Approaches to selective fibroblast growth factor receptor 4 inhibition through targeting the ATP-pocket middle-hinge region

Article abstract of DOI:10.1039/c7md00213k

A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diaminopyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a hemithioacetal adduct with a 2-formyl naphthalene. In addition, the introduction of an acrylamide into a known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family members.

Full text of DOI:10.1039/c7md00213k

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RESEARCH ARTICLE
Approaches to selective fibroblast growth factor
receptor 4 inhibition through targeting the ATP-
pocket middle-hinge region†‡
Cite this: DOI: 10.1039/c7md00213k
*
Robin A. Fairhurst,
Thomas Knoepfel, Catherine Leblanc, Nicole Buschmann,
Christoph Gaul, Jutta Blank, Inga Galuba, Jörg Trappe, Chao Zou, Johannes Voshol,
Christine Genick, Peggy Brunet-Lefeuvre, Francis Bitsch, Diana Graus-Porta and
Pascal Furet
A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches
and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was
consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of
the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diamino-
pyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an
SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a
hemithioacetal adduct with a 2-formyl naphthalene. In addition, the introduction of an acrylamide into a
known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly
conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family
members.
Received 27th April 2017,
Accepted 28th May 2017
DOI: 10.1039/c7md00213k
rsc.li/medchemcomm
A number of pan-FGFR inhibitors have been described
which target the ATP binding site within the kinase domain
1. Introduction
The fibroblast growth factor family consists of four fibroblast
growth factor receptors (FGFRs), of the receptor tyrosine ki-
nase family, which interact with eighteen fibroblast growth
factor ligands (FGFs). With such a large number of ligand–re-
ceptor permutations the FGFs are key components of a wide
range of signaling systems which are involved in regulating
multiple aspects of development and normal physiology, as
well as being implicated as the drivers of a number of malig-
nancies.1 Of particular interest in the context of cancer, the
aberrant FGF19/FGFR4 signaling in hepatocellular carcinoma
(HCC) has been shown to be both an initiating event in pre-
clinical studies,2 and additionally to be required for the main-
tenance of the disease.3 As a consequence, we became inter-
ested in the identifying FGFR4 inhibitors that could be used
for the treatment of FGFR4-driven diseases, and in particular
HCC. In this letter we describe a number of the hit-finding
approaches that were taken to identify selective FGFR4 inhibi-
tor starting points, and our rationale for prioritisation be-
tween them.
of the receptor, and are characterised as being either: biased
towards FGFR 1–3 inhibition, with >10-fold lower FGFR4 po-
tency; or equipotent versus all four family members.4 The
most advanced of these pan-FGFR inhibitors, infigratinib
(NVP-BGJ398) and AZD4547,5,6 fall into the former FGFR 1–3
biased category, and are currently in PhII clinical studies for
the treatment of cancer, Fig. 1. However, for all of these
agents the robust inhibition of FGFR4 is anticipated to also
lead to extensive inhibition of the other three isoforms, and
as a result will be associated with the side effects resulting
Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland.
E-mail: robin.fairhurst@novartis.com
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available. CCDC 1546562. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c7md00213k
Fig. 1 Structures of the pan-FGFR inhibitors infigratinib and AZD4547.
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from FGFR 1–3 inhibition.7 Therefore, the opportunity to
identify selective inhibitors of FGFR4 was investigated with
the anticipation that it would provide better tolerated treat-
ments, and as a result enable more robust inhibition of the
target to be explored.
lent attachment, are anticipated to be limited, and to be very
specific.
In addition to Cys552, the ATP-binding site of FGFR4 also
contains a second poorly-conserved cysteine residue. Cys477
resides on the P-loop of FGFR4 and is also present at the
equivalent position in FGFR 1–3, and five other kinases
within the human kinome, Fig. 2.8 In contrast to the middle-
hinge region, the P-loop is a relatively dynamic portion of the
protein, and as a result Cys477 should be possible to target
for a covalent interaction in multiple conformations.16
To date a number of groups have reported covalent inhibi-
tors based upon variants of the pan-FGFR inhibitor scaffold
PD 166866.17 Inhibitors which target the middle-hinge region
for FGFR4 selectivity have been reported by five groups: the
acrylamides BLU9931, BLU554, H3B-6527, 1 and 2 bind cova-
As part of the feasibility assessment for the project a se-
quence alignment was carried out across the human kinome
looking for differences within FGFR4 that could be exploited
to gain selectivity. This analysis revealed a poorly conserved
cysteine residue within the middle-hinge region of the ATP
binding site of the FGFR4 kinase domain at position 552.
