Fragment-Based Discovery of Potent and Selective DDR1/2 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00143

The DDR1 and DDR2 receptor tyrosine kinases are activated by extracellular collagen and have been implicated in a number of human diseases including cancer. We performed a fragment-based screen against DDR1 and identified fragments that bound either at th

Full text of DOI:10.1021/acsmedchemlett.5b00143

Letter
pubs.acs.org/acsmedchemlett
Fragment-Based Discovery of Potent and Selective DDR1/2 Inhibitors
Christopher W. Murray,*,† Valerio Berdini,† Ildiko M. Buck,† Maria E. Carr,† Anne Cleasby,†
Joseph E. Coyle,† Jayne E. Curry,† James E. H. Day,† Phillip J. Day,† Keisha Hearn,† Aman Iqbal,†
Lydia Y. W. Lee,† Vanessa Martins,† Paul N. Mortenson,† Joanne M. Munck,† Lee W. Page,† Sahil Patel,†
Susan Roomans,† Kirsten Smith,† Emiliano Tamanini,† and Gordon Saxty†
†Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.
S
* Supporting Information
ABSTRACT: The DDR1 and DDR2 receptor tyrosine
kinases are activated by extracellular collagen and have been
implicated in a number of human diseases including cancer.
We performed a fragment-based screen against DDR1 and
identified fragments that bound either at the hinge or in the
back pocket associated with the DFG-out conformation of the
kinase. Modeling based on crystal structures of potent kinase
inhibitors facilitated the “back-to-front” design of potent
DDR1/2 inhibitors that incorporated one of the DFG-out
fragments. Further optimization led to low nanomolar, orally
bioavailable inhibitors that were selective for DDR1 and DDR2. The inhibitors were shown to potently inhibit DDR2 activity in
cells but in contrast to unselective inhibitors such as dasatinib, they did not inhibit proliferation of mutant DDR2 lung SCC cell
lines.
KEYWORDS: discoidin domain receptor, fragment-based drug design, back to front kinase design
iscoidin domain receptors, DDR1 and DDR2, are
DDR2 of approximately 100 nM, and experimental binding
Dtransmembrane receptor tyrosine kinases that are modes in DDR1 were determined.14,16 Gao et al. have
activated by collagen binding to their extracellular domain.1,2
published a series of potent Type II DDR1 inhibitors that are
selective over other kinases, including DDR2.13
DDR1 and DDR2 have been associated with extracellular
In this Letter we describe a fragment based approach to the
discovery of potent and selective DDR1/2 inhibitors. We first
obtained a soakable crystal form of DDR1 suitable for high-
throughput crystallography and fragment screening (see
Supporting Information). Approximately 1500 compounds
from the Astex fragment library were screened against DDR1
using a protein thermal shift assay (TM). Hits from this assay
were progressed into X-ray structure determination, and around
50 fragment−protein complexes were determined. Additionally
protein−ligand complexes were also determined for 10−15
nonselective kinase inhibitors, including dasatinib. Most
fragments bound at the hinge region, but intriguingly, about
10 fragments bound in the back pocket of DDR1, which is only
accessible to the inactive DFG-out form of the protein. In our
experience of many fragment screens against kinases,17−22 it is
somewhat unusual to obtain X-ray structures of fragments
binding in this region because only a small proportion of
kinases support fragment binding here,23 and we have found
such fragments tend to have relatively low solubility.
remodeling, cell adhesion, proliferation and migration, and they
have been linked to a number of human diseases, including
fibrotic disorders, atherosclerosis and cancer.3−5 There has
recently been evidence suggesting that DDR2 inhibitors would
be useful for the treatment of lung squamous cell cancer6,7
although the role of DDR2 may be more complex than first
realized.8−10 Hammerman et al.6,7 have shown that DDR2 is
mutated in approximately 4% of lung squamous cell cancer and
have presented data to suggest that these are gain of function
mutations. Hammerman et al.6 have also shown that cell lines
harboring these mutations are selectively sensitized through
knockdown of DDR2 by RNA interference or by treatment of
the multitargeted kinase inhibitor dasatinib.11 Selective DDR2
inhibitors would therefore be useful to probe the role of DDR2
and may have utility for the treatment of lung cancer.
