From the Research Unit Molecular Cell Biology,
Medical Faculty, Friedrich Schiller University, D-07747 Jena,
Germany, the ¶ Institute of Molecular Biology, Bulgarian
Academy of Sciences, 1113 Sofia, Bulgaria, the
Department of
Medicine/Hematology and Oncology, University of
Münster, D-48149 Münster, Germany, and the
** Institute of Pharmacy, University of Regensburg, D-93040
Regensburg, Germany
Received for publication, September 25, 2002, and in revised form, November 1, 2002
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ABSTRACT |
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The tyrosine kinase inhibitor STI-571 potently
blocks BCR-Abl, platelet-derived growth factor (PDGF) Tyrosine kinase inhibitors have a great pharmacological potential
for the treatment of various forms of cancer and other diseases. Most
of the recent leads competitively bind at the ATP site of the kinase
domain but are nevertheless fairly selective. Some crystal structures
of kinase-inhibitor complexes, e.g. of Hck from the c-Src
family with the ATP site inhibitor PP1/AGL1872 (1) and of the
fibroblast growth factor receptor-1
(FGFR-1)1 tyrosine kinase
with different inhibitors (2, 3), indicate molecular
determinants of inhibitor selectivity. Because of its pharmacological
profile and therapeutic potential, the phenylaminopyrimidine STI-571
(GleevecTM, formerly CGP57148B) has received much
attention. This compound inhibits Abelson tyrosine kinases (c-Abl,
BCR-Abl) platelet-derived growth factor (PDGF) PDGFR- Flt3 (Flk2, STK1) is structurally closely related to the PDGF receptor
kinases and c-Kit. It is overexpressed in various types of leukemia,
including B-lineage acute lymphoblastic leukemia and acute
myeloid leukemia as well as T-lineage acute lymphoblastic leukemia and
chronic myelogenous leukemia blast crisis cells (15-17). Different
activating mutations in the Flt3 gene have been
detected in acute myeloid leukemia patients. Flt3 may, therefore, be a suitable target for therapy of Flt3-dependent leukemias.
Despite its close homology to PDGF receptors, Flt3 kinase is not
inhibited by STI-571.
Multiple sequence alignments and the three-dimensional structure
of the Abl kinase STI-571 complex (6) indicate possible reasons for
selectivity. In some chronic myelogenous leukemia patients who
responded initially to STI-571 but then relapsed, the resistance to the
drug was associated with a single T315I mutation in the Synthesis of STI-571--
Synthesis was done according to
published procedures (21), which contain, however, no details of the
reaction conditions for the individual steps. Reaction conditions were
determined empirically and are given in the synthesis scheme (found in
the Supplemental Material, available online at
http://www.jbc.org).
DNA Constructs--
Human Flt3 and Flt3-ITD cDNAs were
cloned as described previously (22, 23) and subcloned into the
pcDNA3.1 (Invitrogen) eukaryotic expression vector. Versions with a
C-terminal HA tag were constructed and kindly provided by D. Schmidt-Arras (Friedrich Schiller University, Jena, Germany).
cDNAs of the human PDGF Kinase Assays--
The expression constructs of Flt3 and PDGF
In vitro kinase assays for PDGFR activity against an
exogenous peptide substrate were performed in a similar manner to those described previously (25). HEK293 cells, stably transfected with the
HA-tagged PDGFR- Modeling--
Initial computer models of the PDGFR-
Side chains of the models were added using a knowledge-based approach,
taking in account the backbone secondary structure and the side chains
at the corresponding residues of the templates. The structurally
variable regions of the models were constructed by the loop search
algorithm within SYBYL. For each structurally variable region,
appropriate fragments from the binary PDB data base with the same
length are proposed on the basis of the distances and superpositions of
the anchor residues. All fragments finally selected for insertion into
the models were extracted from one of the tyrosine kinase template
structures. The activation loop and the nucleotide binding loop were
refined by additional loop searches (see "Results" and
"Discussion").
