Identification of an Indazole-Based Pharmacophore for the Inhibition of FGFR Kinases Using Fragment-Led de Novo Design

Article abstract of DOI:10.1021/acsmedchemlett.7b00349

Structure-based drug design (SBDD) has become a powerful tool utilized by medicinal chemists to rationally guide the drug discovery process. Herein, we describe the use of SPROUT, a de novo-based program, to identify an indazole-based pharmacophore for the inhibition of fibroblast growth factor receptor (FGFR) kinases, which are validated targets for cancer therapy. Hit identification using SPROUT yielded 6-phenylindole as a small fragment predicted to bind to FGFR1. With the aid of docking models, several modifications to the indole were made to optimize the fragment to an indazole-containing pharmacophore, leading to a library of compounds containing 23 derivatives. Biological evaluation revealed that these indazole-containing fragments inhibited FGFR1-3 in the range of 0.8-90 μM with excellent ligand efficiencies of 0.30-0.48. Some compounds exhibited moderate selectivity toward individual FGFRs, indicating that further optimization using SBDD may lead to potent and selective inhibitors of the FGFR family.

Full text of DOI:10.1021/acsmedchemlett.7b00349

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Letter
Identification of an Indazole-Based Pharmacophore for the
Inhibition of FGFR Kinases Using Fragment-Led De Novo Design
Lewis D Turner, Abbey J Summers, Laura O Johnson, Margaret A Knowles, and Colin W. G. Fishwick
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.7b00349 • Publication Date (Web): 11 Nov 2017
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Identification of an Indazole-Based Pharmacophore for the Inhibition
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of FGFR Kinases Using Fragment-Led De Novo Design
‡
‡
║
┼
Authors: Lewis D. Turner , Abbey J. Summers , Laura O. Johnson , Margaret A. Knowles and Colin W.
G. Fishwick║*
║School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK
┼
Leeds Institute of Cancer and Pathology, University of Leeds
Keywords: SBDD, De Novo, FGFR, Fragment, Indazole
ABSTRACT: Structure-based drug design (SBDD) has become a powerful tool utilised by medicinal chemists to rationally guide
the drug discovery process. Herein, we describe the use of SPROUT, a de novo-based program, to identify an indazole-based phar-
macophore for the inhibition of fibroblast growth factor receptor (FGFR) kinases, which are validated targets for cancer therapy. Hit
identification using SPROUT yielded 6-phenylindole as a small fragment predicted to bind to FGFR1. With the aid of docking mod-
els, several modifications to the indole were made to optimise the fragment to an indazole-containing pharmacophore, leading to a
library of compounds containing 23 derivatives. Biological evaluation revealed that these indazole-containing fragments inhibited
FGFR1-3 in the range of 0.8-90 μM with excellent ligand efficiencies of 0.30-0.48. Some compounds exhibited moderate selectivity
towards individual FGFRs, indicating that further optimisation using SBDD may lead to potent and selective inhibitors of the FGFR
family.
FGFRs are a sub-family of tyrosine kinases (TKs) that are in-
volved in manycellular processes such as cell proliferation, cel-
lular repair and cell migration.1 Aberrant signalling within this
class of kinase has many implications in cancer and particularly
in bladder cancer.2 It has been shown that around 50% of upper-
and lower-urinary tract tumours possess FGFR3 mutations.3 As
well as mutations within FGFR3, overexpression in both
FGFR3 and FGFR1 have been found in urothelial carcinomas
at all grades and stages.4,5 Currently, several FGFR inhibitors
are in clinical use or under clinical development with some act-
ing as selective FGFR inhibitors whilst others are pan-kinase
inhibitors (see Supplemental Section 1.0 (SI-1.0)). There are
currently no examples of molecules that exhibit sub-type selec-
tivity for the FGFRs and the role of sub-type selectivity in the
pharmacological profile of such anticancer agents has not been
established. Development of a pan FGFR inhibitor may be more
clinically beneficial than an inhibitor that only perturbs one
FGFR sub-type, however, unwanted inhibition of FGFR1 and
FGFR3 leads to side effects such as hyperphosphatemia6 under-
lining the need for FGFR2 sub-type selective inhibitors.
CH5183284 is a potent and selective inhibitor of FGFR1-3 ex-
hibiting IC50 values of 9.3, 7.6 and 22 nM7 respectively and this
compound is currently under clinical investigation for the treat-
ment of cancer patients that harbour FGFR genetic alterations.
