pro-
liferation of tumor cell lines that overexpress either EGFR or ErbB-2
ErbB Structure and Receptor Activation. Unlike most receptor
tyrosine kinases, autophosphorylation of the A-loop is not required for
ErbB receptor catalytic activity (30, 31). A variety of evidence sug-
gests that ligand-induced dimerization and subsequent conformational
change trigger activity (reviewed in ref. 32). However, the nature of
the conformational change is not understood.
The conformation of GW572016/EGFR is similar in many ways to
the inactive conformations of Src and Hck (23–25). These enzymes
are multidomain kinases that have NH2-terminal SH2 and SH3 reg-
ulatory domains linked to the catalytic domain. Activity is regulated
by phosphorylation of a specific tyrosine in the COOH-terminal tail.
Intramolecular binding of the SH2 domain to this residue keeps the
enzyme in the inactive state. This regulatory mechanism has been
referred to as a switch–clamp–latch model. Conformational changes
in the C helix and activation loop constitute the switch. The SH3 and
SH2 domains act like a clamp, fixing the relative orientation of the
EGFR BOUND TO GW572016
NH2- and COOH-terminal lobes. The phosphorylated tyrosine in the
COOH-terminal tail is considered the latch.
Activation of EGFR and other ErbB receptors may be similar to Src
in some respects. If the apo-inactive enzyme has a conformation
similar to GW572016/EGFR, then the C helix is in a position that is
not competent for activity. The position of the COOH-terminal tail,
which packs along the hinge, is positioned in such a way that it may
act as a regulatory clamp. Dimerization induced by ligand binding
may result in a shift that releases the COOH-terminal tail and allows
the C-helix and A-loop to adopt an active conformation.
Conclusions. GW572016/EGFR is found in an inactive-like con-
formation. We do not know if the inhibitor binds to a pre-existing pool
of EGFR that is already in this inactive state or if inhibitor binding
induces a change to this conformation. cAbl bound to STI-571 (also
known as Gleevec and Imatinib) also has an inactive-like conforma-
tion (33). Binding to inactive kinase conformations may provide some
advantages for blocking biological activity through mechanisms re-
lated to the overall signal transduction process. Alternatively, binding
to an inactive form of a kinase (or inducing this conformation) may
simply provide an advantage by reducing the rate of inhibitor disso-
ciation, allowing for prolonged effects in biological systems after
inhibitor concentrations have dropped. In support of the latter mech-
anism, we have found that EGFR receptor autophosphorylation re-
covers very slowly in tumor cells after treatment with the slow-off rate
Our results have important implications for protein kinase drug
discovery. GW572016 binds a form of EGFR that is distinct from the
previously described structure of EGFR bound to another drug in
clinical development, OSI-774. Thus, specific drugs may target dif-
ferent forms of the same enzyme. The selectivity of these drugs seems
to be significantly affected by the tertiary structure of the receptor
target. Thus, the primary amino acid sequence of the kinase ATP
binding site may not be a very good predictor of selectivity. The slow
off-rate, bound EGFR structure, and dual ErbB1 and Erb2 inhibition
profile differentiate GW572016 from the other clinical agents tested.
Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was
supported by the members of the Industrial Macromolecular Crystallography
Association through a contract with Illinois Institute of Technology. Use of the
Advanced Photon Source was supported by the United States Department of
Energy, Office of Science, Office of Basic Energy Sciences, under Contract
No. W-31–109-Eng-38. We thank Jon Williams, Wendy White, Craig Wagner,
and Erin Chaney for intact protein LC/MS of inhibitor-treated EGFR samples.
We thank Dr. Gary Smith and Dr. Thomas Meek for critically reading this
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EGFR BOUND TO GW572016