After hydrogens were added, the models were roughly energy-minimized
using the AMBER 4.1 force field (30) with AMBER95 charges (distance-dependent dielectricity constant 1, ~500
cycles, first 50 cycles with constrained backbone, steepest descent
method). The inhibitor STI-571 was extracted from the crystal structure 1iep and provided with AMBER 4.1 atom types by analogy with corresponding amino acid atoms as well as with Gasteiger-Hueckel charges. New parameters describing aromatic carbon-nitrogen and aromatic-sp3 carbon bonds, bond angles, and torsion had to be added to
the AMBER 4.1 force field for STI-571 (derived from the Tripos force
field and from a comparison with similar AMBER parameters). The STI-571
conformation was then docked into the PDGFR- Flt3 Phe-691/PDGFR-
To evaluate whether the formation of a hydrogen bond between PDGFR-
It seems that the residue in The Activation Loop, Another Critical Determinant of Inhibitor
Selectivity--
The STI-571 binding site of murine c-Abl (6, 20)
spans the whole core between both kinase domains and contains 23 residues within 3 Å around the ligand. The perfect complementary fit
of STI-571 to the inactive, nonphosphorylated state of Abl (6) indicates in particular that little spatial scope is left for the most
flexible functional regions, the activation and the nucleotide binding
loop. Activation of Abl by prephosphorylation greatly reduced the
STI-571 sensitivity of the kinase (6). We therefore tested whether
phosphorylation and activation of the PDGFR-
The conformation of the activation loop is a feature that
distinguishes not only between the phosphorylated and the
inactive state of a given species but also between different tyrosine
kinases. Binding of STI-571 at Abl kinase involves interactions with
Asp-381 and Phe-382 in the highly conserved N-terminal anchor region
(the Asp-Phe-Gly motif) of the loop. Schindler et al. (6)
have demonstrated that in the complex between STI-571 and the natural,
autoinhibitory Abl conformation, Tyr-393 mimics a tyrosine residue of
substrate peptides but is not phosphorylated because of the
displacement of Asp-Phe-Gly. Inactivity of STI-571 at tyrosine kinases
of the Src family (Hck, Lck) in both the inactive and active states
follows from collision with this motif. Thus, an appropriate
Asp-Phe-Gly course seems to be essential for STI-571 binding. To model
activation loops of the PDGF-
Table I presents the
Fig. 6 illustrates the Asp-Phe-Gly
courses of the seven kinases together with a volume contour of STI-571
bound to Abl. The model is based on an alignment of C Computer Models of the PDGFR-
As derived previously, the Asp-Phe-Gly courses must be similar
to those in c-Abl to enable binding of STI-571. Modeling approaches based on this assumption, however, have demonstrated that even the
entire activation loop of the targets may follow an Abl-like course.
This implies an autoinhibitory mechanism with Tyr-857 (PDGFR-
The nucleotide binding loops are well ordered in the crystal structures
of Abl and FGFR-1 complexed with the inhibitors STI-571 and SU-5402,
respectively, and adopt similar, specific conformations induced by
ligand fitting. The aromatic side chains of Abl Tyr-253 and FGFR-1
Phe-489 stabilize the down-fold of the loops by van der Waals contacts
with the inhibitors and by a water-mediated hydrogen bond between
Tyr-253 and Asn-322 in the case of Abl, or by oxygen-aromatic
interactions in the case of FGFR-1. A corresponding fold of the
nucleotide binding loops of PDGFR-
On the basis of these considerations, the preliminary SCR models of the
PDGFR-
The binding sites of the PDGFR-
Van der Waals interactions in particular contribute to the
complementary fit of the 4-pyridin-3-yl-pyrimidin-2-ylaminophenyl moiety, involving aromatic and aliphatic side chains from different regions, e.g. Leu-606 (
In summary, the predicted interactions of STI-571 with the PDGFR-- and
-receptors, and c-Kit kinase activity. Flt3, a receptor tyrosine
kinase closely related to PDGF receptors and c-Kit is, however, not
inhibited by STI-571. Sequence alignments of different kinases and
indications from the crystal structure of the STI-571 Abl kinase
complex revealed amino acid residues that are probably crucial for this
activity profile. It was predicted that Flt3 Phe-691 in the
5
strand may sterically prevent interaction with STI-571. The point
mutants Flt3 F691T and PDGF
-receptor T681F were constructed, and
kinase assays showed that the Flt3 mutant but not the PDGF
-receptor mutant is inhibited by STI-571. Docking of STI-571 into computer models
of the PDGF
-receptor and Flt3 kinase domains and comparison with the
crystal structure of the STI-571 Abl kinase complex indicated very
similar binding sites among the three nonphosphorylated kinases, suggesting corresponding courses of their Asp-Phe-Gly
motifs and activation loops. Accordingly, we observed reduced
sensitivity of preactivated compared with nonactivated PDGFR-
for
the inhibition by STI-571. Courses of the activation loop that collide
with STI-571 binding explain its inactivity at other kinases as the
insulin receptor. The binding site models of PDGFR-
and Flt3
were applied to predict structural approaches for more selective
PDGF
-receptor inhibitors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
- and
-receptor
and c-Kit kinase activity with similar potency (4). Based on inhibition
of BCR-Abl, STI-571 has recently been introduced successfully into the
treatment of chronic myelogenous leukemia (5). Further clinical
applications of STI-571 may rest on the inhibition of c-Kit and PDGF
receptors and on the established role of these kinases in certain forms of cancer. Two crystal structures of the murine Abl kinase in complex
with STI-571 and with a smaller variant (6) suggest binding of the
inhibitor to the inactive kinase state with nonphosphorylated Tyr-393,
as observed earlier by others for PP1/AGL1872 and Hck (1).
and -
are members of the class III receptor tyrosine
kinases. Aberrant activation of PDGF receptors has been linked to
several disease states including certain malignancies and
atherosclerosis, restenosis, and fibrotic conditions (7). Selective
PDGF receptor tyrosine kinase inhibitors have therefore been developed.
