Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual inhibitors of EGFR and ErbB-2 protein tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2008.05.024

We herein disclose a novel series of 4-aminopyrimidine-5-carbaldehyde oximes that are potent and selective inhibitors of both EGFR and ErbB-2 tyrosine kinases, with IC₅₀ values in the nanomolar range. Structure-activity relationship (SAR) studies elucidated a critical role for the 4-amino and C-6 arylamino moieties. The X-ray co-crystal structure of EGFR with 37 was determined and validated our design rationale.

Full text of DOI:10.1016/j.bmcl.2008.05.024

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3495–3499
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes
as dual inhibitors of EGFR and ErbB-2 protein tyrosine kinases
*
Guozhang Xu , Lily Lee Searle, Terry V. Hughes, Amanda K. Beck, Peter J. Connolly, Marta C. Abad,
Michael P. Neeper, Geoffrey T. Struble, Barry A. Springer, Stuart L. Emanuel, Robert H. Gruninger,
Niranjan Pandey, Mary Adams, Sandra Moreno-Mazza, Angel R. Fuentes-Pesquera,
Steven A. Middleton, Lee M. Greenberger
Johnson & Johnson Pharmaceutical Research & Development, Medicinal Chemistry, 8 Clarke Drive, Cranbury, NJ 08512, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein disclose a novel series of 4-aminopyrimidine-5-carbaldehyde oximes that are potent and
selective inhibitors of both EGFR and ErbB-2 tyrosine kinases, with IC50 values in the nanomolar range.
Structure–activity relationship (SAR) studies elucidated a critical role for the 4-amino and C-6 arylamino
moieties. The X-ray co-crystal structure of EGFR with 37 was determined and validated our design
rationale.
Received 24 April 2008
Accepted 7 May 2008
Available online 10 May 2008
Keywords:
Receptor tyrosine kinase
ErbB-2
Ó 2008 Elsevier Ltd. All rights reserved.
EGFR
Oxime
Quinazoline
Bioisostere
The epidermal growth factor (ErbB) family of receptor tyrosine
kinases (RTKs) plays an important role in the regulation of cell
growth, differentiation, and survival.1 The ErbB family consists of
four receptors: epidermal growth factor receptor (EGFR or ErbB-
1), ErbB-2 (Her-2), ErbB-3, and ErbB-4.2 Dysregulation of the ErbB
signaling pathways has been observed in numerous solid tumors
(e.g., breast, lung, colon, and prostate).3 The increased ErbB signal-
ing observed in these tumors usually results from receptor overex-
pression, gene amplification, mutation, and/or elevated levels of
ErbB ligands. Both EGFR and ErbB-2 have been targeted for inhib-
itor development by the pharmaceutical industry,4 and over a dec-
ade of research has culminated in the approval of gefitinib 1 and
erlotinib 2 for the treatment of non-small cell lung cancer follow-
ing prior chemotherapeutic intervention (Fig. 1).5 Lapatinib 3, a po-
tent dual EGFR/ErbB-2 inhibitor, for the treatment of advanced or
metastatic ErbB-2 positive breast cancer is currently in Phase II
and III trials for the treatment of other solid tumors.6 These agents
belong to the 4-anilinoquinazoline class of inhibitors and the key
features between the receptor and this template have been re-
vealed as follows.7,8 (1) The quinazoline moiety fits into the ATP
binding pocket of the kinase domain, where the N-1 nitrogen of
the quinazoline nucleus interacts with the backbone NH of Met-
769 via a hydrogen bond, and a water mediated hydrogen bonding
is observed between the N-3 of the quinazoline and the Thr-766
side chain. (2) The aniline ring fills an adjacent lipophilic pocket.
(3) The solubilizing side chains at C-6 and/or C-7 of the quinazoline
core act to improve physical properties and confer a more favor-
able pharmacokinetic profile.
We envisioned that replacement of the quinazoline found in
molecules like 1, 2, and 3 with our recently reported 4-aminopy-
rimidine-5-carbaldehyde oxime scaffold9,10 could lead to the iden-
tification of potent EGFR/ErbB-2 dual inhibitors (Fig. 2). The pseudo
six-membered ring formed by the intramolecular NH. . .N@C
hydrogen bond of 5 would function as a mimic of the phenyl ring
of the quinazoline. In this report, we describe the synthesis and
structure–activity relationship (SAR) of a novel series of dual
EGFR/ErbB-2 inhibitors designed around this hypothesis. An X-
ray co-crystal structure of a representative compound in complex
with EGFR is also described.
