Structure-based approach for the discovery of pyrrolo[3,2- d ]pyrimidine-based EGFR T790M/L858R mutant inhibitors

Article abstract of DOI:10.1021/ml300327z

The epidermal growth factor receptor (EGFR) family plays a critical role in vital cellular processes and in various cancers. Known EGFR inhibitors exhibit distinct antitumor responses against the various EGFR mutants associated with nonsmall-cell lung cancer. The L858R mutation enhances clinical sensitivity to gefitinib and erlotinib as compared with wild type and reduces the relative sensitivity to lapatinib. In contrast, the T790M mutation confers drug resistance to gefitinib and erlotinib. We determined crystal structures of the wild-type and T790M/L858R double mutant EGFR kinases with reversible and irreversible pyrrolo[3,2-d]pyrimidine inhibitors based on analogues of TAK-285 and neratinib. In these structures, M790 adopts distinct conformations to accommodate different inhibitors, whereas R858 allows conformational variations of the activation loop. These results provide structural insights for understanding the structure-activity relationships that should contribute to the development of potent inhibitors against drug-sensitive or -resistant EGFR mutations.

Full text of DOI:10.1021/ml300327z

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Approach for the Discovery of
Pyrrolo[3,2‑d]pyrimidine-Based EGFR T790M/L858R Mutant
Inhibitors
Satoshi Sogabe,*,‡ Youichi Kawakita,‡ Shigeru Igaki,‡ Hidehisa Iwata, Hiroshi Miki, Douglas R. Cary,
Terufumi Takagi, Shinji Takagi, Yoshikazu Ohta, and Tomoyasu Ishikawa*
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa
251-8555, Japan
S
* Supporting Information
ABSTRACT: The epidermal growth factor receptor (EGFR) family plays a
critical role in vital cellular processes and in various cancers. Known EGFR
inhibitors exhibit distinct antitumor responses against the various EGFR
mutants associated with nonsmall-cell lung cancer. The L858R mutation
enhances clinical sensitivity to gefitinib and erlotinib as compared with wild type
and reduces the relative sensitivity to lapatinib. In contrast, the T790M
mutation confers drug resistance to gefitinib and erlotinib. We determined
crystal structures of the wild-type and T790M/L858R double mutant EGFR
kinases with reversible and irreversible pyrrolo[3,2-d]pyrimidine inhibitors
based on analogues of TAK-285 and neratinib. In these structures, M790 adopts distinct conformations to accommodate
different inhibitors, whereas R858 allows conformational variations of the activation loop. These results provide structural
insights for understanding the structure−activity relationships that should contribute to the development of potent inhibitors
against drug-sensitive or -resistant EGFR mutations.
KEYWORDS: EGFR T790/L858 mutant inhibitors, NSCLC, drug sensitivity and resistance, crystal structure,
pyrrolo[3,2-d]pyrimidine derivatives, TAK-285
treatments of NSCLC patients. To date, several irreversible
he signal transduction pathway based on binding of
Tepidermal growth factor (EGF) to EGF receptor (EGFR) inhibitors such as neratinib (HKI-272),8 afatinib (BIBW-
2992),9 and WZ400210 have been developed to target a
on the cell surface regulates cell proliferation, differentiation,
cysteine (C797) residue located at the lip of the adenosine
triphosphate (ATP)-binding cleft. These compounds were
rationally designed to make a covalent bond with C797, which
provides the potential to inhibit EGFR-T790M mutation.7
However, such irreversible inhibitors have potential safety
concerns due to poor selectivity with respect to other kinases as
a result of their chemical reactivity with other cysteine
residues.11 Therefore, it is a worthy goal to discover novel,
potent, noncovalent, reversible inhibitors against both the wild-
type and/or the T790M/L858R double-mutated EGFR.
Herein, we present a structural approach for the development
of novel reversible pyrrolo[3,2-d]pyrimidine inhibitors of the
EGFR double mutant.
We previously reported potent dual inhibitors of HER2/
EGFR based on a pyrrolo[3,2-d]pyrimidine scaffold with
significant in vivo antitumor efficacy.5,12 Crystallographic
analysis showed that the pyrrolo[3,2-d]pyrimidine compounds
bind to the inactive conformation of EGFR, as has been
reported for lapatinib.13 Against the T790/L858R mutant,
and migration. The EGFR family plays a critical role in vital
cellular processes and in various cancers and is a proven target
in the treatment of cancer.1 Several EGFR kinase inhibitors
have been approved for clinical use or are currently in clinical
development. These include gefitinib,2 erlotinib,3 lapatinib,4
and TAK-285 (Figure 1).5
Several specific mutations of EGFR have been clinically
identified in nonsmall-cell lung cancer (NSCLC) patients.6
Known EGFR kinase inhibitors such as gefitinib, erlotinib, and
lapatinib show distinct inhibitory profiles against these EGFR
mutants. The most frequent single activation-loop mutation
(L858R) increases inhibitory activity for gefitinib and erlotinib
as compared with the wild-type EGFR and reduces the
sensitivity to lapatinib.2 After treatment with these inhibitors,
however, emergence of an additional gate keeper mutation
(T790M) has been reported as a key factor of drug resistance.