These include phenylaminopyrimidines (8) such as STI-571,
phenylbenzimidazoles (9, 10), quinoxalines (11, 12),
6,7-dimethoxyquinolines (13), and bis(1H-2-indolyl)
methanones (14).
5 strand of
the Abl kinase domain (18). The side chain of Thr-315 is assumed to
form a critical O-HN hydrogen bond with the pyrimidinylamino group of
STI-571. Replacement of Abl Thr-315 by IRK Met-1076 has been suggested
as responsible for the inactivity of the drug at the nonphosphorylated
insulin receptor kinase (6). In a similar manner, the selectivity of the ATP site inhibitor PP1/AGL1872 to p60c-Src versus v-Src
kinase is related to the replacement of p60c-Src Thr-338, which
corresponds to Abl Thr-315, by a v-Src Ile residue (19). Abl Thr-315 is equivalent to Thr-681 in the PDGF
receptor kinase and to Phe-691 in
Flt3. Considering the 23 residues within 3 Å around STI-571 in the Abl
kinase crystal structure, 1iep (20), this Thr
Phe replacement in
5 is indeed the only significant difference between the STI-571 binding kinases, Abl and PDGFR, on the one hand, and the
nonbinding kinase Flt3, on the other hand. To investigate whether
sterical hindrance by the Phe-691 side chain prevents inhibition of
Flt3 by the drug, cross-wise amino acid exchanges between Flt3 and
PDGFR-
were performed and analyzed for inhibitor sensitivity in the
present study. Computer models of the STI-571 kinase complexes, based
on sequence alignments and homology modeling using crystal structures
of four different tyrosine kinases, provided further details of the
binding mechanism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
receptor (M21616) and human FGFR-1 were
kindly provided by Drs. C. H. Heldin and L. Claesson-Welsh (Uppsala,
Sweden), respectively, and subcloned into pcDNA3.1. A C-terminal HA
tag was introduced into the PDGFR-
sequence as described previously
(24). A cDNA of the human insulin receptor in pRK5RS was kindly
provided by Dr. A. Ullrich (Martinsried, Germany). Point mutations were
introduced with the Quick-Xchange kit (Stratagene) according to the
instructions of the manufacturer and were verified by DNA sequencing
(MWG Biotech).
receptor and the corresponding mutants were transfected into HEK293
cells as described previously. The transfected cells were
starved overnight in 0.5% fetal calf serum/Dulbecco's modified
Eagle's medium. Incubation with STI-571 (or with
Me2SO as solvent control, final concentration 1%)
was performed at 37 °C for 30 min, and then the cells were stimulated with Flt3 ligand (FL, PeproTech) or PDGF-BB (PeproTech) at
room temperature in a concentration of 100 ng/ml for 5 min or 50 ng/ml
for 10 min, respectively. The cells were lysed in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM EDTA, 2 mM EGTA, 10 mM
sodium pyrophosphate, 50 mM NaF, 5 µg/ml leupeptin, 20 µM zinc acetate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.3 µM
aprotinin, and 1 mM benzamidine, and cell extracts were
subjected to affinity purification with wheat germ agglutinin-agarose
beads (Amersham Biosciences) as described previously. Tyrosine
phosphorylation of the overexpressed receptors was detected by
immunoblotting with monoclonal anti-phosphotyrosine antibodies (4G10,
Upstate Biochemicals, Inc.). Receptor loading was controlled by
immunoblotting with anti-Flt3 antibody C-20 (Santa Cruz Biotechnology)
or anti-HA-antibody (BabCO, Richmond, CA). For in vitro
kinase assays, the lysates of transfected HEK293 cells were subjected
to immunoprecipitation (5 µg of anti-HA antibody or 5 µg of
anti-Flt3 and 100 µl of protein A-Sepharose/10-cm dish), and the
immunoprecipitates were washed three times in lysis buffer and once
with kinase buffer containing 50 mM HEPES, pH 7.4, 5 mM MnCl2, and 0.1 mM sodium
orthovanadate. The immunoprecipitates of each dish were suspended,
divided into six aliquots in new tubes, resedimented, and resuspended
in 20 µl of kinase buffer. STI-571 or Me2SO solvent
control (1% final concentration) was added for 30 min on ice, and then
[
-32P]ATP was added (2-3 µCi/sample in 5 µl) and
the kinase reaction was allowed to proceed at 30 °C for 20 min. The samples were boiled with SDS-PAGE sample buffer and analyzed
by SDS-PAGE and autoradiography.