More specifically, this residue is situated two positions be-
yond the gate-keeper residue towards the C-terminus of the
protein (GK+2 position), Fig. 2. Making a comparison based
upon the size of the amino-acid side-chain at this position in
FGFR4: only twelve other kinases in the kinome have similar
sized, or smaller, residues at the GK+2 position. Additionally,
the other three members of the FGFR family all contain a
larger tyrosine residue at this position. Alternatively, the po-
tential to target the nucleophilic character of Cys552, for a
targeted covalent interaction, revealed only four other kinases
sharing a cysteine at the GK+2 position.8 Therefore, either
targeting the larger sub-pocket adjacent to the hinge, based
upon size, or for covalent attachment, has the potential to
gain selectivity over the other FGFR family members, and
over 97%, or 99%, of the kinome respectively. Similar analy-
ses for targeting FGFR4 selective inhibitors have also been
reported by others.9–15
lently to Cys552.10–14
A structurally unrelated series of
imidazolopyridines, exemplified by 3, are reported to target
the size difference in this region of the ATP pocket to achieve
FGFR4 selectivity.15 In addition, the P-loop cysteine in the
FGFR family has been targeted for pan-FGFR inhibition in an
irreversible-covalent manner with the acrylamide containing
inhibitors FIN-1 and PRN1371,18,19 and in a reversible-
covalent manner with the structurally-related cyano-acrylam-
ides, as exemplified by 4, Fig. 3.20
2. Results and discussion
The differences within the middle-hinge region of the FGFR
family are highlighted in Fig. 4, which shows the X-ray struc-
ture determined for infigratinib bound to FGFR1.5 In this fig-
ure the GK+2 tyrosine, present in FGFRs 1–3, and the cysteine
in FGFR4 are overlaid.21 One hypothesis which was generated
from this structure suggested that substitution of the phenyl
ring of infigratinib in the position ortho to the secondary ani-
line, which resides in the hydrophobic channel of the ATP-
pocket, would provide access to the GK+2 sub-pocket, as indi-
cated in Fig. 4. Such a strategy was appealing as a number of
kinase scaffolds have an aromatic group occupying this ‘hy-
drophobic channel’ region of the ATP pocket, and hence of-
fered the opportunity to readily explore this hypothesis.
At the beginning of the project a number of hit-finding ac-
tivities were initiated to identify FGFR4-selective starting
points, including: a high-throughput screening campaign, in
which hits were counter-screened against FGFR2 to gain an
early insight into selectivity; data mining in combination
with de novo design, to identify known ATP-pocket binders
that could be modified to increase FGFR4 selectivity, and the
rational incorporation of electrophiles for targeted covalent
binding. One goal was to evaluate which type of starting
point would provide the best opportunity to deliver a selective
FGFR4 inhibitor candidate, by any means, but with a particu-
lar interest in those in which the selectivity involved an inter-
action with the middle-hinge region of the ATP-pocket. Such
opportunities were considered to provide a high probability
for kinome-wide selectivity based upon the above sequence
and structural evaluation.
The low conformational flexibility in this region of the ki-
nase domain, adjacent to the hinge, was also anticipated to
facilitate selectivity: substituents fitting well into this rela-
tively rigid region of the protein should differentiate clearly,
as any changes in protein conformation would be anticipated
to be highly disfavoured.16 Additionally, for a covalent-
binding approach the side chain of Cys552 occupies a posi-
tion deep within the middle-hinge region of the protein. As a
result, the shape and size of electrophile, and the possible
approach vectors which could lead to the formation of a cova-
Fig. 2 Positions of Cys552 and Cys477 within the ATP-pocket of the
kinase domain of FGFR4. Two possible conformations of the kinase
P-loop are represented: in beige, the typical hairpin conformation and
in magenta, a disordered conformation observed in the crystal struc-
ture of FGFR4 in complex with compound 8, vide infra.
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Fig. 3 Structures of PD 166866, the FGFR4 selective inhibitors BLU9931, BLU554, H3B-6527, 1, 2 and 3, and the FGFR family P-loop cysteine
targeting FIN-1, PRN1371 and 4.