Synthesis of the target molecules is illustrated in Scheme 1.
Treatment of 4,6-dichloropyrimidine-5-carboxaldehyde11 6 with
either NH3 gas or CH3NH2 in toluene gave 7, followed by reac-
tion with an appropriately substituted aniline to give pyrimi-
dine-5-carboxaldehyde 8. High conversion of 7–8 was usually
achieved when the base (DIEA) was added first and the aniline
was added last. Reversal of the order of addition sometimes
resulted in the Schiff base formation with the C-5 aldehyde
* Corresponding author. Tel.: +1 609 655 6915; fax: +1 609 655 6930.
E-mail address: gxu4@prdus.jnj.com (G. Xu).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.024

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G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3495–3499
F
O
H N
N
Cl
H N
N
N
O
O
O
O
N
O
O
N
Erlotinib (2)
Potent EGFR TK inhibitor
Gefitinib (1)
Potent EGFR TK inhibitor
O
F
H N
N
Cl
H
N
N
O
S
O
O
Lapatinib (3)
Potent ErbB-2/EGFR TK inhibitor
Figure 1. Examples of EGFR and ErbB-2 receptor tyrosine kinase inhibitors in clinical use: Gefitinib 1; Erlotinib 2; Lapatinib 3.
oro-benzene in the presence of PPh3, CuI, and prolinol afforded
Ar
Ar
N
2-substituted benzo[b]furan 11,14 which was reduced to 12 via
hydrogenation.
HN
HN
6
4
O
O
O
1
N
R
N
H
3
5
R
R
To ascertain whether the 4-aminopyrimidine-5-carboxalde-
hyde oxime derivatives were capable of affording potent EGFR/
ErbB-2 inhibition, we selected the 1-(3-fluorobenzyl)indazol-5-
amino group as the C-6 side chain since it has been shown in
both pyrrolotriazine15 and quinazoline16 scaffolds to provide
optimal dual EGFR and ErbB-2 kinase inhibitions. We were de-
lighted to find that compound 13 (Table 1) displayed potent inhi-
bition against both EGFR and ErbB-2 with IC50 = 8 and 12 nM,
respectively.17 However, methyl substitution of the C-4 amino
group (14) abrogated both EGFR and ErbB-2 activities. Such a
pronounced drop in activity against the enzyme suggested that
C-4 NH2 is necessary for binding, or that the methyl group pre-
vents the molecule from attaining a proper conformation for
binding to the enzyme.
2
N
N
3
2
4
N
1
H
5
4
Figure 2. Quinazoline and 4-amino-6-arylaminopyrimidine-5-carbaldehyde oxime
templates.
moiety. Subsequent condensation of 8 with an O-substituted
hydroxylamine provided the desired pyrimidine oxime 9. It
should be pointed out that the E-isomer was either the major
or exclusive product obtained.
Anilines used to prepare analogues 20–24, 26 and 27 were syn-
thesized by published procedures.12,13 Aniline 12 required for the
synthesis of analogue 30 was prepared according to Scheme 2.
Pd/C-catalyzed reaction of 2-iodophenol 10 with 1-ethynyl-3-flu-
We next turned our attention to determining the effect of vari-
ous modifications at the C-6 aniline position on EGFR and ErbB-2
Ar
N
Ar
N
HN
HN
Cl
N
Cl
N
O
R2
N
O
N
N
O
O
a
b
c
HN
R1
N
HN
R1
N
HN
R1
Cl
8
9
7
6
Scheme 1. Reagents and conditions: (a) R1-NH2, toluene (90%); (b) DMSO, DIEA, Ar–NH2, 100 °C (65–80%); (c) R2-ONH2ÁHCl, MeOH, 75 °C (70–90%).
O2N
H2N
O2N
I
b
a
O
O
OH
F
F
12
11
10
Scheme 2. Reagents and conditions: (a) 1-ethynyl-3-fluoro-benzene, 10% Pd/C, PPh3, CuI, s-prolinol/H2O (80%); (b) H2, 5% Pd/C (95%).

G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3495–3499
3497
Table 1
such as methoxy at the 3-position (22) exhibited greatly reduced
potency against EGFR. The 4-fluorobenzyl analogue 24 also proved
to have suboptimal potency. Indole-substituted analogue 25 main-
tained potency against both enzymes, whereas the 2,3-dihydro-in-
dole compound 27 showed much weaker potency against EGFR
and ErbB-2. Changing the indazole group to benzoimidazole (26),
which also displaces the 3-fluorobenzyl group to the 2-position
of the heterocycle, resulted in 6-fold lower potency against both
enzymes. Alternative extended anilines (28 and 29) gave compara-
ble, high EGFR potency but different selectivity versus ErbB-2. The
benzofuran analogue 30, which may be viewed as a conformation-
ally restricted version of 29, was inactive in both EGFR and ErbB-2
assays.