(kindly provided by Dr. B. Markova), were starved
overnight in medium containing 0.5% fetal calf serum, stimulated with
PDGF-BB (for obtaining preactivated receptor) or left unstimulated, and
were then extracted. PDGFR-
was immunoprecipitated with anti-HA
antibodies. The immunoprecipitate from PDGF-stimulated cells was
treated under shaking with 1.2 mM ATP at 30 °C for 15 min in 50 mM Hepes, pH 7.5, 5 mM
MnCl2, 0.1 mM sodium orthovanadate (kinase
buffer). The immunoprecipitate from nonstimulated cells was treated
likewise in buffer without ATP. The immunoprecipitates were washed
three times in ice-cold kinase buffer and were then aliquoted. Aliquots
were incubated with STI-571 at different concentrations on ice for 20 min. KY751 peptide (25) was added to a final concentration of 2 mM, and the kinase reaction was initiated by the addition of [
-32P]ATP (2.5 µCi/sample) and allowed to
proceed for 20 min at 30 °C. The reaction was stopped by adding EDTA
to a final concentration of 100 mM, and peptide
phosphorylation was evaluated as described (25).
kinase
and the Flt3 F691T mutant, excluding the kinase insert regions, were
generated using the program COMPOSER (26), part of the molecular
modeling package SYBYL, version 6.8 (Tripos Inc., St. Louis,
MO). Eight crystal structures from the SYBYL binary Protein Data Bank
(PDB) library were selected as templates by overall sequence identity: VEGFR-2 (PDB code 1vr2, chain A (27), identity 54.6% with PDGFR, 52.9% with Flt3); FGFR-1 (PDB codes 1fgi, chain A, 1agw, chain
A (2), 1fgk, chain A (28), and 2fgi (3), identity 51.4-53.6% with
PDGFR and 49.3-51.8% with Flt3); murine c-Abl kinase (PDB code 1fpu,
chain A (6), 1iep, chain A (20), identity 37.7% with PDGFR and 40.4%
with Flt3), inactive insulin receptor kinase (1irk (29), identity
36.1% with PDGFR and 38.2% with Flt3). On the basis of optimal
sequence alignments, the structurally conserved regions (SCR) (see Fig.
1) and an average C
framework structure of the template SCRs were
determined by an iterative approach, improving both the multiple
alignment and the subsequent SCR framework by pairwise Needleman and
Wunsch dynamic programming procedures with a similarity matrix
constructed from inter-C
distances. The backbone of each SCR of the
PDGFR-
kinase and the Flt3 F691T mutant was then built by fitting
the corresponding SCR from one of the known homologs (namely that with
the highest block sequence identity, mostly VEGFR-2) to the appropriate
region of the framework. The least-squares fits are inversely weighted
by the variation of the residue positions across the known structures.
The average r.m.s. distance of the eight templates (pairwise fits of
SCR C
atoms) amounts to 1.07 Å (s = 0.50 Å), and
the poorest fits are with the insulin receptor kinase (r.m.s.
1.53-1.74 Å). The r.m.s. distances of the initial PDGFR-
kinase
(0.67 (FGFR 1fgi) to 1.65 Å (1irk)) and Flt3 F691T mutant (0.56 (FGFR
1fgk) to 1.51 Å (1irk)) models are in the same range. This approach
provides a sufficient degree of diversity in constructing the SCRs of
the models and avoids an arbitrary focus on Abl.
kinase and the
Flt3 F691T mutant models together with those water molecules from the
1iep Abl kinase structure lying within 6 Å around the co-crystallized
inhibitor. The complexes were roughly preoptimized (200 cycles,
steepest descent with constrained backbone), and finally minimized up
to an r.m.s. gradient of 0.05 kcal/mol Å (Powell conjugate gradient).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Thr-681 as a Critical
"Switch" for Inhibitor Sensitivity--
According to the sequence
alignment (Fig. 1) and the crystal
structures of the Abl kinase, Phe-691 of Flt3 may prevent binding of
STI-571. To test this prediction, mutant variants of Flt3 and PDGFR-
were generated and analyzed with respect to inhibition by STI-571.
Phe-691 in Flt3 was replaced by Thr, the corresponding residue in the
PDGFR-
kinase. When expressed in HEK293 cells, the Flt3 F691T
variant has a somewhat reduced kinase activity compared with wild-type
Flt3. Importantly, STI-571 inhibits Flt3 F691T with an IC50
of 0.1-0.3 µM (Fig.
2A, lower panel),
which is very close to the known IC50 of PDGFR-
kinase
inhibition in intact cells (Ref. 4; see also Fig.
3). As shown previously (4), Flt3 wild
type is refractory to STI-571 inhibition (Fig. 2A,
upper panel). The in vitro immunocomplex assays (Fig.
2B) confirmed these results, although higher concentrations
of STI-571 are required to obtain complete inhibition of Flt3 F691T.
The strong difference in susceptibility compared with Flt3 wild type is
still obvious. Corresponding results were obtained with a
pathologically relevant, constitutively active Flt3 variant harboring
an internal tandem duplication (ITD) in the juxtamembrane domain.
Although Flt3ITD is resistant to inhibition (Fig. 2C,
upper panel), the Flt3ITD F691T variant was potently inhibited by
STI-571 (Fig. 2C, lower panel). In agreement with
the suggested Abl-like binding mode, the replacement of Phe-691 with
Thr removes the sterical constraints for binding of STI-571. As an
additional indication of this mechanism, replacing Thr-681 by Phe in
the corresponding position of the PDGFR-
kinase should lead to
inactivity of STI-571. This was indeed the case; although the wild-type
PDGFR-
kinase was potently inhibited by STI-571 (Fig.