CH5183284 was discovered using the conventional approach of
high throughput screening (HTS). As a prominent approach in
lead discovery, HTS allows rapid screening of large compound
libraries but is limited to the chemical diversity of that library.
Typically, an HTS library may contain approximately one mil-
lion compounds; a fraction of the total theoretical ‘drug-like’
chemical space which is predicted to be between 1060-10100
compounds.8 SBDD is a useful addition tool for lead identifica-
tion that can be used alone or in conjunction with HTS to initiate
and facilitate a drug discovery program. The application of
SBDD can take a number of different forms such as the use of
virtual HTS, template matching, and de novo molecular design.
A de novo approach can produce compounds ‘from scratch’.
They are predicted to bind to a target and in theory, access lim-
itless untapped chemical space that may not be present in cur-
rent compound libraries. De novo design was first used in the
1980s with numerous programs offering access to novel chem-
ical entities from which a number of pharmacologically active
molecules have resulted.9,10,11 One such program, SPROUT,
was developed by Johnson et al in the early 90s and is designed
for constrained structure generation.12 Structure generation can
be simplified into two steps: the first part is formation of a mo-
lecular ‘skeleton’ that must satisfy the steric constraints of a
binding pocket; the second part is generation of a molecule(s)
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by atom-substitution to the skeleton.13 The constraints of the
on the different tools and modules within SPROUT that are
binding pocket are usually defined using the X-ray crystal data
of a target. However, if X-ray crystal data is unavailable, it is
possible to design novel inhibitors based purely on a pharmaco-
phore hypothesis.13 Once the constraints have been defined it is
possible to choose interaction sites within the protein e.g. amino
acids within binding pockets for endogenous ligands. Comple-
mentary atoms or fragments from available libraries within the
program are chosen to interact with these sites and are linked
together using spacer fragments. The resulting solutions are
clustered using a range of parameters including estimated
binding affinity or molecular complexity.13 Further information
used for structure generation can be found in supplementary in-
formation (SI-4.1). Here, we describe the use of SPROUT to
generate an active indazole-based pharmacophore for the inhi-
bition of FGFR kinases. The crystal structure of inhibitor
CH5183284 co-crystallised within FGFR1 (PDB code: 3WJ6)
was loaded into SPROUT (v6.4.10) and visualised using
PyMol14 (Figure 1a). Compound CH5183284 occupies the
adenosine triphosphate (ATP) binding site within FGFR1 and
several interactions are observed. Two H-bonds form with the
benzimidazole moiety; one with the backbone nitrogen of
Asp641 and the other with a side chain carboxy oxygen of
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fg
b)
Glu531
O
O
H2
N
H
NH
N
N
N
NH
O
H1
H
H
H
O
N
N
O
N
H
O
Asp641
Ala564
Glu562
Figure 1.a) Co-crystal structure of CH5183284 bound within
Figure 2.a) De novo designed fragment 6-phenylindole (1) docked
within the active site of FGFR1 using Glide. An H-bond is pre-
dicted to form between the indole NH and the backbone carbonyl
of Glu562. b) Schematic of binding pose of 6-phenylindole core
within FGFR1 with modifications outlined in orange.
FGFR1. H-bonding interactions are indicated using cyan dashes
'
and hydrophobic pockets are indicated by H1 and H2. b) Sche-
matic of binding pose of CH5183284 within FGFR1 showing in-
termolecular interactions. Amino acids, H-bonds and hydrophobic
pockets are shown in green, red and blue respectively.
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Glu531 (Figure1). Another H-bond forms between the pyrazole
Compounds 1-6 were readily prepared using classic Suzuki
chemistry using available aryl bromides and boronic acids
(SI-1.1).