The analysis of the Novartis internal kinase panel revealed
a small number of compounds exhibiting some level of selec-
tivity for FGFR4 within the FGFR family, and the broader
kinome. Of these potential starting points, the diamino-
pyrimidine 5, Fig. 5, was of particular interest, having been
originally prepared as part of a project targeting inhibitors of
focal adhesion kinase,22 and possessing greater than 100-fold
selectivity within the FGFR family. Biochemical data for the
inhibition of the kinase activities of FGFRs 1–4 are shown in
Tables 1 and 2.21
mode within the FGFR4 kinase domain to be assigned for 5
with a high degree of certainty.23 Based upon this binding
mode, the phenyl group in 5 would occupy the same position
in the hydrophobic channel as shown for infigratinib in
Fig. 4. As a result the isopropoxy group would be orientated
into the larger sub-pocket created by the Cys552 residue.
Thus, enabling the FGFR4 selectivity for 5 to be readily
rationalised based upon this specific interaction, as shown in
Fig. 4. Further support for this rationale was obtained with 6,
the methoxy analogue of 5, in which the smaller methoxy res-
idue can be accommodated in the middle-hinge region by all
four FGFRs, and as a result the compound is a potent inhibi-
tor across all the FGFR family members.
Starting from a well described ATP-competitive scaffold,
such as the diaminopyrimidines, also enabled the binding
Taking the diaminopyrimidine 5 as the starting point, the
structure activity relationships from the further optimisation
of the series were consistent with the proposed binding
mode, which included exploring a diverse range of substitu-
ents on the phenyl ring targeting the GK+2 sub-pocket. This
Fig. 4 Comparison of Cys versus Tyr at the GK+2 of the FGFR family
based upon the X-ray structure of infigratinib (shown in beige) bound
into the ATP binding site of FGFR1 (PDB code: 3TT0): the GK+2 Tyr563
of FGFR1 is shown in white, the GK+2 Cys552 side-chain of FGFR4 is
modeled into the structure and shown in magenta. The hydrogen-
bonds forming the hinge interaction are shown as dotted lines. The or-
tho position of infigratinib, with access to the middle-hinge sub-
pocket, is highlighted by the green arrow.
Fig. 5 Structures of the diaminopyrimidines 5, 6 and 7.
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Table 1 Biochemical FGFR 1–4 activities
Biochemical IC50 (nM)
Compound
FGFR4 (388–802)
FGFR1 (407–822)
FGFR2 (406–821)
FGFR3 (411–806)
Infigratinib
AZD4547
BLU9931
5
6
7
8
9
71 34
56 21
14 10
29 2.1
n.d.
2.2 0.9
1.5 1.0
53 18
57 10
0.8 0.4
0.8
3200
6350 2665
19 6.3
280
4.5 4.9
>10 000
>10 000
1.2 0.8
1.1 0.1
1700 0.42
4433 1650
n.d.
363 172
2.2 2.0
>10 000
>10 000
1.8 1.2
5.2
780
8000 1709
85 50
3500
1.7 0.5
>10 000
>10 000
10
n.d. = not determined; data are from single experiments, or expressed as mean SD, where 2 to 9 repeated experiments were performed.
Table 2 Biochemical FGFR4 activities with the non-phosphorylated wild-type and C552A/C477A variants
FGFR4 biochemical IC50 values (nM)
Compound
Wild-type (442–753)
C552A (442–753)
C477A (442–753)
Infigratinib
AZD4547
BLU9931
6
7
8
9
10
11
12
13
14
62 23
103 46
0.82
64 21
67 17
9.4 0.5
655 118
>10 000
1920 200
8.4 3.2
3.6 1.2
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
11
16
1
3
20
3
6.9 3.6
0.8 0.2
32 11
65 49
>10 000
>10 000
>10 000
>10 000
7.5 0.5
443 94
21
1
43 10
>10 000
>10 000
>10 000
>10 000
Data are from single experiments, or expressed as mean SD, where 2 to 8 repeated experiments were performed.
optimisation yielded analogues with improved FGFR4 po-
tency and a high level of kinase selectivity, including >100-
fold selectivity versus the other FGFR family members, as ex-
emplified by 7.21 The isopropoxy starting point for the GK+2
targeting moiety in 5 was found to be close to optimal for
this diaminopyrimidine series, and the phenyl ring in the
optimised analogue 7 is substituted with the closely related
isobutoxy substituent. Compound 7 was shown to be a useful
non-covalent FGFR4 inhibitor tool compound, and is shown
modelled into the kinase domain of FGFR4 in Fig. 6. Key in-
teractions include: hydrogen bonds to the hinge residue
Ala553; and key hydrophobic contacts between the isobutoxy
group and Cys552, and the methylene-bridge of the bicyclic
moiety and a hydrophobic cleft formed between Val481 and
Gly474.