Structure–activity relationships for the C-4 position
O
N
H
N
F
NHR1
5
6
4
1
3
N
N
N
N
2
a
a
Compound
R1
EGFR IC50 (lM)
ErbB-2 IC50 (lM)
13
14
H
CH3
0.008
0.012
(39% inh at 10 lM)
(48% inh at 2 lM)
To study the SAR at the oxime side chain (R2 group), a series of
unsubstituted and O-alkyl oximes were prepared and tested. The
inhibitory activity of the resultant compounds is listed in Table 3.
In general, all modifications led to potent activity against EGFR.
However, compounds with bulky and hydrophobic substituents
(33, 35, and 36) are less potent in the ErbB-2 assay. The cellular
activity of these compounds was evaluated in proliferation assays
using the SKBR3 and BT474 cell lines,19 both of which overexpress
ErbB-2. Compounds 13, 32, 34, 35, and 36 showed submicromolar
cellular antiproliferative potency, whereas compound 31 had re-
duced activity even at 10 lM, suggesting that it does not penetrate
the cell membrane effectively.
In order to determine the kinase selectivity for this series, com-
pounds 25 and 28 were evaluated against a panel of 102 kinases at
the concentration of 3 lM in the presence of 100 lM ATP20 and
found to be highly selective for the EGFR subfamily. In pharmaco-
kinetic evaluation in Sprague–Dawley rats, compound 28 exhibited
high plasma levels, low volume of distribution, and low clearance
(Table 4). Its bioavailability is 15%.
To further validate our design rationale, an X-ray crystal struc-
ture of EGFR with compound 37 bound at the ATP site was solved
at a resolution of 2.3 Å.21 Figure 3 illustrates the complex and
shows that the pyrimidine ring nitrogen N3 interacts with the
backbone NH of Met793 in the kinase hinge region, whereas the
amino group at the C-4 position is engaged in a second hydrogen
bond with the backbone C@O of Met793. This binding mode ex-
plains the finding that N-methylation at C-4 amino position was
detrimental for activity. The 1-(3-fluorobenzyl)indazolylamino
group is oriented deep in the back of the ATP binding site and
makes predominantly hydrophobic interactions with the protein.
The 3-fluorobenzyl group occupies a pocket formed by the side
chains of Met766, Leu777, Thr790, Thr854, and Phe856. The 3-flu-
oro on the benzyl group is hydrogen bonded to both backbone NH
groups of Arg776 and Thr790, and is important for optimally po-
tent inhibition of EGFR. This also explains why replacement by
chlorine, cyano, or methoxy group resulted in less potent com-
pounds (20, 21, and 22), because they cannot occupy this binding
pocket without displacing key residues. The N-2 atom of the inda-
zole ring forms a weak hydrogen bond to the backbone C@O of Leu
788 (3.7 Å). The aniline nitrogen and the N-1 atom of the indazole
ring are not involved in any direct hydrogen bonding interactions
with the protein. The methoxyethoxy moiety appended to the
oxime is extended to the solvent region, which supports the find-
ing that most of the analogues with alkyl oxime substituents give
favorable inhibitory potency against both EGFR and ErbB-2. The
C-4 amino group forms an intramolecular hydrogen bond with
the oxime nitrogen atom as we anticipated, therefore mimicking
the quinazoline phenyl ring.
a
Mean values of at least three experiments are used for enzyme assays of ErbB-2/
17
EGFR, IC50 values reported as lM concentrations. See Ref. for assay conditions.