3A, upper panel) with an IC50 of
0.1-0.3 µM, the PDGFR-
T681F mutant had an unaltered kinase activity but was unresponsive to STI-571 inhibition (Fig. 3A, lower panel). These findings were reconfirmed
by in vitro kinase assays with corresponding
immunoprecipitates (Fig. 3B).
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Fig. 1.
Sequence alignment of the targets PDGFR-
and Flt3 with the four tyrosine kinases c-Abl, FGFR-1, IRK, and
VEGFR-2. The alignment results from the COMPOSER algorithm
implied in SYBYL 6.8, considering three-dimensional superposition
(similarity matrix was constructed from inter-C
distances). The
numbering of residues in the second column corresponds to
the PDB files (see "Experimental Procedures"). Bars
delineate secondary structure elements and functional loops.
SCR, structurally conserved regions used for construction of
the model frames in COMPOSER. Bold (additionally, Abl is
underlined) residues depict the STI-571 binding site (3 Å around STI-571 according to the 1iep crystal structure with hydrogens
added). An asterisk denotes the position of the critical
residue, Thr-681, in PDGFR-
.
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Fig. 2.
Flt3 F691T is sensitive to
inhibition by STI-571. Flt3 or the indicated Flt3 variants were
expressed in HEK293 cells. A, the cells were treated with
STI-571 in the indicated concentrations and lysed, and the
overexpressed receptor tyrosine kinases were isolated by wheat germ
agglutinin affinity precipitation. Tyrosine phosphorylation and
receptor expression levels were evaluated by immunoblotting with
anti-phosphotyrosine or anti-Flt3 antibodies, respectively.
WB, Western blot; PY, phosphotyrosine;
Wt, wild type. B, Flt3 was immunoprecipitated
from overexpressing HEK293 cells with anti-Flt3 antibodies. The
immunoprecipitated kinase variants were treated with STI-571 at the
indicated concentrations and subsequently subjected to an
autophosphorylation reaction in the presence of
[ -32P]ATP. The Flt3 autophosphorylation level was
analyzed by autoradiography. C, the experiment was performed
as described in A, except that HA epitope-tagged Flt3ITD
variants were used. The numbering of the Thr/Phe-691 of Flt3,
maintained for simplicity, does not consider the ITD insert.
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Fig. 3.
T681F mutation renders
PDGF -R insensitive to inhibition by
STI-571. HA-tagged PDGFR-
or PDGFR-
T681F were expressed in
HEK293 cells. A, the cells were treated with STI-571 in the
indicated concentrations and lysed, and the overexpressed receptor
tyrosine kinases were isolated by wheat germ agglutinin affinity
precipitation. Tyrosine phosphorylation and receptor expression levels
were evaluated by immunoblotting with anti-phosphotyrosine or anti-HA
antibodies, respectively. WB, Western blot; PY,
phosphotyrosine; Wt, wild type. B, PDGFR-
was
immunoprecipitated from overexpressing HEK293 cells with anti-HA
antibodies, and the immunoprecipitated kinase variants were treated
with STI-571 at the indicated concentrations and subjected subsequently
to an autophosphorylation reaction in the presence of
[
-32P]ATP. The PDGFR-
autophosphorylation level was
analyzed by autoradiography.
Thr-681 and the STI-571 pyrimidinylamino group is important for STI-571
binding, we also tested a PDGFR-
T681A mutant. Interestingly, this
mutant was not less, but even somewhat more, susceptible to STI-571
inhibition than the PDGFR-
wild type (mean IC50, 0.18 µM in five experiments versus 0.34 µM in six experiments, respectively), indicating no
independent contribution of the hydrogen bond at least in the case of
PDGFR-
. Possibly, there is no net gain of binding energy because the
H-bond formation may be preceded by the displacement of a water
molecule H-bonded to Thr-681. This is not the case in the Ala mutant,
which, however, enables a similar or even better spatial fit of
STI-571.
5 corresponding to PDGFR-
Thr-681
critically determines the susceptibility to STI-571 and probably other
phenylaminopyrimidines, mainly by sterical constraints. Taken together
with previous observations (31, 32), the atypical variability of this
position in different kinases suggests that it may be a
general key switch for obtaining selective inhibitors. We, therefore,
tested whether other receptor tyrosine kinases could also be sensitized
to STI-571 by mutating the corresponding residue. However, as shown in
Fig. 4, neither the FGFR-1 V561T mutant
(Fig. 4A) nor the insulin receptor M1103T mutant (Fig. 4B) was inhibited by STI-571. Thus, further structural
determinants must prevent binding of STI-571 to these kinases.
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Fig. 4.
Replacement of FGFR-1 Val-561 and insulin
receptor Met-1103 with Thr is insufficient to allow sensitivity to
STI-571 inhibition. A, FGFR-1 wild type or FGFR-1 V561T
were overexpressed in HEK293 cells. Analysis of STI-571 susceptibility
was performed as described for PDGFR- in the legend to Fig.