The optimisation leading to the identification of
7
highlighted that the differences in the size of the GK+2 resi-
due could be exploited by a non-covalent inhibitor to gain an
acceptable level of selectivity for FGFR4 within both, the
FGFR family, and also the broader kinome. In parallel, hit-
finding activities for potential targeted-covalent binders re-
vealed a number of putative electrophile-containing hits,
suggesting a ‘reactive-cysteine’ residue, or residues, were
present within the FGFR4 kinase domain. Of these, the three
hits 8, 9 and 10, shown in Fig. 7, proved to be the most inter-
esting and demonstrated the breadth of covalent-binding in-
teractions that were identified with FGFR4, vide infra.
To better understand any putative covalent-interactions
with FGFR4 biochemical assays were established in which ei-
ther the middle-hinge Cys552, or the P-loop Cys477, were re-
placed by alanine and compared to the wild-type protein.
These data are included in Table 2, and also differ from the
FGFR4 data in Table 1 in that shorter non-phosphorylated
Fig. 6 Model of 7 bound to the kinase domain of the FGFR4.
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Fig. 7 Structures of the FGFR4 covalent binders 8, 9 and 10.
kinase-domain constructs were used in these assays.21 Com-
parison of the IC50 values between the wild-type FGFR4 as-
says in Tables 1 and 2 indicated comparable behavior for the
two proteins, across all the compounds. Additionally, both
FGFR4 alanine variants in Table 2 showed similar bio-
chemical behavior to the wild-type enzyme with respect to:
ATP-binding affinity and the ability to phosphorylate the pep-
tide substrate.21 Thus, supporting no gross changes having
occurred to the kinase domain as a result of these cysteine to
alanine modifications.
One striking difference with the alanine modified FGFR4s
was the greatly reduced activity of the methoxy-substituted di-
aminopyrimidine 6 with the C552A variant, when compared
to the isobutoxy analogue 7. Molecular modeling revealed a
plausible hypothesis for this difference.21 In the case of 6, in
which the smaller alanine side-chain residue is interacting with
the smallest ether residue, the hydrophobic surface shielding
the hinge binding interaction is interrupted. As a result this
key element of the binding interaction is weakened due to an
increased exposure of the hydrogen bonding network to sol-
vent. In contrast, the larger isobutoxy moiety in 7 maintains
the hydrophobic surface, shielding the hinge interaction with
the Ala552 residue present, and as a result exhibits no de-
crease in potency.
The acrylamide 8 was identified by the rational introduc-
tion of an electrophile into a known FGFR ATP-site-binding
pharmacophore. Starting from the pan-FGFR inhibitor
AZD4547, the phenyl ring of the benzamide moiety resides in
the hydrophobic channel of the ATP-binding site in a similar
position to the phenyl ring of 7, as shown in Fig. 6.24 Thus,
the introduction of an acrylamide moiety into either position
ortho to the carbonyl group on the phenyl ring was antici-
pated to present an electrophilic center proximal to Cys552,
or to Cys477 if the P-loop adopted a disordered conforma-
tion. These two alternatives are freely interchangeable via an
approximately 180° rotation about the amide carbonyl to
phenyl-ring bond. However, the conformer in which the acryl-
amide is orientated towards Cys477 is the most stable due to
an intramolecular hydrogen-bond between the acrylamide
N–H and benzamide carbonyl groups.25
when compared to the non-covalent parent-compound
AZD4547, Table 1, strongly supporting a covalent interaction.
Furthermore, comparing the data between the wild type and
FGFR4 variants in Table 2, a 400-fold loss in activity with the
C477A variant strongly supported this cysteine to be the pri-
mary site of reaction with the acrylamide. In contrast, only a
5-fold lower IC50 was determined for the middle-hinge C552A
variant which suggested a much lower level of covalent inter-
action, if any, at this position for 8.
To confirm the putative covalent-interactions of 8, a series
of mass spectrometry (MS) studies were conducted with the
wild type and the cysteine to alanine FGFR4 variants.21 Com-
pound 8 was found to covalently bind to all three proteins to
give the mass of the kinase domain plus 420 Daltons, consis-
tent with the anticipated Michael addition reaction, when
compound and protein where incubated at equivalent con-
centrations. When higher concentrations of 8 were used, no
multiple addition products were observed (highest ratio: 1 to
10). Therefore, confirming that reaction had taken place,
likely with either the GK+2, or P-loop, cysteine. However, five
other cysteines are present in the shorter FGFR4 442–753
kinase-domain constructs which are anticipated to be
‘unreactive cysteines’ based upon their involvement in inter-
actions defining the secondary and tertiary protein structure.