Table 2
Structure–activity relationships for the C-6 aniline substitution
O
N
H
5
6
N
1
NH2
Ar
4
3
N
N
2
Compound
Ar
EGFR
IC50 (lM)
ErbB-2
IC50 (lM)
a
a
15
16
17
18
19
20
21
22
13
23
(3-Cl-4-F)phenyl
3-Br-phenyl
(3-Cl-4-F)benzyl
Indazol-5-yl
1-benzyl-indazol-5-yl
1-(3-Cl-benzyl)indazol-5-yl
1-(3-CN-benzyl)indazol-5-yl
1-(3-OCH3-benzyl)indazol-5-yl
1-(3-F-benzyl)indazol-5-yl
3-Cl-4-(3,5-F2-benzyloxy)phenyl
0.070
0.024
1.123
0.306
0.044
0.023
0.020
0.267
0.008
0.033
0.204
0.557
5.22
4.123
0.022
0.030
0.044
NTb
0.012
(52% inh
at 10 lM)
0.189
24
25
26
27
28
29
30
1-(4-F-benzyl)indazol-5-yl
0.104
1-(3-F-benzyl)indol-5-yl
0.013
0.027
2-(3-F-benzyl)benzoimidazol-5-yl
1-(3-F-benzyl)-2,3-dihydro-indol-5-yl
(3-Cl-4-benzyloxy)phenyl
0.048
0.057
0.016
0.076
0.255
0.153
3-Cl-4-(3-F-benzyloxy)phenyl
2-(3-F-phenyl)benzofuran-5-yl
0.012
(<10% inh
at 2 lM)
0.018
(<10% inh
at 10 lM)
a
Mean values of at least three experiments are used for enzyme assays of ErbB-2/
EGFR.
b
Not tested.
kinase inhibitory activities (Table 2). Analogues with small C-6 ani-
lines (15 and 16) or simple fused 5,6-bicyclic heterocycles (18)
were selective EGFR inhibitors. However, this activity was dramat-
ically decreased if the aromatic ring was displaced by one carbon
(17). Appending a lipophilic benzyl group (19) to the C-6 indazol-
ylamine increased ErbB-2 kinase inhibition without reducing EGFR
potency. A similar trend was also reported in the quinazoline series
where increasing size at the C-4 aniline position substantially im-
proved ErbB-2 activity.18 Both m-chlorobenzyl (20) and m-cyanob-
enzyl (21) analogues showed comparable potency to compound
19. Interestingly, adding a fluorine atom at the 3-position of the
benzyl group (13) resulted in a 5-fold increase in inhibitory po-
tency against EGFR and an approximate 2-fold increase against
ErbB-2, highlighting the potential interaction of fluorine with the
enzyme (vide infra). There was slight increase in EGFR potency
for the 3,5-difluorobenzyl compound 23. However, larger groups
In summary, 4-amino-6-arylaminopyrimidine-5-carbaldehyde
oxime derivatives showed potent inhibition of ErbB-2/EGFR ki-
nase activities and antiproliferative effects toward ErbB-2 over-
expressing SK-BR-3 and BT474 cell lines. An unsubstituted
amino group at the C-4 position is essential for potent inhibitory

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G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3495–3499
Table 3
Structure–activity relationships for the C-5 side chain
R2
O
N
H
N
F
5
6
NH2
4
1
3
N
N
N
N
2
a
a
a
a
Compound
R2
EGFR IC50 (lM)
ErbB-2 IC50 (lM)
SKBR3 IC50 (lM)
BT474 IC50 (lM)
13
31
32
33
34
35
36
37
CH3
H
CH2CH3
CH2CH(CH3)2
CH(CH3)2
benzyl
2-OCH3-benzyl
(CH2)2OCH3
0.008
0.012
0.005
0.032
0.014
0.023
0.047
0.014
0.012
0.042
0.007
0.173
0.025
0.095
0.435
0.006
0.26
0.25
(41% inh at 10 lM)
0.27
1.88
0.71
0.18
0.13
(34% inh at 10 lM)
0.062
1.03
0.54
0.13
0.21
(84% inh at 10 lM)
(60% inh at 10 lM)
a
Mean values of at least three experiments are used for enzyme assays of ErbB-2/EGFR, IC50 values reported as lM concentrations.
Table 4
Pharmacokinetic parameters for compound 28, determined after dosing to Sprague–Dawley rats at 2 mg/kg iv (vehicle = 10% solutol in D5W) and 10 mg/kg po (vehicle = 0.5%
hydroxypropyl methylcellulose)
Compound
Vdss (L/Kg)
Cl (ml/min/Kg)
3.56
po Cmax (lM)
po Tmax (h)
0.63
po T1/2 (h)
8.4
po AUC (lM.h)
Bioavailability %
15
28
0.17
5.7
19.3
Acknowledgment
Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the Ad-
vanced Photon Source was supported by the companies of the
Industrial Macromolecular Crystallography Association through a
contract with the Center for Advanced Radiation Sources at the
University of Chicago.
References and notes
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Figure 3. Interactions between EGFR and compound 37. Compound 37 is shown in
green, protein residues are colored gray and atoms are colored by element for both
molecules with nitrogen blue, oxygen red and fluorine light green.
6. Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.; Alligood, K. J.; Rhodes, N.;
Keith, B. R.; Murray, D. M.; Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer
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Oppenheimer, S.; Ruediger, E.; Tarrant, J.; Tokarski, J. S.; Vite, G. D.; Vyas, D.
activity. This result was supported by the X-ray crystallographic
elucidation of the complex of 37 with EGFR, where the NH2
made key hydrogen bonding interaction with backbone C@O of
Met 793. The C-6 aniline portion determines the potency and ki-
nase selectivity, and this conclusion parallels the literature SAR
at the C-4 aniline position reported for quinazoline-based RTK
inhibitors.22,23 The oxime side chain is oriented toward
the solvent front, consistent with the observation that a variety
of substituents were tolerated. These studies suggest that
4-amino-6-arylaminopyrimidine-5-carbaldehyde oxime scaffold
effectively mimics the well-known quinazoline kinase template.
Future reports will detail the design of potent dual EGFR/ErbB-
2
inhibitors with improved bioavailability that demonstrate
antitumor efficacy in preclinical disease models.

G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3495–3499
3499
M.; Wong, H.; Wong, T. W.; Zhang, H.; Zhang, G. Bioorg. Med. Chem. Lett. 2007,
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0.8 lm vacuum filter and mixed to
a
TALON metal affinity resin (BD
Biosciences) overnight at 4 °C. The lysate-TALON mixture was loaded into a
column and washed with 10 column volumes of Buffer A (50 mM Tris pH 8.0,
500 mM NaCl, 5 mM imidazole.) EGFR was eluted using a 10 column volume
linear gradient going from Buffer A to Buffer B (50 mM Tris pH 8.0, 300 mM
NaCl, 250 mM Imidazole.) Fractions containing EGFR, as assayed by SDS–PAGE,
were pooled. Thrombin was added to the pooled protein to remove the
histidine tag (0.005 U of thrombin/lg EGFR). The reaction was dialyzed
overnight against 50 mM Tris buffer pH 8.0, 250 mM NaCl, 1 mM DTT.
16. Cockerill, S.; Stubberfield, C.; Stables, J.; Carter, M.; Guntrip, S.; Smith, K.;
MaKeown, S.; Shaw, R.; Topley, P.; Thomsen, L.; Affeck, K.; Jowett, A.; Hayes, D.;
Willson, M.; Woollard, P.; Spalding, D. Bioorg. Med. Chem. Lett. 2001, 11, 1401.
17. The reaction was incubated for 60 min at 30 °C for ErbB-2 in (60 nM Hepes pH
7.5, 3 nM magnesium chloride, 3 mM manganese chloride, 0.003 mM sodium
vanadate, 1.2 mM DTT, 50 g/mL PEG 20,000, 0.001 mM ATP, 1.5 ng/mL
biotinylated polyGluTyr and 0.2 lCuries 33P-c-ATP) and for EGFR in (50 mM
Tris pH 8.0, 10 mM manganese chloride, 0.1 mM sodium vanadate, 1 mM DTT,
0.005 mM ATP, 1.5 ng/mL biotinylated polyGluTyr and 0.2 lCuries 33P-c-ATP)
in streptavidin coated FlashPlates (NEN, Boston, MA). Plates were sealed and
read on the TopCount scintillation counter. Each measurement was performed
at least in duplicate and the IC50 values were calculated with standard
deviation from 2 to 8 separate experiments.
18. (a) Bhattacharya, S. K.; Cox, E. D.; Kath, J. C.; Mathiowetz, A. M.; Morris, J.; Moyer,
J. D.; Pustilnik, L. R.; Rafidi, K.; Richter, D. T.; Su, C.; Wessel, M. D. Biochem. Biophys.
Res. Commun 2003, 307, 267; (b) Brignola, P. S.; Lackey, K.; Kadwell, S. H.;
Hoffman, C.; Horne, E.; Carter, H. L.; Stuart, J. D.; Blackburn, K.; Moyer, M. B.;
Alligood, K. J.; Knight, W. B.; Wood, E. R. J. Bio. Chem. 2002, 277, 1576.
19. Antiproliferative activity of dual EGFR/ErbB-2 kinase inhibitors was assessed in
monolayer cultures by 14C-thymidine incorporation into cellular DNA as
described Emanuel, S. et al Mol. Pharm. 2004, 66, 635. except that total time in
which cells were exposed to drug was 96 h.