To date, most DDR1/2 inhibitors have been derived from
cross-screening of existing kinase inhibitors.12 Initial com-
pounds often lacked selectivity over homologous enzymes such
as Bcr-Abl, but a number of recent papers have described more
selective inhibitors which all bind to the DFG-out form of the
enzyme.13−15 Gray and co-workers have identified DDR1/2
inhibitors by screening a library of compounds that had
previously been designed by mixing and matching motifs from
known Type II kinase inhibitors.14 The resulting compounds
were reported to be selective with IC50 values versus DDR1 and
We were particularly interested in fragment 1 (see Figure
1(a) and Figure 2), which places a chlorophenyl in the back
Received: April 7, 2015
Accepted: June 4, 2015
© XXXX American Chemical Society
A
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Figure 1. Crystal structures of compounds bound to DDR1 (PDB Codes: 5bvk, 5bvw, 5bvn and 5bvo). (a) Fragment 1 in DDR1 with pockets
annotated. (b) Superimposition of the DDR1−dasatinib cocomplex and the DDR1−fragment 1 cocomplex. (c) Compound 4 superimposed on the
crystallographic positioning of fragment 1. The four hydrogen bonding residues are marked. (d) Compound 9 in DDR1.
pocket region and a pyridyl in the selectivity pocket proximal to
the small gatekeeper residue (Thr701 in DDR1/2). The linker
region of fragment 1 contains a urea moiety which hydrogen
bonds to the side chain of Glu672 and to the backbone N−H of
Asp784. The molecule is similar in structure and binding mode
to fragments of a number of Type II kinase inhibitors (e.g.,
sorafenib) but is unusual in containing a benzylic methylene in
the linker region (Figure 2). Fragment 1 is larger and more
lipophilic than the vast majority of compounds in our current
fragment library and is a relatively weak inhibitor (>100 μM)
with poor ligand efficiency (LE <0.3).24 However, we believed
that fragment 1 could be improved rapidly because (i) it did
not optimally fill the selectivity pocket and (ii) it lacked a hinge
binding moiety. Additionally we believed that the incorporation
of the unusual benzylic methylene could potentially lead to
novel and selective DDR inhibitors. The approach we adopted
is known as “back-to-front” kinase design, reflecting the fact
that design begins in the back pocket of the kinase and works
its way to the hinge binding region at the “front” of the ATP
Figure 2. Schematic of the design process. Molecules are colored
binding cleft (Figure 1(a)). This approach has only rarely been
according to the binding mode in DDR1: blue = back pocket region,
attempted,25−28 although the discovery of one of the earliest
red = linker region, green = selectivity pocket, magenta = hinge
and most influential DFG-out inhibitors, BIRB796, started from
binding group.
an HTS hit that occupied the back pocket of p38 MAP
kinase.27
B
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a
b
Table 1. Assay , Pharmacokinetic and Property Data for Compounds from the Text
a
Assay data given in IC50 or percentage inhibition at specified concentration. More details on assays including error estimates are provided in the
b
Supporting Information. Where quoted LE values are given in kcal/mol/(heavy atom). IV dosing in mouse at 1 mg/kg and oral dosing in mouse at
5 mg/kg. More details on pharmacokinetic data including additional in vitro data are provided in the Supporting Information. No inhibition value
c
was determined in this case.
The first stage of the design process was to identify potential
hinge binding motifs that could be grafted onto fragment 1
without significantly perturbing its binding mode. Given the
large number of internal DDR1 structures and internal/public
domain kinase structures, we chose to employ a computational
tool, AstexMerge, to suggest potential designs. AstexMerge is
an internal implementation of the program, BREED, described
by Pierce et al.29 The method starts from a pre-existing web
page of proteins in complex with small molecules superimposed
in the same frame of reference; this is often the same web page
used by medicinal chemists and modelers to visualize all the
relevant crystal structures associated with a particular project.
The user then selects a starting molecule/fragment, and
AstexMerge looks for bonds in all other molecules that
superimpose well with the starting molecule. When a suitable
hit molecule is located, two merged molecules are displayed,
each containing part of the starting fragment and part of the hit
molecule. The merged molecules are scored according to the
quality of bond and neighboring atom superimposition. The
user then visualizes a selection of the best scoring merged
molecules. Fragment 1 was used as the starting fragment in
AstexMerge, and a number of promising designs were
suggested, including design A (Figure 2), based on the
superimposition with dasatinib (Figure 1(b)). This design
was further modified based on our previous knowledge of
FGFR inhibitors,21 suggesting that the thiazole hinge binder of
dasatinib could be replaced by an imidazopyridine (design B in
Figure 2).