To establish if either, or both, the anticipated sites in wild-
type FGFR4 were reacting with 8 a trypsin digest was made
following the incubation with protein.21 From these studies
both the anticipated compound-modified oligo-peptides
could be detected, supporting that both sites were contribut-
ing to the observed activity of 8. However, although not quan-
titative, the signal strength for the P-loop modified oligo-
peptide was much stronger, with the GK+2 modified oligo-
peptide being close to the lower limit of quantification with
the wild-type protein. This observation was in line with the
biochemical IC50 values in Table 2, indicating that 8 inhibits
FGFR4 mainly through binding to the P-loop cysteine, with
only a small contribution from binding at the GK+2 position.
Although biochemical assays remain a satisfactory way to
compare the activity of irreversible-covalent binding com-
pounds, the IC50 values determined in this way are highly de-
pendent on the assay conditions, and the ratio of kinact/Ki rep-
resents a superior, and invariant, measure of potency in this
Based upon biochemical activity, 8 showed a 50-fold in-
crease in the IC50 value for the inhibition of wild-type FGFR4
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setting.26 Applying this approach with 8, by comparing the
wild-type and C477A kinase domain variant, indicated a
1000-fold faster reaction with the P-loop cysteine compared
to the middle-hinge cysteine, data are shown in Table 3.21
To further characterise the covalent binding of 8, co-
crystallisation studies were performed following reaction with
the wild-type FGFR4 kinase domain. Purification of the major
addition-product prior to crystallization provided the X-ray
co-crystal structure in which the anticipated Michael addition
with 8 had occurred with the P-loop Cys477, as shown in
Fig. 8.21 This structure shows that 8 remains bound within
the ATP-pocket following covalent attachment, and continues
to make the key hinge, and back-pocket, interactions made
by the parent compound AZD4547 consistent with the design
hypothesis.24
Fig. 8 X-ray crystal structure of 8 covalently bound to the P-loop
Cys477 of the FGFR4 kinase domain (PDB ID: 5NWZ).
The dipyridylamine 9 was identified as a singleton from a
high throughput screening campaign and was suspected to
be a covalent binder due to such a relatively small molecule
delivering a high level of FGFR4 potency and selectivity. Man-
ual docking of 9 into the ATP pocket of FGFR4 led to a hy-
pothesis in which the 5-trifluoromethyl pyridyl group consti-
tuted the hinge-binding element. As a consequence, the
6-chloropyridyl moiety would be positioned proximal to
Cys552, due to an internal hydrogen bond between the
2-aminopyridyl N–H and nitro groups, and ideally situated for
a nucleophilic aromatic substitution (SNAr) reaction in which
the 6-chloro substituent would be displaced by the thio-
methyl side-chain. Strengthening this hypothesis, the inhibi-
tory activity of 9 was completely lost with the C552A middle-
hinge variant of FGFR4, when the putative nucleophile was
removed.
teines’. The P-loop cysteine was classed as a ‘reactive
cysteine’ based upon it being positioned on a flexible region
of the protein, and not being involved in strong interactions
within the protein structure. Interestingly, 9 is unable to impact
the kinase activity of the C552A variant, Table 2, even though
the MS studies indicate covalent modification of the P-loop cys-
teine would have likely occurred in the biochemical assay. Con-
sistent with this observation, molecular modeling of the
P-loop modified protein identified no interactions that would
retain 9 within the ATP-site, to prevent ATP binding, and no
interactions that would impede the phosphoryl transfer when
the P-loop is in the active hairpin-conformation.21
For 9, the kinact/Ki ratio was determined to be 3.0 × 104
M−1 s−1 with the wild-type FGFR4 construct and indicated a
170-fold slower rate of reaction compared to the acrylamide
8, although each compound targets primarily a different cys-
teine residue within the ATP-pocket for inhibition.
MS studies were consistent with the anticipated SNAr reac-
tion between 9 and Cys552: the mass of kinase domain plus
352 Daltons was observed when equivalent concentrations of
protein and compound were used. No covalent adducts were
observed under these conditions with 9 and the C552A vari-
ant, confirming the primary site for reaction with the FGFR4
kinase domain. When higher compound concentrations were
used (10-fold excess over protein), a double addition of 9 was
observed with the wild-type protein, indicating some non-
specificity for this electrophile. In contrast, no further change
was seen with the C477A variant, beyond the mono-addition
product, when the compound concentration was increased to
the same extent. Additionally, at the higher compound con-
centrations a mono-addition product started to form with the
C552A variant at a similar rate to the second addition to the
wild-type protein. Taken together these data support the pri-
mary site of reaction for 9 to be Cys552, and that the com-
pound can react in a non-specific manner with ‘reactive cys-
The X-ray co-crystal structure of 9 with the wild-type
FGFR4 kinase domain was also obtained in the same manner
as described above for compound 8, and is shown in Fig. 9.