The secondary T790M mutation reduces sensitivity to gefitinib
and erlotinib.7 The differential susceptibility of NSCLC to
various inhibitors gave rise to a number of questions regarding
the relation between EGFR mutation and clinical efficacy of
EGFR inhibitors.
Received: October 10, 2012
Accepted: December 18, 2012
The development of EGFR inhibitors to target drug-resistant
mutants has become a significant unmet need for the
© XXXX American Chemical Society
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Figure 1. Structures of EGFR inhibitors.
TAK-285 (1) has no inhibitory activity, as shown in Table 1.
We initially designed an irreversible inhibitor (2) based on the
than that found for the erlotinib complex due to a
conformational shift of the N-lobe to accommodate the bulky
substituent of M790 adjacent to the regulatory αC-helix. The
salt-bridge interaction between two highly conserved residues
(K745 and E762), which is a canonical feature in the active
conformation, is elongated due to a slight outward shift of the
αC-helix (Figure 2a). The N-1 nitrogen on the pyrrolo[3,2-
d]pyrimidine ring makes a hydrogen bond with the main-chain
nitrogen of Met793 of the hinge region between the N- and the
C-lobes. The trifluoromethyl-phenoxy group is positioned in a
hydrophobic space, defined predominantly by I759, M766,
L777, L788, and M790, in the vicinity of the αC-helix, as
discussed below.
We also determined a crystal structure of the T790M/L858R
mutant in complex with TAK-285 (Figure S1 in the Supporting
Information). The binding conformation of TAK-285 with the
T790M/L858R mutant is almost identical to that of compound
2, suggesting that the interaction with the hinge region is
independent of the covalent bond with C797. The irrever-
sibility of the covalent bond seems not to contribute to the
binding affinity against the T790M/L858R mutant for TAK-
285.
On the other hand, structural comparison with the complex
of TAK-285 bound to wild-type EGFR (Figure 2b) reveals that
the T790M/L858R mutant adopts a different conformation
than when bound to compound 2 or TAK-285. The orientation
of the trifluoromethyl-phenoxy group is flipped between the
wild-type and the mutant EGFR structures due to a change in
the αC-helix conformation. The trifluoromethyl group is
exposed to solvent region in both the mutant structures. For
the T790M/L858R mutant, the pyrrolo[3,2-d]pyrimidine ring
Table 1. IC50 Values for Compounds in the EGFR Enzyme
Assays
a
EGFR IC50 (nM) (95% CI)
compd
wild-type
T790M/L858R
1
2
3
4
4.0 (2.9−5.6)
4.6 (3.3−6.3)
2.5 (1.4−4.3)
6.9 (4.6−10)
8400 (6700−11000)
8100 (6300−11000)
66 (43−100)
19 (12−32)
a
IC50 values and 95% confidence intervals (CI) were calculated by
nonlinear regression analysis of the percentage inhibitions (n = 2).
scaffold of compound 1 with a pendant Michael acceptor to
bind covalently at C797 in an attempt to provide inhibitory
activity against the mutant protein. However, compound 2
shows no significant inhibition of the T790M/L858R mutant,
nor does it show any time dependency with an incubation time
of up to 1 h (data not shown). We believed that structural
analysis of compound 2 bound to mutant EGFR would provide
valuable insights into the key interactions at the mutation sites
and would help facilitate the rational design of potent EGFR
mutant inhibitors. Therefore, we determined a crystal structure
of the T790M/L858R double mutant in complex with
compound 2 (Figure 2a).
The crystal structure of T790M/L858R mutant adopts the
activelike conformation, similar to that of the complex with
erlotinib.3 Compound 2 forms a covalent bond with C797, as
expected, and occupies the ATP binding cleft, which is wider
Figure 2. (a) Crystal structure of compound 2 bound to the T790M/L858R mutant, colored in green. Two mutation sites and C797 are labeled. (b)
Crystal structure of TAK-285 (1) with wild-type EGFR (PDB code: 3POZ), colored in pink. (c) Superposition of compound 2 (cyan) with TAK-
285 (blue) with key residues surrounding the binding site. Hydrogen bonds found in a and b are indicated by orange and yellow dashed lines,
respectively.