NH2 and the backbone carbonyl of Glu562. An H-bond can also
be seen between the ketone moiety and the backbone NH of
Ala564.7 De novo design was carried out targeting the
CH5183284 binding region within the crystal structure of
FGFR1 using SPROUT. Three interaction sites (Asp641,
Glu531 and Glu562) were chosen and complementary frag-
ments were selected (H-bond acceptors for Asp641 and H-bond
donors for Glu531 and Glu562 respectively). Spacer templates
consisting of aryl, alkyl and amide groups were chosen to link
these various moieties together consecutively. The resulting
solutions were then considered visually in terms of percieved
ease of synthesis of analogues, identifying 6-phenylindole as a
particularly attractive fragment. As an independent check on the
validity of 6-phenylindole as a starting point for further
inhibitor design, 6-phenylindole was re-docked into the FGFR1
crystal structure using Glide15 and the binding pose analysed
(Figure 2a). This fragment is predicted to bind in a similar way
to that of CH5183284 with the indole NH forming an H-bond
with the backbone carbonyl of Glu562. Further modelling indi-
cated that several structural modifications could be made in or-
der to increase the number of bonding interactions between the
inhibitor and FGFR1. Specifically, substitution from an indole
to an indazole would open up the opportunity for an H-bond to
form between the indazole N-2 and the backbone NH of
Ala564. Additionally, modification of the 6-phenyl ring to a 6-
pyridyl derivative could also introduce an H-bond between the
pyridine nitrogen and the backbone NH of Asp641. Further-
more, placement of a small hydrophobic group in the meta-po-
sition of the 6-phenyl ring was predicted to allow occupation of
the sub-site H2 pocket, hypothesised to further increase binding
affinity (Figure 2). This led to us considering an initial fragment
library of varying indole/indazole scaffolds which appeared to
offer modest binding potential in the enzyme and would also
readily allow us to test the initial design hypotheses (Figure 3).
Biological Evaluation
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Compounds 1-6 were screened against FGFR1 at an initial con-
centration of 500 µM using a fluorescence resonance energy
transfer (FRET)-based Z’-lyte® assay16 (SI-3.1). IC50 measure-
ments for compounds 1-6 were determined using a 10-point ti-
tration experiment with 3-fold serial dilutions starting from a
concentration of 500 µM. The results are outlined (Table 1):
Table 1. Biological results for compounds 1-6 when
screened against FGFR1.
Compound
No.
% Inhibitiona
500 µM
Structure
IC50 (µM)
LE
1 ± 0.0b
16 ± 0.5
21 ± 3.5
53 ± 0.0b
66 ± 1.5
73 ± 1.0
NTc
N/A
1
2
3
4
5
6
NT
N/A
N/A
0.38
0.38
0.39
>500
77 ± 0.9
90 ± 0.9
36 ± 0.9
a % Inhibition and IC50 values are given as the mean ± SD of all data points. b No differ-
ence in measured data points (N=2). c NT = not tested.
Interestingly, indole-based fragments 1-3 were found to be es-
sentiallyinactive with >50% less inhibition againstFGFR1 than
their corresponding indazole counterparts 4-6. This was con-
firmed following IC50 measurements with the indazole deriva-
tives having a potencyin the range of36-90 µM against FGFR1.
This is consistent with the design hypothesis which requires the
2-position nitrogen present in the indazole compounds to be in-
volved in H-bonding with the backbone NH of Ala564 and
would appear to be crucial for inhibition of FGFR1. These re-
sults are also consistent with those demonstrated by Liu et al
who have recently reported inhibitors of FGFR1 containing in-
dazole cores.17 The indazole ligands 4-6 possess a ligand effi-
ciency (LE) of >0.35 which is an excellent start for fragment
Cl
N
N
N
H
H
H
N
N
1
2
3
6
a
N
N
Cl
N
N
N
N
H
H
H
N
N
4
5
Figure 3. Initial fragment compound library
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optimisation.18 Compound 6 is the most active with an IC50
The results obtained for single point analyses were carried out
value of 36 µM. Compound 4 is more active than compound 5
suggesting that the pyridine nitrogen has a detrimental effect
upon the binding affinity of these fragments to FGFR1. Ration-
alization of these observed activities was investigated using
docking models (SI-4.2).
at a concentration of 100 µM. The observed activities for com-
pounds 8-10 indicate that substituting larger groups in the 3-
position of the phenyl ring increases potency, with compound
10 having an IC50 value of 2.0 µM and an excellent LE of 0.44.
The increase in potency could be due to the ethoxy group occu-
pying the H1 pocket as predicted using modelling (SI-4.2). The
phenol moiety present in compound 11 has the potential to be
an H-bond donor to residue Glu531 that lies deep within the
ATP binding pocket whereas compound 12-13 lack this ability.
Interestingly, compound 11 has an IC50 of 12 µM whereas com-
pounds 12-13 are inactive. This suggests that the H-bond donor
potential of the phenol is crucial for inhibition of FGFR1 and
modelling reinforces this hypothesis (SI-4.2). Substitution at
the 6-position substituent on the indazole core from a six mem-
bered ring to a five membered ring results in a loss of activity,
shown by the results obtained for compounds 15-16. Compound
17 is completely inactive; revealing that a 3,5-substitution pat-
tern around the 6-position phenyl ring is detrimental to activity
suggesting there may be a steric limit to what can be tolerated
around this ring.