The structure shows the anticipated SNAr reaction with
Cys552 having taken place at the 2-pyridyl position to dis-
place chloride. Following covalent attachment the bound
Table 3 kinact/Ki measured for 8 with the non-phosphorylated wild-type
and C477A FGFR4 kinase domain variants
kinact/Ki values (M−1 s−1
)
Fig. 9 X-ray crystal structure of compound 9 covalently bound to the
middle-hinge Cys552 of the FGFR4 kinase domain. Hydrogen bonds to
the hinge residues are indicated by dotted lines (PDB ID: 5NUD).
FGFR4 wild-type
FGFR4 C477A
5.1 × 106
5.1 × 102
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dipyridylamine retains the hypothesised interactions within
the hinge region: a hydrogen bond with residue Ala553, and
an aromatic C–H pseudo hydrogen bond with the backbone
carbonyl group of residue Glu551, both interactions involving
the trifluoromethylated pyridine ring.
Somewhat surprisingly this mode of covalent binding has
only been sparsely reported to date,27 even though SNAr reac-
tions offer multiple opportunities to modulate reactivity by
changing the nature of the aromatic group, the leaving
group, and the steric and electronic properties of the ring
substituents.
Additionally, in this X-ray structure the 2-formyl group adopts
an anti-conformation with respect to the quinoline ring-
nitrogen which was calculated to be the lowest energy confor-
mation, and 4.9 kcal mol−1 more stable than the syn con-
former. Although ground state conformations do not control
reaction outcomes,28 in this instance the addition of the low-
est energy anti-conformer of 10 results in the epimer with the
(R)-configuration at the newly formed hemithioacetal center.
However, both epimeric addition products could be modeled
with a hydrogen bond between the newly formed hydroxyl
group and Val500, and these hydrogen-bonding interactions
were also anticipated to stabilise both transition states.
Therefore, an a priori prediction of the stereochemistry of the
major addition product could not readily be made. Fig. 11
shows a model of 10 bound to FGFR4 based upon the above
binding hypothesis in which the hinge interaction involves a
hydrogen bond between the pyridyl N and the backbone N–H
of Ala553, and the hemithioacetal hydroxyl group is shown in
the (R)-configuration making a hydrogen bond with the car-
bonyl of Val500. In addition, as observed in the crystal struc-
ture of compound 9 bound to FGFR4, the model suggests the
possibility of an aromatic C–H pseudo hydrogen bond be-
tween the pyridiyl ring and the backbone carbonyl of Glu551.
Support for the hypothesised covalent-binding mode of 10
was derived from the complete loss of FGFR4 activity with
the C552A variant, Table 2. This result highlights the critical
role of the Cys552 thiol functionality for the binding of
FGFR4 to 10. Additionally, analogues 11–13, Fig. 12, in which
the 2-quinolinyl aldehyde was replaced by a proton, carboxylic
acid, or a hydroxymethyl group were all found to be inactive.
These results highlight the critical role of the 2-formyl group
for the interaction of 10 with FGFR4. The 2-formylquinoline
analogue 14, in which the hinge-binding interaction was
destabilised by exchanging the pyridine for the correspond-
ing phenyl analogue, also resulted in a complete loss of
FGFR4 activity. Taken together, the data with analogues
10–14 provided consistent support for the hypothesised cova-
lent interaction between 10 and FGFR4.
