Bioorganic & Medicinal Chemistry Letters
Fragment-based discovery of focal adhesion kinase inhibitors
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Ulrich Grädler , Jörg Bomke, Djordje Musil, Verena Dresing, Martin Lehmann, Günter Hölzemann,
Hartmut Greiner, Christina Esdar, Mireille Krier, Timo Heinrich
Merck KGaA, Merck Serono Research, Frankfurter Str. 250, 64293 Darmstadt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon res-
onance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was
confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography
revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural compar-
ison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up
optimization leading to improved binding affinity.
Received 26 April 2013
Revised 17 July 2013
Accepted 23 July 2013
Available online 31 July 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Fragment screening
Surface plasmon resonance
Focal adhesion kinase
X-ray structure
DFG-out
The nonreceptor tyrosine kinase FAK (EC 2.7.10.2) is composed
by an N-terminal FERM domain (Four-point-one, ezrin, radixin,
moesin), which regulates the enzymatic activity of its C-terminal
kinase domain (KD).1,2 Direct interaction of the FERM domain with
the kinase C-lobe occludes the ATP-binding site and maintains an
auto-inhibited conformation also by protecting the activation loop
from phosphorylation by Src kinase. The auto-inhibitory state can
be released upon interaction with FERM binding partners such as
integrins and growth factors, underlining the important role of
FAK in integrating diverse cellular signalling pathways. Disruption
of the FERM–KD interaction leads to auto-phosphorylation of
Tyr397 in the linker region and exposure of the activation loop.
This is followed by binding of Src and phosphorylation of Tyr576
and Tyr577 within the FAK activation loop resulting in full catalytic
activation of the enzyme.3,4 Apart from playing a key role in regu-
lation of normal cellular activities such as adhesion, migration and
survival, FAK is also involved in cancer cell invasion, metastasis
and survival. Therefore, FAK has been reported as attractive target
for oncology and small molecule inhibitors are already in clinical
phase-1 testing.5–7
Fragments allow the sampling of a greater portion of chemical
diversity and exploration of more binding motifs within the target.
Even weak binding interactions of fragments to the protein target
can be detected by applying high compound concentrations in bio-
physical methods such as NMR, surface plasmon resonance (SPR),
or X-ray crystallography.8–10
In our FBLD campaign, we screened a library of 1920 fragments
against the immobilized kinase domain of FAK using SPR (Fig. 1a).11
Fragments were screened at a fixed concentration of 2 mM to re-
move ‘sticky’ and other bad behaving compounds.12 At such high
fragment concentrations, 80% of the fragments showed binding
to the FAK surface. Therefore, the remaining fragments were tested
again at 2 mM but in the presence of FAK-inhibitor I (Fig. 1b),
which has been published by Roberts et al. at Pfizer (PF-562,271)
as high-affinity ATP-competitive binder (IC50 = 1.5 nM).13 The
number of ‘false-positive’ hits (e.g., unspecific binders) could be
dramatically decreased by using the FAK-inhibitor I for SPR compe-
tition experiments and fragments binding specifically within the
ATP-pocket were identified. As a result, 180 fragments with a cor-
responding reduction in their binding levels in the presence of the
competitor were selected as primary hits. The primary fragment
hits were evaluated in further detail by SPR in titration series of
Fragment-based lead discovery (FBLD) is considered as valuable
technology within the pharmaceutical industry and has been re-
ported to deliver lead series for a variety of drug targets.8–10 FBLD
uses a diverse library (ꢀ103 molecules) of small molecules with
MW typically below 250 Da for screening in contrast to high-
throughput screening of large libraries (ꢀ106 molecules).
11 concentrations (1.95 lM to 2.0 mM). Steady state dissociation
constants (KD,ss) were determined by plotting the binding levels
at the end of the association phase against the concentration (see
Supplementary Fig. 3S for details) and then fitting the data to a sin-
gle-site binding isotherm.
The affinity determination by SPR confirmed 105 hits with KD,ss
values in the range of 13 lM to 3.5 mM, which were considered as
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Corresponding author. Tel.: +49 6151 725975; fax: +49 6151 72915975.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.