Cleaved EGFR was filtered through
a 0.2 lm SFCA cartridge filter,
concentrated, and loaded onto Superdex 200 HR 10/30 column (GE
a
Healthcare,) preequilibrated with 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM
DTT. Fractions containing EGFR, as assayed by SDS–PAGE, were pooled and
dialyzed against 10 mM Tris pH 8.0, 1 mM DTT, 1 mM sodium azide, 0.1 mM
benzamidine.; (d) Crystallization, data collection, molecular replacement, and
structure refinement of the EGFR-compound 37 complex. Purified human EGFR
was concentrated to 4 mg/ml, complex with compound 37 in a 1:2 ratio and
crystallized from a solution containing 2 M Na/K3PO4, 0.1 M Caps pH 9.0 and
0.2 M Li2SO4.16 X-ray diffraction data to a resolution of 2.0 Å were collected at
the IMCA-CAT ID-17 beamline at the Argonne National Laboratory. Diffraction
data were indexed, integrated, and scaled using the HKL2000 suite. The EGFR
crystals belong to the P212121 space group, with unit cell parameters
a = 45.50, b = 67.69, c = 103.25 Å. The structure was determined by molecular
replacement with CNX [Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L.,
Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N.
S., Read, R. J., Rice, L. M., Simonson, T., Warren, G. L (1998) Acta Crystallogr D54,
905–921] using the structure of EGFR complexed with Lapatinib as a search
model (PDB id 1XKK).16 All model building was done using O [Jones, T. A., Zou,
J. Y., Cowan, S. W., Kjeldgaard, M. (1991) Acta Crystallogr A47, 110–119] and
refinement and map calculations were carried out using CNX [Brunger, A. T.,
Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang,
J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T.,
Warren, G. L. (1998) Acta Crystallogr D 54, 905–921]. The final structure was
refined to an Rfactor of 22.7 and Rfree of 26.7.
20. Upstate Cell Signaling Solutions, Charlottesville, VA.
21. (a) PDB code is 3BEL.; (b) EGFR clonning. The DNA sequence encoding amino
acids S671-G998 of human EGFR mature protein (Genbank Accession #NM
005228) was amplified by polymerase chain reaction (PCR) and inserted into
the vector pDEST8 (Invitrogen) modified to include sequences encoding an in-
frame hexameric histidine sequence and
a thrombin cleavage site. The
resulting construct thus encoded MHHHHHHVDLVPRGSHMA-(EGFRS671-G998
)
which was confirmed by DNA sequencing. This expression construct was then
integrated into bacmid DNA and a recombinant isolate used to tansfect SF9
insect cells as recommended by the vendor. The resulting baculovirus was then
used to infect additional Sf9 cells to increase viral titer and volume.; (c) EGFR
purification. All purification processes were carried out on an ÄKTA FPLC
system (GE Healthcare) at 4 °C. The purification protocol was performed as
described previously [J. Biol. Chem. (2002), 277, p. 46265–46272,]. Briefly,
frozen cells were thawed and resuspended in 50 mM Tris pH 7.5, 200 mM NaCl,
1% glycerol, 5 mM BME, 1 X complete EDTA-free protease inhibitor cocktail
(Roche). Resuspended cells were dounce homogenized and mechanically lysed
with an Emulsiflex-C5 (Avestin) at 10,000–15,000 psi. The lysate was clarified
by centrifugation at 40,000g for 1 h. The supernatant was filtered through a
22. Rusnak, D. W.; Affleck, K.; Cockerill, S. G.; Stubberfield, C.; Harris, R.; Page, M.;
Smith, K. J.; Guntrip, S. B.; Carter, M. C.; Shaw, R. J.; Jowett, A.; Stables, J.;
Topley, P.; Wood, E. R.; Brignola, P. S.; Kadwell, S. H.; Reep, B. R.; Mullin, R. J.;
Alligood, K. J.; Keith, B. R.; Crosby, R. M.; Murray, D. M.; Knight, W. B.; Gilmer, T.
M.; Lackey, K. Cancer Res. 2001, 61, 7196.
23. Gaul, M. D.; Guo, Y.; Affleck, K.; Cockerill, G. S.; Gilmer, T. M.; Griffin, R. J.;
Guntrip, S.; Keith, B. R.; Knight, W. B.; Mullin, R. J.; Murray, D. M.; Rusnak, D.
W.; Smith, K.; Tadepalli, S.; Wood, E. R.; Lackey, K. Bioorg. Med. Chem. Lett. 2003,
13, 637.