A small set of closely related 2-formylquinoline amides
were identified as part of the screening campaign, of which
10 is a representative member. Once more, the high potency
for the inhibition of FGFR4 from a relatively small inhibitor,
combined with excellent FGFR 1–3 selectivity, suggested an
interaction with the middle-hinge region was driving the ac-
tivity, and likely a covalent interaction with Cys552. Addition-
ally, the wider kinase-selectivity for 10 was also excellent
when measured against a panel of >50 kinases.21 MK2
(MAPKAPK2) was the kinase inhibited to the greatest extent,
when screened at a concentration of 10 μM, and for which an
IC50 value of 2.3 μM was determined. This observation fur-
ther implicated a key interaction with Cys552 for 10, as MK2
is one of the four other kinases within the human kinome
containing a GK+2 cysteine residue.8
A binding hypothesis for 10 was generated in which the
2-aminopyridyl moiety constituted the hinge-binding element,
with an equivalent hydrogen-bonding interaction to that
shown in Fig. 6 for 7. Due to the strong intramolecular hydro-
gen bond in 10, between the amide N–H and quinoline nitro-
gen, the 2-formyl quinoline group would then be ideally posi-
tioned for an addition reaction to occur with the thiol of
Cys552 to generate a hemithioacetal adduct.25 The topogra-
phy of the protein in the middle-hinge region ensures that
this addition reaction can only occur with a single face of the
aldehyde when the hinge interaction is in place, or being
formed. Fig. 10 shows the single crystal X-ray structure of 10,
in which the intramolecular hydrogen bond is highlighted.21
Fig. 10 ORTEP plot of 10 determined by X-ray crystallography. The
quinoline-nitrogen to amide N–H distance in this structure is deter-
mined to be 1.9 Å, consistent with an intramolecular hydrogen-bond.
Fig. 11 Model of 10 covalently bound to the middle-hinge Cys552 of
the FGFR4 kinase domain. Hydrogen bonds are represented as dotted
lines.
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the potential for protein adducts to remain long after the
compound concentration has been depleted. Additionally,
one further benefit of reversible-covalent inhibitors is that
they can be envisioned to be less immunogenic. In contrast
to irreversible-covalent binders, the cysteine affinity of
reversible-covalent binders will be greatly reduced for the
oligo peptides that arise following protein degradation.20
With no possibility to disconnect efficacy from exposure
in a favorable way, none of interesting irreversible-covalent
starting points, as exemplified by 8 and 9, were evaluated fur-
ther. However, the non-covalent and the reversible-covalent
opportunities remained as interesting possibilities for further
study. In particular, 10 highlighted the potential for high
FGFR4 potency, and excellent selectivity, in combination with
the expectation that efficacy would be regulated following a
standard exposure effect relationship. Although some ques-
tions remained concerning the presence of the aryl aldehyde
group in 10,34 the favorable potency and selectivity made this
an interesting series for further investigation.
Fig. 12 Structures of the analogues 11–14 exploring the key elements
of the pharmacophore of 10.
Previously a number of aliphatic aldehydes have been de-
scribed to react with cysteine thiol groups, in particular in
the area of protease inhibitors.29 The C–S bond formed be-
tween inhibitor and protein in these hemithioacetal interme-
diates constitutes a relatively weak covalent-bond, typically
considered to be involved in reversible interactions.30 Consis-
tent with the notion of reversibility, mass spectral studies
with the 2-formylquinoline 10 revealed no evidence for stable
addition products under the same ionisation conditions in
which covalent adducts had been observed with the irrevers-
ible inhibitors 8 and 9. Taken together the above observa-
tions strongly supported a reversible-covalent interaction to
be occurring between compound 10 and FGFR4.
In parallel to the identification of covalent starting points
for lead optimisation, the resynthesis-rates of FGFR4 in two
FGF19/FGFR4 positive HCC cell lines (HUH7 and Hep3B)
were determined. Using a stable isotope labelling with amino
acids (SILAC) approach revealed a relatively rapid resynthesis
half-life of <2 h in both cell lines.21,31 The inclusion of a fully
efficacious concentration of a selective FGFR4 inhibitor in
these SILAC experiments was found not to change the
resynthesis rates, suggesting the half-life of FGFR4 is also in-
dependent of the level of pathway activation in these HCC
cell lines.