Compounds 2 and 3 were the first compounds that we
synthesized in the imidazopyridine series (Table 1). Gratify-
ingly, addition of the hinge binding element in compound 2 led
to a >300-fold increase in affinity for DDR2, and inclusion of
C
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the methyl group in 3 gave a further 30-fold improvement in
affinity. Limited further SAR exploration led to compound 4,
which, after further characterization, became the initial lead
molecule in this series. Compound 4 showed promising in vitro
PK and in vivo PK in mice (Table 1 and Table S5). The
selectivity profile of compound 4 was also encouraging (Table 1
and Table S1) because, in contrast to dasatinib, it showed
selectivity over c-src. Figure 1(c) shows the experimental
binding mode of 4 in DDR1, superimposed on the starting
fragment 1. The binding mode is in agreement with the original
design hypothesis, and the imidazopyridine forms the
anticipated hydrogen bonds with the hinge.
Compound 4 showed off-target activity against a number of
tyrosine kinases and in particular was a potent c-kit inhibitor
(19 nM). Inspection of the crystal structure for compound 4
indicated that there was space to grow off the benzylic
methylene. It was hypothesized that this change might improve
selectivity because our analysis of kinase literature indicated
that branched sp3 centers were poorly precedented in this
region for type II kinase inhibitors. The α-methyl compounds,
5, 6 and 7 were synthesized and the assay data (Table 1)
showed the S-isomer was tolerated whereas, in agreement with
the structure, the R-isomer was considerably less active. The
compounds showed improved selectivity profiles (see Table 1
and Table S1).
Further design assessed the scope to replace the urea linker.
It has been suggested that 3-trifluorobenzamide is a privileged
group for Type II inhibitors,30 and so we incorporated this idea
into the branched series resulting in the potent amide, 8 (Table
1). Benzoxazoles have been employed as urea replacements in
Type II inhibitors of VEGFR2,31 and so it was also interesting
to explore this change within the branched DDR series.
Compound 9 showed good activity in the assay and exhibited
the expected experimental binding mode in DDR1 (Table 1
and Figure 1(d)). Compounds 8 and 9 show promising
pharmacokinetics in mouse (Table 1 and Table S5) and good
selectivity (Table 1 and Table S1)
The properties of the lead compound 4 were assessed in
cells. Attempts to confirm inhibition of DDR2 phosphorylation
(pDDR2) in mutant-DDR2 lung squamous cell cancer cell lines
(NCI-H2286 and HCC-366) were unsuccessful because DDR2
expression could not be detected in these cell lines (Figure S2).
HEK293 cells were therefore stably transfected with either wild
type DDR2 or mutant DDR2 (I638F), leading to over-
expression of DDR2 in these cells. Compound 4 significantly
reduces basal and collagen I-stimulated DDR2 phosphorylation
in both wild type DDR2- and mutant DDR2-expressing
HEK293 cells (Figure 3(a)). The multikinase inhibitors,
dasatinib and nilotinib, also reduce pDDR2 in this experiment;
however, the relatively selective c-src inhibitor, saracatinib, only
inhibits pDDR2 at the highest concentration. The inhibition of
DDR2 activity conferred by compound 4 was quantified using a
MesoScale Discovery (MSD) assay, and an EC50 of 8.7 nM was
determined (Figure S1).
Figure 3. (a) Knockdown of DDR2 phosphorylation (pY) in HEK293
cells stably transfected with wild type (upper blots) or mutant DDR2
(I638F) (lower blots). Knockdown is reported with specified
compounds for collagen I stimulated cells (right) or unstimulated
cells (left). (b) Effect of compounds on proliferation of two mutant
DDR2 cell lines. More experimental details are provided in the
Supporting Information.
these cell lines are unlikely to be activating. Recent papers in
the literature have also questioned whether DDR2 mutants in
lung cancer are activating.8−10 We note that DDR1 and/or
DDR2 inhibitors may be useful as anti-invasive or antimeta-
static agents,13 or indeed that combination with other kinase
inhibitors may be beneficial.14 However, the lack of a direct
antiproliferative phenotype in mutant-DDR2 squamous cell
lines meant that the project was halted in favor of more
attractive targets.
As previously shown by Hammerman et al.,6 dasatinib and
nilotinib inhibit proliferation in DDR2 mutant cell lines (Figure
3(b)). Despite potently inhibiting pDDR2 in cells, compound 4
has no effect on the cell proliferation (Figure 3(b)), thus
suggesting that the antiproliferative effects conferred by
dasatinib and nilotinib are not attributable to DDR2 inhibition.