2A. WB, Western blot; PY,
phosphotyrosine. B, insulin receptor wild type (IR
Wt) or insulin receptor M1103T was overexpressed in HEK293 cells.
Tyrosine phosphorylation of the receptor was analyzed in cell lysates.
The phosphorylated insulin receptor precursor as well as the
phosphorylated mature
-subunit are detectable (indicated by
arrows).
would likewise affect
STI-571 susceptibility. This was indeed the case. When assayed against
an exogenous peptide substrate, preactivated PDGFR-
required a one
order of magnitude higher concentration of STI-571 for inhibition than
unstimulated PDGFR-
(IC50 0.63 µM
versus 5.05 µM, respectively; Fig.
5). In line with these experiments, the
PDGFR-
Y857F mutant, which lacks the tyrosine whose phosphorylation is critical for kinase activation, is slightly better inhibited than
the wild type when expressed in intact cells (IC50 0.25 versus 0.34 µM, respectively). It should be
noted here that effective inhibition of the stimulated wild-type kinase
in intact cells occurs because the susceptible, inactive kinase
conformation is regenerated by the action of protein tyrosine
phosphatases (33). This is most likely the reason for the relatively
small difference in inhibition of wild type and the PDGFR-
Y857F
mutant in intact cells.
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Fig. 5.
Preactivation of PDGFR-
reduces sensitivity to STI-571 inhibition. HEK293 cells
overexpressing PDGFR-
were stimulated with PDGF-BB; PDGFR-
was
immunoprecipitated, and autophosphorylation was allowed in the presence
of unlabeled ATP. Excess ATP was removed by washing, and a kinase assay
was performed with the synthetic peptide KY751 and
[
-32P]ATP as substrates. To obtain nonactivated
PDGFR-
, cell stimulation was omitted, and prephosphorylation was
replaced by mock treatment. Inhibition of kinase activity against the
KY751 peptide is depicted (means of three independent experiments
performed in duplicate).
receptor kinase and Flt3, this
assumption was checked by analyzing the course of additional kinases.
and
backbone
angles of the Asp-Phe-Gly motif and the preceding residue from seven
tyrosine kinase crystal structures. The courses are determined mainly
by the Asp
angles, discriminating between inactive states of Abl
and insulin receptor kinase (IRK) on the one hand, of Hck, VEGFR-2, and
FGFR-1 kinase, as well as active states of IRK and Lck on the other
hand. In the second group, typical Asp
and Phe
values separate
active (IRK, Lck) from inactive conformations (Hck, VEGFR-2) and
indicate, together with specific Gly angles, an individual course for
the FGFR-1 activation loop, corresponding to a special autoinhibitory mechanism (28). Although the autoinhibitory activation loops of Abl and
IRK, both with a substrate-mimicking tyrosine, are alike, IRK Gly-1149
preceding Asp induces an individual course of the Asp-Phe-Gly motif
because the
angle of 170° is possible only in glycine
residues.
Courses of the Asp-Phe-Gly motif in different tyrosine kinases
and
angles in crystal
structures. inact., inactive; act., active. PDB codes are in
parentheses.
atoms of
7
and
8 (r.m.s. distance 0.25-0.36 Å in pairwise fits to Abl). Three
principal groups become obvious: 1) Abl and inactive IRK; 2) inactive
FGFR-1, VEGFR-2, and Hck; 3) active IRK and Lck. However, the course of inactive IRK is markedly steeper than that of Abl, leading to a
complete overlap of the Phe-1151 side chain with the pyrimidinylamino moiety of STI-571. This overlap might contribute to the
inactivity of the inhibitor at IRK. The courses of the other five
kinases are completely incompatible with STI-571 docking because they all cross the benzylpiperazinyl moiety of the ligand.
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Fig. 6.
Asp-Phe-Gly courses in crystal structures of
tyrosine kinases. Murine Abl (PDB code 1iep (20)), FGFR-1
(1fgk (28)), VEGFR-2 (1vr2 (27)), inactive IRK (1irk (29)), active IRK
(1ir3 (35)), inactive Hck (1qcf (1)), and active Lck (3lck (36))
together with a volume contour of STI-571 bound to Abl. Alignment: C
atoms of
7 (not shown) and
8. Backbones and Phe side
chains are depicted for Abl and inactive IRK.
Kinase and the Flt3 F691T
Mutant--
The simple reversal of the wild-type PDGFR-
kinase
selectivity of STI-571 into Flt3 selectivity of the F691T mutant with potencies close to those for inhibition of Abl requires more
detailed investigations and interpretations by means of
three-dimensional computer models of the complexes. PDGFR-
kinase
and Flt3 F691T mutant models were derived from template crystal
structures and the alignment in Fig. 1 as described (see
"Experimental Procedures"). The kinase insert regions were ignored.
The SCRs (see Fig. 1) and, except for the nucleotide binding and the
activation loops, the remaining regions of the models (structurally
variable regions) were inserted from one of the eight templates.
