X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan–McDermid Syndrome

Article abstract of DOI:10.1002/cmdc.201800344

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan–McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Full text of DOI:10.1002/cmdc.201800344

Accepted Article
Title: X-ray structures and feasibility assessment of CLK2 inhibitors for
Phelan McDermid syndrome
Authors: Andreas Markus Lerchner, Joerg Kallen, Christian Bergsdorf,
Bertrand Arnaud, Mario Bernhard, Murielle Brichet, Amanda
Cobos-Correa, Azeddine Elhajouji, Felix Freuler, Ivan
Galimberti, Christel Guibourdenche, Simon Haenni, Sandra
Holzinger, Juerg Hunziker, Aude Izaac, Markus Kaufmann,
Lukas Leder, Hans-Joerg Martus, Peter von Matt, Valery
Polyakov, Patrik Roethlisberger, Guglielmo Roma, Nikolaus
Stiefl, and Marianne Uteng
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201800344
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X-ray structures and feasibility assessment of CLK2 inhibitors for
Phelan McDermid syndrome
[b]
Joerg Kallen [a], Christian Bergsdorf [a], Bertrand Arnaud [c], Mario Bernhard [b], Murielle Brichet
,
Amanda Cobos-Correa [b], Azeddine Elhajouji [b], Felix Freuler [b], Ivan Galimberti [b], Christel
Guibourdenche [c], Simon Haenni [b], Sandra Holzinger [b], Juerg Hunziker [b], Aude Izaac [b], Markus
Kaufmann [b], Lukas Leder [b], Hans-Joerg Martus [b], Peter von Matt [c], Valery Polyakov [c], Patrik
Roethlisberger [b], Guglielmo Roma [b], Nikolaus Stiefl [c], Marianne Uteng [b], and Andreas Lerchner *[c]
[a]
[b]
[c]
Dr. J. Kallen, Dr. C. Bergsdorf, Novartis Institutes for BioMedical Research, Novartis Campus CH-4002 Basel Switzerland; these two first authors
contributed equally to this work
M. Bernhard, M. Brichet, Dr. A. Cobos-Correa, Dr. A. Elhajouji, F. Freuler, Dr. I. Galimberti, S. Haenni, S. Holzinger, A. Izaac, M. Kaufmann, Dr. L. Leder,
Dr. H-J Martus, P. Roethlisberger, Dr. G. Roma, , Dr. M. Uteng, Novartis Institutes for BioMedical Research, Novartis Campus CH-4002 Basel Switzerland
Dr. A. Lerchner, B. Arnaud, Dr. C. Guibourdenche, Dr. J. Hunziker, Dr. P. von Matt, Dr. V. Polyakov, Dr. N. Stiefl, Global Discovery Chemistry, Novartis
Institutes for BioMedical Research, Novartis Campus CH-4002 Basel Switzerland; E-mail: andreas.lerchner@novartis.com
Abstract
Several pan-CLK inhibitors with acceptable ADME properties
have been described to be selective against most other kinases
(apart from CLK1-4/DYRK1A family) and in particular the benzo-
thiazole derivative TG003 has been shown to restore synaptic
impairments in SHANK3 deficient neurons [1-2]. While it seems to
be sufficient to restore spine density with the selective
knockdown of CLK2 [1], a pan-CLK inhibitor might lead to higher
safety concerns in comparison to the inhibition of only one CLK
family member. Therefore, we investigated whether it was
possible to identify CLK2 inhibitors with high selectivity over
CLK1, CLK3, CLK4, and/or DYRK1A. Due to biochemical
potency data reported in recent publications [3], we anticipated
that this would be difficult to achieve and would require an in-
depth analysis. Here we present the identification of a new CLK
inhibitor scaffold, found in a biochemical CLK2 Caliper assay
screen with 30k compounds that were selected by an in silico
approach. We show seven new CLK-family X-ray structures
(including the first X-ray structure for CLK4), for the complexes
with TG003 [2], with a novel potent in house CLK2 inhibitor
(Indazole1) and with two tool compounds. These novel CLK-
family X-ray structures are the basis for compound
design/optimization and address questions concerning
selectivity within the CLK1-4/DYRK1A kinase family.