This relatively rapid FGFR4 protein resynthesis rate in
combination with an emerging pharmacodynamic (PD)/effi-
cacy relationship, which suggested complete and continuous
inhibition of FGFR4 would be required for maximum anti-
tumor efficacy,32 resulted in a re-evaluation of the suitability
of the irreversible-covalent approach for the treatment of
HCC. The concern being that to achieve this level of inhibi-
tion in vivo would minimally require an efficacious level of
compound to be maintained throughout the dosing interval,
to continually engage newly synthesised protein. Achieving
this exposure profile was considered to be challenging for an
irreversible-covalent modality. However, acrylamide based in-
hibitors have been described with respectable in vivo half-
lives, including BLU554.11 Irreversible-covalent inhibition
was also considered to be the situation most likely to incur
undesirable side-effects due to non-specific reactivity.33 In
particular, in the gut for compounds administered orally at
the relatively high doses that were anticipated to be required
to achieve sustained PD modulation. In this setting even
highly selective irreversible-covalent binders are anticipated
to bind non-specifically due to the initial high gut-levels, with
Conclusion
In conclusion, structurally diverse series of selective FGFR4
hits have been identified through screening and de novo de-
sign. Although the screening approaches were unbiased, the
origin of the selectivity for the most interesting examples can
be rationalized to arise from key interactions within the
middle-hinge region of the ATP pocket. This observation was
consistent with a selectivity hypothesis generated at the start
of the project from a kinase-domain sequence-alignment
which was centered on the cysteine residue at the GK+2 posi-
tion of FGFR4. This hypothesis also enabled the rational in-
troduction of acrylamides into known FGFR inhibitor scaf-
folds to generate covalent inhibitors targeting both of the
reactive cysteines within the ATP pocket of FGFR4. Of greater
interest, potent and highly selective FGFR4 inhibitors were
identified through screening which interacted covalently with
Cys552 through less well established mechanisms of action.
However, the rapid FGFR4 resynthesis-rate, and temporal re-
quirements for FGFR4 inhibition in HCC, made irreversible-
covalent inhibition a less attractive approach compared to
the non-covalent and reversible-covalent options. Of these op-
tions the 2-formylquinoline amides, represented by 10,
proved to be particularly interesting. The further evaluation
of this hit series, which is presumed to be inhibiting through
a reversible-covalent mechanism of action, will be the subject
of future disclosures.
Author contributions
R. A. F., T. K., C. L., N. B., C. G., J. B., J. T., J. V., C. G., F. B.,
D. G-P. and P. F. analysed data and designed the experi-
ments. R. A. F., T. K., C. L., N. B. and C. G. conducted the
synthetic and medicinal chemistry. P. F. carried out the com-
putational chemistry. J. B., I. G. and J. T. developed and
conducted the biochemical assays. C. Z. conducted the
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FGFR4 co-crystallisation studies. C. G., P. B-L. and F. B.
designed and conducted the mass spectroscopy studies. J. V.
designed and analysed the SILAC experiments. R. A. F., T. K.,
N. B. and P. F. wrote and reviewed the manuscript.
growth factor receptor tyrosine kinase family, Cancer Res.,
2012, 72, 2045–2056.
7 R. Dienstmann, J. Rodon, A. Prat, J. Perez-Garcia, B. Adamo,
E. Felip, J. Cortes, A. J. Iafrate, P. Nuciforo and J. Tabernero,
Genomic abberations in the FGFR pathway: opportunities
for targeted therapies in solid tumors, Ann. Oncol., 2014, 25,
552–563.
Acknowledgements
The authors would like to thank Thomas Huerlimann, Da-
mien Hubert, Elvira Masso, Michel Niklaus, Pierre Nimsgern,
Sebastien Ripoche, Karin Ryffel and Jasmin Wirth for techni-
cal assistance in the preparation of the compounds; Binesh
Shrestha, Sihame Haddad, Magdelana Maschlej, Sandra
Kapps and Aurelie Winterhalter for the cloning, expression
and purification of the proteins; Sylvia Buhr for conducting
the biochemical assays; Nina Baur, Bruno Inverardi and
Astrid Pornon for carrying out the SILAC experiments; Ina Dix
for performing small molecule X-ray crystallography. The in
vivo pharmacokinetic experiments were performed by Michael
Kiffe, Sandrine Desrayaud, Peter Wipfli, Melih Altin and Marc
Fischer, according to the regulations effective in the Canton
Basel-Stadt, Switzerland, all procedures and protocols were
reviewed and approved by the local veterinary authorities of
the Canton Basel-Stadt, specifically according to experimental
license No. BS1587.
8 E. Leproult, S. Barluenga, D. Moras, J.-M. Wurtz and N.
Winssinger, Cysteine mapping in conformationally distinct
kinase nucleotide binding sites: application to the design of
selective covalent inhibitors, J. Med. Chem., 2011, 54,
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9 E. Lesca, A. Lammens, R. Huber and M. Augustin, Structural
analysis of the human fibroblast growth factor receptor 4
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10 M. Hagel, C. Miduturu, M. Sheets, N. Rubin, W. Weng, N.
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Shutes, C. Winter, C. Lengauer, N. E. Kohl and T. Guzi, First
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11 C. V. Miduturu, M. Hagel, M. Sheets, N. Rubin, W. Weng, N.
Bifulco, L. V. Dipietro, J. Kim, N. Brooijmans, B. L. Hodous,
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