) and
Tyr-842 (Flt3) pointing inward toward the catalytic site and mimicking
substrate binding (see Fig. 7). The
reduced sensitivity of preactivated PDGFR-
to STI-571 inhibition
discussed above further supports such a structure. The predicted
PDGFR-
activation loop conformation may be stabilized by four
intramolecular interactions: H bonds or electrostatic forces between
the side chains of Arg-853 and Asp-691 (
D), Asn-856 and Arg-830
(catalytic loop), and Tyr-857 and Asp-826 (catalytic loop) and a
hydrophobic cluster of Leu-847, Ile-851, and Met-852. The first
interaction is not possible in Flt3 (Ser-838 instead of Arg). In the
crystal structure of c-Abl kinase, only the corresponding
Tyr-393-Asp-363 H bond and hydrophobic Leu-Leu-Met cluster are
obvious. Thr-392 (instead of PDGFR-
Asn-856) does not approach
Arg-367 but contacts Met-388 and Pro-402 via hydrophobic or van
der Waals interactions. Very recent results (34) have indicated that
Ala mutants in the human c-Abl kinase at positions corresponding to
Met-388 and Thr-392 in the c-Abl crystal structure show higher levels
of tyrosine phosphorylation. Thus, the inactive state of the activation
loop, which protects the substrate-mimicking tyrosine from
phosphorylation, must be stabilized by several intramolecular
interactions and/or an inhibitor like STI-571 to freeze the natural
flexibility of this region.
View larger version (37K):
[in a new window]
Fig. 7.
Overview of the refined
PDGFR- kinase model with STI-571 and suggested
conformation of the activation loop. Upper panel,
ribbon-tube representation of the backbone (
-strands are
shown in blue,
-helices in green, loops in
gray, the nucleotide binding loop in yellow, the
catalytic loop in purple, and the activation loop in
orange). Inhibitor is shown as MOLCAD separation surface
with color scale from red (closest contacts between ligand
and binding site) to gray (farthest distance). Lower
panel, scaled up from the gray frame in the upper
panel in a slightly different view; the activation loop and
surrounding structural elements (coloring of ribbons, tubes, and carbon
atoms of displayed residues is the same as in the upper
panel, with ligand omitted). Atoms participating in the suggested
interactions are shown as balls. Cyan,
hydrophobic cluster of the side chains of Leu-847, Ile-851, and
Met-852; magenta, substrate-mimicking Tyr-857.
and the Flt3 F691T mutant may
likewise be stabilized; Phe-611 and Phe-621, respectively, replacing
Abl Tyr-253, could be involved in perpendicular van der Waals contacts
with the pyridylpyrimidinylamino moiety and in oxygen-aromatic
interactions with an Asp residue in place of Abl Asn-322. In
conclusion, the similar inhibitory activity of STI-571 at all three
kinases, Abl, PDGFR-
, and Flt3 F691T, suggests a resemblance
among their nucleotide binding loops.
kinase and the Flt3 F691T mutant were completed, provided
with the STI-571 conformation and surrounding water molecules from the
Abl kinase crystal structure 1iep (20), and refined by energy
minimization. Fits of the final models (C
atoms of SCRs) with the
templates resulted in r.m.s. distances from 0.87 (FGFR 1fgk) to 1.69 Å (1irk) for PDGFR-
kinase, and from 0.92 (FGFR 1fgi) to 1.72 Å (1irk) for the Flt3 F691T mutant. Fig. 7 presents an overview of the
PDGFR-
kinase model and the predicted conformation of the activation
loop. The separating surface between STI-571 and the binding site
describes a nearly complete lock-and-key shape, with only one edge of
the piperazinyl moiety not in contact with site atoms.
kinase and the Flt3 F691T mutant
were defined by the 23 amino acids aligned to the corresponding Abl
kinase residues (PDB code 1iep) within 3 Å around STI-571 (see
Fig. 1). Fits of the backbones of these 23 residues resulted in r.m.s.
distances of 1.01 (PDGFR-
versus 1iep), 0.92 (Flt3 versus 1iep), and 0.54 Å (PDGFR-
versus
Flt3). Fig. 8 shows models of the binding
sites, pointing to essential interactions and to putative targets for
selectivity. The discussion can be generalized in terms of the
PDGFR-
kinase model (Fig. 8A), because only 2 of the 23 residues are different in the Flt3 F691T mutant (Ile-654
versus Flt3 Met-664, Ile-679 versus Flt3
Leu-689). The network of hydrogen bonds in the Abl STI-571 complex is
preserved in the models. The pyridine nitrogen interacts with the
backbone NH of Cys-684 after
5 (Abl Met-318) like the N1 nitrogen of
ATP. The side chain oxygen of Thr-681 (
5) is attached to the
pyrimidinylamino NH of STI-571. Fig. 8B demonstrates that
Phe-691 interferes in all reasonable conformations with STI-571 binding
to the Flt3 wild type. Equal inhibition of the PDGFR-
wild type and
the T681A mutant (see above) indicates that indeed sterical hindrance
is the main determinant of inactivity. Glu-651 (
C) of the PDGFR-
kinase forms a hydrogen bond with the amide NH of STI-571 and an ion
pair with Lys-634 typical of many tyrosine kinases. The backbone oxygen
of Val-823 in the catalytic loop may be attached to the protonated N4
of the piperazinyl ring, and the backbone NH of Asp-844 (Asp-Phe-Gly
motif) is involved in a hydrogen bond with the amide oxygen of the
ligand.