CLK2 inhibition has been proposed as a potential mechanism to
improve autism and neuronal functions in Phelan-McDermid
syndrome (PMDS). Herein, we report the discovery of a very
potent indazole CLK inhibitor series, and the CLK2 X-ray
structure of its most potent analog. This new indazole series was
identified via a biochemical CLK2 Caliper assay screen with 30k
compounds that were selected by an in silico approach. Novel
high resolution X-ray structures of all CLKs, including the first
CLK4 X-ray structure, bound to known CLK2 inhibitor tool
compounds (e.g. TG003, CX-4945), are also shown and yield
insights into inhibitor selectivity in the CLK family. Efficacy of our
new CLK2 inhibitors from the indazole series was demonstrated
in the mouse brain slice assay, and potential safety concerns
were investigated. We show genotoxicity findings in the human
lymphocyte MNT assay using two structurally different CLK
inhibitors, which reveals a major concern for pan-CLK inhibition
in PMDS.
Introduction
Phelan-McDermid syndrome (PMDS) is a congenital disease
caused by loss of the terminal segment of chromosome 22, with
concomitant loss of the SHANK3 gene, a deficiency that is
associated with intellectual disability and autism. In a recent
Results and Discussions
work, inhibition of the serine-threonine kinase CLK2 was shown
X-ray structure reveals the binding mode of TG003
complexed with CLK2 kinase domain
[1]
to rescue spine density in SHANK3 knockdown neurons
.
Therefore, inhibition of CLK2 was proposed as an interesting
new target that could potentially ameliorate autistic features in
PMDS. Obviously, in order to assess the therapeutic concept of
CLK inhibitors to treat PMDS, it was especially important to
investigate some safety relevant aspects of CLK inhibition. In
particular, it was already known that CLKs regulate alternative
splicing by phosphorylation of serine- and arginine rich (SR)
When we initiated our work, one CLK2 X-ray structure was
publicly available, deposited by the SGC consortium (pdb
entry 3NR9, 2.89 Å resolution). As the binding mode of TG003
was unknown, and there was the possibility that TG003 was
an allosteric and/or covalent inhibitor, we embarked on
determining its CLK2 X-ray co-crystal structure (see Fig. 1 for
the chemical structures of all compounds described in this
paper). A human CLK2 kinase domain construct containing
amino acids R130–D496 was co-expressed with λ-
proteins, which promote the assembly of the spliceosome [2]
.
1
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phosphatase in E. coli, affinity-purified and determined to be
un-phosphorylated by LC-MS. Optimized co-crystals with
TG003 diffracted to 2.6 Å (supporting information, Table S2).
The electron density clearly showed that TG003 binds non-
covalently in the ATP site (supporting information, Fig. S1A)
and that the N- and C-terminal kinase lobes are packed tightly,
contributing to the overall binding affinity (Fig. 2A). The benzo-
thiazole core of TG003 is sandwiched between A191, V177 of
the N-terminal lobe and L297, V326 of the C-terminal lobe
(Fig. 2B).
tool compounds in complex with various CLKs (notably, no
CLK4 X-ray structure was publicly available). Two publications
about CX-4945 by Kim et al. describe this compound in its role
as a highly potent CLK inhibitor [5] and also in its role as a highly
potent DYRK1A inhibitor; DYRK1A inhibition is proposed as a
potential treatment for Down syndrome [6]. The high potency of
CLK inhibitor Cpd-2 is described in a publication by Araki et al. [7]
For CLK4 we designed a construct based on a sequence
alignment with CLK2. All four CLK kinase domains were
expressed and purified, using the constructs CLK1(H148–I484),
CLK2(S136–D496), CLK3(Q275–T632) and CLK4(H146–K480).
All in house X-ray structures turned out to be the un-
phosphorylated forms, even though the protein batches used in
crystallization for CLK1 and CLK4 were ca. 50%
phosphorylated and 50% un-phosphorylated (CLK2 and CLK3
batches were 100% un-phosphorylated).
.