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Library Expansion
In order to expand the structure activity relationships (SARs)
for the active indazole pharmacophore 7, a larger focus library
of target compounds was developed. Compounds were readily
synthesised using the conditions outlined in SI-1.1 and sub-
jected to biological evaluation (Table 2).
Table 2: Reaction yields and biological results for compounds
8-17 when screened against FGFR1.
Compound
No.
% Inhibitiona
100 µM
IC50
Yield
(%)b
Structure
LE
(µM)
19 ± 1.5
32 ± 0.5
85 ± 0.0c
83 ± 3.5
11 ± 2.0
12 ± 4.0
20 ± 3.0
29 ± 0.5
19 ± 0.5
-5 ± 0.5
NTd
N/A
35
18
39
5e
8
NT
N/A
0.44
0.43
N/A
N/A
N/A
N/A
N/A
N/A
9
SAR Exploration of Compounds 10 and 11
2 ± 0.4
12 ± 1.6
NT
10
11
12
13
14
15
16
17
Compounds 10 and 11 were taken forward to further optimise
focusing on substitution around the phenyl ring. Based on mod-
elling (SI-4.2) it became apparent that further substitution of
small hydrophobic groups in addition to the existing substituent
on the 6-position phenyl ring could result in tighter binding to
FGFR1. A further library was therefore developed, synthesised
using the conditions outlined in SI-1.1, and biologically evalu-
ated (Table 3). Compounds 21-24 were synthesised using an al-
ternative route (SI-1.1.1). In addition to FGFR1, the activity of
these compounds against FGFR2/3 was also evaluated. Inspec-
tion of the data in Table 3 reveals some interesting points. In
addition to FGFR1, compound 10 was also found to inhibit
FGFR2/3 with IC50 values of 0.8 and 4.5 µM respectively. In
general, addition of small hydrophobic substituents to the phe-
nyl ring of compound 10 results in a decrease in potencyagainst
FGFR1-3. Compound 18 exhibits a complete loss of activity for
FGFR3, a 42-fold decrease in activity for FGFR1, and a 15-fold
decrease for FGFR2 when compared to compound 10. A similar
trend can be seen for compounds 19 and 20. This suggests that
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43
NT
NT
NT
NT
NT
a % Inhibition and IC50 values are given as the mean ± SD of all data points. b Reaction yields for
Suzuki chemistry (SI-1.1). c No difference in measured data points (N=2). d NT = not tested. e Low
yield explained by propensity of 2/4-hydroxyphenylboronic acids undergoing rapid protode-
boronation.19
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Table 3: Biological results for compounds 10-24 when screened against FGFR1-3.
IC50 (µM)
FGFR
LE
5
6
7
8
Compound No.
10
Structure
Yield (%)a
FGFR
2
1
2
3
1
3
2.0 ± 0.4
0.8 ± 0.3
4.5 ± 1.6
0.44
0.47
0.42
39
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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28
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30
31
32
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34
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83 ± 0.9
9.7 ± 1.1
>100
12 ± 0.9
6.4 ± 0.9
52 ± 0.9
>100
>100
>100
0.30
0.37
N/A
0.36
N/A
N/A
N/A
42
18
2
18
19
20
0.38
0.30
12 ± 1.6
9.9 ± 0.7
3.0 ± 1.0
5.4 ± 0.9
51 ± 1.2
>100
0.43
0.41
0.48
0.43
0.38
N/A
5
11
21
23*
14 ± 0.8
7.3 ± 1.1
NTb
0.40
0.43
N/A
72*
22
6.4 ± 0.8
8.8 ± 0.7
NT
NT
NT
NT
0.43
0.42
N/A
N/A
N/A
N/A
13*
20*
23
24
% Inhibition and IC50 values are given as the mean ± SD of all data points. a Reaction yields for Suzuki chemistry (SI-1.1). b NT = not tested. *Yield for Suzuki
step only- see SI-1.1.1 for full synthetic detail.
the requirements to inhibit FGFR3 are more stringent than
FGFR1/2 with FGFR2 being the most tolerant to further substi-
FGFR1 and compound 18 shows that although the potency has
dropped the difference in selectivity has increased to ~7-fold by
addition of a fluorine atom in the 5-position of the 6-position
phenyl ring. As these compounds are fragments (MW ~250) we
hypothesise that structural expansion of these molecules will re-
sult in an increase in the small selectivity difference between
FGFR1/2 that compounds 10 and 18 currently exhibit.