View larger version (38K):
[in a new window]
Fig. 8.
Models of STI-571 binding to the
PDGFR- kinase and Flt3. A,
PDGFR-
kinase STI-571 complex showing 23 residues corresponding to
amino acids within 3 Å around the inhibitor in the Abl crystal
structure, 1iep. Colors of carbon and some hydrogen atoms:
white, STI-571; orange, identical
residues, magenta, mutated residues compared with murine Abl
kinase. The isolated red balls are suggested water oxygens,
and hydrogen atoms marked as balls participate in
suggested hydrogen bonds. Transparent tubes:
blue,
-strands; green,
-helices;
gray, loops. B, docking of STI-571 into Flt3
derived from the model of the Flt3 F691T mutant STI-571 complex. The
depiction of residues and the coloring scheme are as described in
A, except for carbon and some hydrogen atoms:
orange, identical residues, green,
mutated residues compared with PDGFR-
kinase. The green
MOLCAD surface of the Phe-691 side chain is drawn in the two common
positions of the
1 torsion angle: 180°, opaque;
60°, transparent.
1), Phe-611 (nucleotide binding
loop), Val-614 (
2), Ile-679 (
5), Tyr-683 (after
5), Leu-833
(
7), and Phe-845 (Asp-Phe-Gly motif), as well as the alkyl chain of Lys-634 (
3). The piperazin-1-ylmethylbenzamide moiety aligns with
three residues in
C (Ile-654, Met-655, and Leu-658) and with Cys-822
(catalytic loop), Cys-843 (after
8), and Asp-844 (Asp-Phe-Gly motif).
kinase and the Flt3 F691T mutant closely resemble those in the complex
with the Abl kinase. This is not self-evident because, apart from the
activation and the nucleotide binding loop (see above), no regions of
the c-Abl kinase crystal structures were explicitly used for model
building. The fact that similar binding sites resulted without
additional constraints is again an indication of the perfect surface
complementarity with STI-571. Its minimized conformation in the
PDGFR-
kinase model strongly corresponds to that in the Abl 1iep
structure (r.m.s. distance of 0.64 Å when fitting the heavy atoms).
Fig. 8 shows that only 7 of the 23 binding site residues differ between
the PDGFR-
and the Abl kinase. Some of these residues are potential
targets to obtain selectivity of STI-571-like inhibitors for PDGFR-
.
For example, the side chain of PDGFR-
Ile-654 (Abl Val-289, Flt3
Met-664) may be in close van der Waals contact with the
methylpiperazinyl moiety, which is aligned perpendicularly with the SH
group of Cys-822 (Abl Phe-359, Flt3 Cys-807) in the model. This
cysteine should form a weak hydrogen bond with an appropriate
substituent. PDGFR-
Tyr-683 (Abl Phe-317, Flt3 Tyr-693) may be
approached by hydrogen acceptor groups at the pyridinyl moiety.
Finally, the SH group of PDGFR-
Cys-843 preceding the Asp-Phe-Gly
motif (Abl Ala-380, Flt3 Cys-828) could interact with small
substituents at the central phenyl ring. The high activity at the Flt3
F691T mutant suggests that STI-571 may even serve as a template for the
design of Flt3 wild-type inhibitors.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dirk Schmidt-Arras and to Drs. Claesson-Welsh, Heldin, Lammers, Markova, and Ullrich for providing various reagents and also to Antje Trümpler for generating some of the receptor mutants.
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FOOTNOTES |
---|
* This work was supported in part by grants from Deutsche Krebshilfe, e.V. (10-1717-Do I to S. D., F. D. B., and S. M.), the Deutsche Forschungsgemeinschaft (Se 600/2-4), and Interdisziplinäres Zentrum für Klinische Forschung and Fonds Innovative Medizinische Forschung Münster (to H. S.).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.
The on-line version of this article (available at
http://www.jbc.org) contains the
synthesis scheme for STI-571.
§ To whom correspondence should be addressed: Research Unit Molecular Cell Biology, Drackendorfer Str. 1, D-07747 Jena, Germany. Fax: 49-3641-304462; E-mail: i5frbo@rz.uni-jena.de.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209861200
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ABBREVIATIONS |
---|
The abbreviations used are: FGFR, fibroblast growth factor receptor; IRK, insulin receptor kinase; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor; HA, hemagglutinin; PDB, Protein Data Bank; SCR, structurally conserved region; r.m.s., root mean square; ITD, internal tandem duplication.
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