Taken together with the observation that the DDR2 mutant
cell lines NCI-H2286 and HCC-366 lack detectable DDR2
expression or activity, we conclude that the DDR2 mutations in
In conclusion, we have described novel inhibitors of DDR1
and DDR2 that contain a substituted sp3 center in the linker
region of Type II kinase inhibitors. The inhibitors were
generated using fragment based drug design starting from
fragments binding in the back pocket of the kinase (so-called
back-to-front design). Structural biology and computational
modeling tools were employed to guide the design of the
inhibitors. We have demonstrated the sp3 center in the linker
region can be used in conjunction with a variety of linker
D
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S.; Hanna, M.; Hatton, C.; Eck, M. J.; Janne, P. A.; Johnson, B. E.;
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the DDR2 kinase gene identify a novel therapeutic target in squamous
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Gray, N. S.; Bass, A. J.; Wong, K. K.; Meyerson, M.; Hammerman, P.
S. Acquired resistance to dasatinib in lung cancer cell lines conferred
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groups, including ureas, amides and benzoxazoles. The resulting
compounds not only are potent and selective but also display
promising pharmacokinetic properties. Biological data on our
DDR2 inhibitors were not supportive of an antiproliferative
role for DDR2 inhibitors in mutant-DDR2 squamous cell lung
cancer. However, the compounds should be useful chemical
probes for assessing the potential role of DDR1 or DDR2
inhibitors in other therapeutic indications.
ASSOCIATED CONTENT
* Supporting Information
(8) Iwai, L. K.; Payne, L. S.; Luczynski, M. T.; Chang, F.; Xu, H.;
Clinton, R. W.; Paul, A.; Esposito, E. A.; Gridley, S.; Leitinger, B.;
Naegle, K. M.; Huang, P. H. Phosphoproteomics of collagen receptor
networks reveals SHP-2 phosphorylation downstream of wild-type
DDR2 and its lung cancer mutants. Biochem. J. 2013, 454, 501−513.
(9) Bai, Y.; Kim, J. Y.; Watters, J. M.; Fang, B.; Kinose, F.; Song, L.;
Koomen, J. M.; Teer, J. K.; Fisher, K.; Chen, Y. A.; Rix, U.; Haura, E.
B. Adaptive Responses to Dasatinib-Treated Lung Squamous Cell
Cancer Cells Harboring DDR2 Mutations. Cancer Res. 2014, 74,
7217−7228.
■
S
Assay conditions and error estimates; more extensive selectivity
data; additional data and methods on in vivo and in vitro
pharmacokinetics; experimental synthetic procedures and
characterization data for novel compounds and intermediates;
additional cell data and associated methods; crystallographic
details. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acsmedchemlett.5b00143.
(10) Payne, L. S.; Huang, P. H. Discoidin domain receptor 2
signaling networks and therapy in lung cancer. J. Thorac. Oncol. 2014,
9, 900−904.
AUTHOR INFORMATION
Corresponding Author
*(C.W.M.) Tel: 44-1223-226228. Fax: 44-1223-226201. E-
mail: chris.murray@astx.com.
■
(11) Das, J.; Chen, P.; Norris, D.; Padmanabha, R.; Lin, J.; Moquin,
R. V.; Shen, Z.; Cook, L. S.; Doweyko, A. M.; Pitt, S.; Pang, S.; Shen,
D. R.; Fang, Q.; de Fex, H. F.; McIntyre, K. W.; Shuster, D. J.; Gillooly,
K. M.; Behnia, K.; Schieven, G. L.; Wityak, J.; Barrish, J. C. 2-
Aminothiazole as a Novel Kinase Inhibitor Template. Structure−
Activity Relationship Studies toward the Discovery of N-(2-Chloro-6-
methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- piperazinyl)]-2-methyl-4-
pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (Dasatinib, BMS-
354825) as a Potent pan-Src Kinase Inhibitor. J. Med. Chem. 2006,
49, 6819−6832.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge Chris Johnson and
David Rees for useful comments on the manuscript, Brent
Graham for help with the cell assays, and Torren Peakman and
Sharna Rich for help with in vitro pharmacokinetics data.
(12) Li, Y.; Lu, X.; Ren, X.; Ding, K. Small Molecule Discoidin
Domain Receptor Kinase Inhibitors and Potential Medical Applica-
tions. J. Med. Chem. 2015, 58, 3287−3301.
(13) Gao, M.; Duan, L.; Luo, J.; Zhang, L.; Lu, X.; Zhang, Y.; Zhang,
Z.; Tu, Z.; Xu, Y.; Ren, X.; Ding, K. Discovery and optimization of 3-
(2-(Pyrazolo[1,5-a]pyrimidin-6-yl)ethynyl)benzamides as novel selec-
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ABBREVIATIONS
■
DDR, discoidin domain receptor; pDDR2, phosphorylated
DDR2; LE, ligand efficiency; MW, molecular weight; HTS,
high throughput screening
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DOI: 10.1021/acsmedchemlett.5b00143
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