B
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Figure 3. (a) Crystal structure of neratinib (3) bound to the T790M/L858R mutant, colored in green. The two mutation sites and Cys-797 are
labeled. (b) Crystal structure of neratinib (3) bound to the T790M mutant (PDB code: 2JIV), colored in gold. (c) Overlaid structures of neratinib
with key residues surrounding the binding site. Hydrogen bonds found in a and b are indicated with orange and yellow dashed lines, respectively.
Figure 4. (a) Crystal structure of compound 4 bound to the T790M/L858R mutant, colored in green. Two mutation sites and Cys-797 are labeled.
(b) Crystal structure of compound 4 bound to wild-type EGFR, colored in pink. (c) Overlaid structures of compound 4 with key residues
surrounding the binding site. Hydrogen bonds found in a and b are indicated by orange and yellow dashed lines, respectively.
is shifted downward by steric hindrance from the αC-helix
(Figure 2c). The hydrogen-bonding pattern with the hinge
region seems not to be optimal, resulting in weak inhibitory
activity against the T790M/L858R mutant. As described above,
the L858R mutant confers sensitivity to inhibitors that bind to
the active conformation but decreases the affinity for inhibitors
that bind to the inactive conformation. Our structural data
suggest that the loss of structural flexibility and stabilization of
the activelike conformation would interfere with optimal
binding for compound 2 or TAK-285. As a consequence, in
the case of these pyrrolo[3,2-d]pyrimidine derivatives, the
covalent bond with C797 was not effective at improving the
EGFR mutant inhibitory activity. To clarify the binding mode
of an irreversible inhibitor with the T790M/L858R mutant, we
determined the structure of its complex with neratinib (3),
which is an irreversible inhibitor against both the wild-type and
the T790M/L858R mutant of EGFR (Table 1).
and length of the acceptor substituent to accommodate
covalent binding. The quinoline nitrogen of neratinib forms a
hydrogen bond to the M793 main-chain nitrogen of the hinge
region. Despite a similar binding mode found in the complexes
of both compound 2 and of neratinib, a distinct conformation
change for M790 was observed in the vicinity of these
compounds. The side-chain rearrangement of M790 is
necessary to accommodate binding of the cyano group for
neratinib. The steric interaction with M790 appears to facilitate
optimal interaction of the quinoline ring with the hinge region,
whereas no steric effect of M790 was observed for pyrrolo[3,2-
d]pyrimidine 2. The terminal pyridyl group of neratinib roughly
occupies the same space as the trifluoromethyl-phenoxy group
of compound 2. The pyrrolo[3,2-d]pyrimidine compound with
the same anilino substituent of neratinib [3-chloro-4-(pyridin-
2-ylmethoxy)phenyl group] shows weak inhibitory activity
although the compound covalently binds in the same fashion as
that of compound 2 (data not shown). Therefore, we
concluded that the covalent interaction with C797 alone is
insufficient to produce potent activity for the pyrrolo[3,2-
d]pyrimidine scaffold.
The crystal structure of the T790M/L858R EGFR complex
of neratinib (3) revealed that neratinib binds in the ATP-
binding cleft with a covalent bond to C797 in a fashion similar
to that observed in the complex with compound 2 (Figure 3a).
The C797 side-chain orientation is associated with the position
C
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The crystal structure of neratinib bound to single mutant
T790M EGFR has been reported.7 Structural comparison with
the single mutant structure indicates that the pyridyl-methoxy
group occupies the hydrophobic pocket adjacent to the αC-
helix with different binding conformations (Figure 3b,c). It was
speculated that the L858R mutation prevents the kinase from
adopting the inactive conformation by stabilizing the activation
loop, resulting in a constitutively active form of the protein. On
the other hand, in the T790M single mutant structure, the
activation loop around L858 forms a short helix, and the αC-
helix is oriented outward to facilitate the inactive conformation.
The activation-loop conformation of the T790M/L858R
mutant is very similar to that of other L858R mutants.2,14
Although the binding conformation of neratinib with the
T790M/L858R mutant may not be optimal, the covalent bond
with C797 and the hydrogen-bonding interaction with the
hinge region result in the retention of kinase inhibitory activity.
This finding was conducive to further chemical modification of
the pyrrolo[3,2-d]pyrimidine derivatives.
To enhance the EGFR T790M/L858R double mutant kinase
inhibitory activity, we designed a back-pocket urea linker with a
hydrophobic moiety to interact with the activation loop and the
αC-helix, because the urea moiety should be able to engage in
structural rearrangement of the DFG-motif conformation, such
as that found for VEGFR2 inhibitors.15 This approach resulted
in the discovery of compound 4, which exhibits potent kinase
inhibitory activity against both the wild-type EGFR and the
T790M/L858R double mutant (Table 1).