tution around the 6-position phenyl ring. Compound 11 was
found to exhibit IC50 measurements of 3.0 and 51 µM against
FGFR2/3 respectively. Addition of small hydrophobic groups
onto the 6-position phenyl ring in compound 11 generally re-
sults in an increase in potency with a preference for substitution
in the 2-position. Compounds 21 and 23 show an increase in
size of the 2-position substituent from fluorine to methyl re-
spectively which results in an increase in potency against
FGFR1, this trend is also seen for compounds 22 and 24. Sub-
stitution of small hydrophobic groups in the 3-position of the 6-
position phenyl ring is less well tolerated when compared to
2-position substituted rings. These fragments show small selec-
tivity differences for individual sub-type FGFRs. Compound 10
exhibits a 2.5-fold selectivity preference for FGFR2 over
Conclusion
We have identified an indazole-based pharmacophore that
shows encouraging levels of inhibition against FGFR kinases
using a denovo-based design approach to identify the initial hit.
Optimisation of this hit led to a library of fragments whereby
SARs were established and specific structural aspects of the
molecules upon which sub-type selectivity appear to depend,
have been identified. Current efforts are focused on designing
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larger compounds in order to increase potency and, more im-
5.
Tomlinson, D. C.; Lamont, F. R.; Shnyder, S. D.;
Knowles, M. A., Fibroblast Growth Factor Receptor 1
Promotes Proliferation and Survival via Activation of the
Mitogen-Activated Protein Kinase Pathway in Bladder
Cancer. Cancer Res. 2009, 69 (11), 4613-4620.
portantly, selectivity for the individual FGFR sub-types.
ASSOCIATED CONTENT
Supporting Information
6.
Ornitz, D. M.; Itoh, N., The Fibroblast Growth
Factor signaling pathway. Wires Dev Biol 2015, 4 (3), 215-
The Supporting Information is available free of charge on the ACS
Publications website.
Current FGFR Inhibitors. Synthetic routes. All experimental details
– general procedures and instrumentation, general methods, com-
pound characterisation, 1H NMR and 13C NMR spectra for all com-
pounds. FRET-based Z’ lyte assay details – assay conditions, IC50
curves. Computational details – SPROUT, docking models (PDF).
266.
7.
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Nakanishi, Y.; Akiyama, N.; Tsukaguchi, T.; Fujii,
T.; Sakata, K.; Sase, H.; Isobe, T.; Morikami, K.; Shindoh,
H.; Mio, T.; Ebiike, H.; Taka, N.; Aoki, Y.; Ishii, N., The
Fibroblast Growth Factor Receptor Genetic Status as a
Potential Predictor of the Sensitivity to CH5183284/Debio
1347, a Novel Selective FGFR Inhibitor. Mol Cancer Ther
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AUTHOR INFORMATION
Corresponding Author
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Simmons, K. J.; Chopra, I.; Fishwick, C. W. G.,
Structure-based discovery of antibacterial drugs. Nat Rev
Microbiol 2010, 8 (7), 501-510.
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strategy for practical lead identification. Med Res Rev 2003,
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Showalter, R.; Pelletier, L. A.; Lewis, C.; Tucker, K.;
Moomaw, E.; Parge, H. E.; Villafranca, J. E., Design,
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*Colin Fishwick - email: C.W.G.Fishwick@leeds.ac.uk. Address:
School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK
Honma, T., Recent advances in De novo design
Author Contributions
The manuscript was written through contributions of L.D. Turner,
M.A. Knowles and C.W.G. Fishwick. M.A. Knowles and C.W.G.
Fishwick provided supervisory support throughout this work. Pro-
ject development and experimental work was carried out princi-
pally by L.D. Turner. ‡These authors contributed equally through
experimental lab work. All authors have given approval to the fi-
nal version of the manuscript.
Babine, R. E.; Bleckman, T. M.; Kissinger, C. R.;
Funding Sources
11.
Iwata, Y.; Naito, S.; Itai, A.; Miyamoto, S., Protein
Medical research council-doctoral training grant.
structure-based de novo design and synthesis of aldose
reductase inhibitors. Drug Des Discov 2001, 17 (4), 349-59.
12.
Williams, P., SPROUT - A Program for Structure
Generation. J. Comput.-Aided Mol. Des. 1993, 7 (2), 127-
153.
ACKNOWLEDGMENT
The authors thank Dr. Martin McPhillie, Medical Research Council
(MRC) and Life Technologies Ltd.
Gillet, V.; Johnson, A. P.; Mata, P.; Sike, S.;
ABBREVIATIONS
13.
Gillet, V. J.; Newell, W.; Mata, P.; Myatt, G.; Sike,
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