The cocrystal structures of wild-type EGFR and the T790M/
L858R mutant bound to reversible pyrrolo[3,2-d]pyrimidine
inhibitor 4 are shown in Figure 4. The structures reveal several
interesting features, which may explain the drug sensitivity and
resistance of the EGFR mutant. Compound 4 is bound to the
ATP binding cleft, and the protein adopts the inactive
conformation (Figure 4a,b). The overall fold of the protein is
similar to that observed in the wild-type EGFR/lapatinib
structure.4 However, there are distinct differences in the
orientation of the N- and C-lobes between the two structures
with the compound 4. These differences are caused by a
conformational rearrangement of the N-lobe, resulting from the
T790M mutation. The cyclohexyl-urea moiety makes a water-
mediated hydrogen bond with the G857 carbonyl oxygen of the
DFG motif in the case of the T790M/L858R mutant, whereas
it forms direct hydrogen bonds with the F856 and G857
carbonyl oxygens of the wild type (Figure 4c). In the wild-type
structure, L858 forms a short distorted helix that inserts into a
hydrophobic pocket beside the αC-helix and interacts with the
cyclohexyl group. On the other hand, in the mutant structure,
the activation loop is elongated because R858 is solvent
exposed with conformational flexibility. The binding con-
formation of the cyclohexyl-urea moiety allows the activation
loop to occupy the hydrophobic space by displacement of the
αC-helix. These findings indicate that the L858R mutation is a
key factor in determining the conformation adopted by the N-
terminal segment of the activation loop. Hydrophobic moieties
such as a short helix for the wild-type and a bulky substituent
for the double mutant are implicated in giving rise to potent
inhibitory activity. At the gate keeper position, M790 of the
mutant forms closer contacts with the phenoxy-chloroanilino
group than those made by T790 in the wild-type protein.
However, the pyrrolo[3,2-d]pyrimidine scaffold interacts with
the hinge region in the same manner for both structures. This
suggests that M790 can optimize its conformation to
accommodate the bound ligand. A bulky substituent such as
the cyclohexyl-urea group presumably facilitates the optimal
positioning of the inhibitor in the ATP binding cleft for the
T790M/L858R mutant.
In summary, structural analysis provided valuable insights
into drug sensitivity (or insensitivity) caused by EGFR
mutation. Irreversible covalent bond formation with the
cysteine residue C797 is well-known to overcome drug
resistance caused by T790M mutation. However, such covalent
bond formation with C797 was ineffective for a slowly
dissociating inhibitor such as TAK-285. The steric config-
uration between the cysteine and the gate keeper residues is
critical for the interaction of inhibitors with the hinge region to
give optimal binding in the ATP binding cleft. Therefore, the
T790M mutation gives rise to differential binding affinity for
different inhibitors based on structural rearrangement of the
protein. The L858R mutation stabilizes the active conformation
due to the presence of the larger side chain. Substituents such
as the trifluoromethyl-phenoxy group of TAK-285 and the
pyridyl group of neratinib are insufficient to exclude the αC-
helix because the activation loop is incapable of forming a short
helix to make a hydrophobic cluster with the N-lobe and αC-
helix. Larger substituents such as the cyclohexyl-urea moiety
require the short-helix position to adopt the inactive
conformation. Ultimately, we discovered the novel reversible
pyrrolo[3,2-d]pyrimidine inhibitor 4 with activity against both
EGFR wild-type and T790M/L858R double mutant. Although
compound 4 exhibited weak inhibition of tumor cell growth
(IC50 = 4.8 μM in an EGFR mutant H1975 cell line), our
results have important implications for further structure-guided
development of more potent noncovalent reversible inhibitors
of drug-resistant EGFR mutants with minimal safety concerns.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and characterization of synthesized
compounds, protein expression and purification methods,
biological enzymatic assay methods, X-ray crystallography,
and Figure S1. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81-467-32-2839. Fax: +81-466-29-4484. E-mail: sato-
shi.sogabe@takeda.com (S.S.). Tel: +81-466-32-1155. Fax:
+81-466-29-4449. E-mail: tomoyasu.ishikawa@takeda.com
(T.I.).
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Gyorgy Snell and Weston Lane for data collection at
Advance Light Source and members of the Structural Biology
Research Center for data collection at Photon Factory.
ABBREVIATIONS
■
EGFR, epidermal growth factor receptor; NSCLC, nonsmall-
cell lung cancer; ATP, adenosine triphosphate
D
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S.; Kobayashi, T.; Tanaka, T.; Chen, L.; Liu, Z. J.; Wang, B. C.;
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Miki, H. Biochemical characterization of a novel type-II VEGFR2
kinase inhibitor: Comparison of binding to non-phosphorylated and
phosphorylated VEGFR2. Bioorg. Med. Chem. 2011, 19, 5342−5351.
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