X-ray structures reveal the binding mode of CX-4945
complexed with CLK2, CLK3, and CLK4 kinase domains
We managed to obtain well-diffracting co-crystals of CX-4945
with CLK2, CLK3 and CLK4. The crystals for CLK4/CX-4945
diffracted to 2.46 Å (supporting information, Table S4). The three
overall kinase domain structures turned out to be similar. The X-
ray structure for CLK4/CX-4945 showed the details of the
interactions in the ATP binding site (Fig. 3A). The benzo-
naphthyridine core is sandwiched between A189, V175 of the N-
terminal lobe and L295, V324 of the C-terminal lobe. It also
makes an aromatic interaction with the gk residue F241. In the
hinge region, the 2-N of the naphthyridine accepts a hydrogen
bond (2.9 Å) from NH-L244. The carboxylate of CX-4945 makes
a strong saltbridge interaction (2.8 Å) with the catalytic lysine
NZ-K191. Interestingly, this carboxylate oxygen is at the same
position as the water molecule which mediates the (weaker)
interactions with NZ-K193 for CLK2/TG003 (Fig. 2B). The
carboxylate of CX-4945 also makes water mediated hydrogen
bonds with NH-F323, OE2-E206. Importantly, the 3-Cl of the Cl-
phenyl group makes an edge interaction (3.4 Å) with F172. A
comparison of the X-ray structures for CLK4/CX-4945 and
Figure 1. Chemical structures of CLK2 inhibitors described in this paper.
[4]
F243 is the gatekeeper (gk) and L246 the gk + 3 residue
.
The methoxy oxygen accepts a hydrogen bond (3.2 Å) from
NH-L246. The keto-oxygen of the propan-2-one group accepts
a hydrogen bond (3.2 Å) from the catalytic lysine NZ-K193 and
makes water mediated hydrogen bonds with NH-D327 and
OE2-E208. Overall, the interactions of TG003 with CLK2 as
determined by its X-ray structure can explain well, why such a
small molecule can still reach decent potency on target
(mCLK2 IC50 = 200 nM published by Muraki et al. [2]).
CLK2/CX-4945 (1.95
Å resolution) revealed no important
differences of interactions and no significant side chain
differences in the ATP binding site. As a consequence we
reasoned that selectivity for CLK2 versus CLK4 with an inhibitor
in the ATP binding site will be very difficult to obtain. By contrast,
a comparison of the X-ray structures for CLK4/CX-4945 and
CLK3/CX-4945 (2.29 Å resolution) revealed a key difference (Fig.
3B). For CLK3, the small sidechain of residue A319 underneath
provides less vdW-interactions (van der Waals interactions) with
the aromatic core of the inhibitor than the larger V324 for CLK4
(V324 for CLK1, V326 for CLK2). The X-ray structures thus
predict that the affinity of CX-4945 should be lowest for CLK3,
and that in general selectivity favouring CLK1, CLK2, CLK4
versus CLK3 could be envisaged in compound optimization. In
order to verify this prediction, CX-4945 was then submitted for
testing in CLK1-4/DYRK1A radiometric protein kinase assays by
ProQinase (see supporting information for assay description),
which showed IC50 values of 12 nM (CLK1), 7 nM (CLK2), 143
nM (CLK3), 11 nM (CLK4), and 14 nM (DYRK1A). These results
confirmed the prediction based on CLK X-rays to reach the
highest selectivity over CLK3 within this family.
Figure 2. X-ray structure shows that TG003 binds in the ATP-binding
site of CLK2. A, X-ray structure at 2.60 Å resolution of the CLK2 kinase
domain shown as ribbon diagram (color ramping from blue: N-terminus to
red: C-terminus) complexed with TG003 (white). B, details of interactions
between CLK2 (carbons in yellow, nitrogens in blue, oxygens in red) and
TG003 (carbons in cyan, computed hydrogen positions in white). Water
molecules are shown as white spheres. Selected interactions are shown as
green dotted lines. The coordinates for CLK2 complexed with TG003 have
been deposited in the PDB databank (PDB access code = 6FYI)
Endeavor to achieve high resolution X-ray structures for
CLK1, CLK2, CLK3 and CLK4 kinase domains
In order to increase our structural understanding of potential
selectivity within the CLK-family and possibly versus DYRK1A,
we embarked to determine X-ray structures for further selected
2
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Figure 4. Comparison of interactions made by Cpd-2 in the ATP-binding
sites of CLK1 and CLK3: Structural basis for selectivity against CLK3. A,
X-ray structure at 2.32 Å resolution of the kinase domain of CLK1 (carbons in
yellow, nitrogens in blue, oxygens in red) complexed with Cpd-2 (carbons in
cyan, computed hydrogen positions in white). Water molecules are not shown.
Selected interactions are shown as green dotted lines. B, Superposed on the
X-ray structure of CLK1/Cpd-2 (coloring as in A) is the X-ray structure at 1.42
Å resolution of CLK3 (carbons in white) complexed with Cpd-2 (carbons in
magenta). Upper/lower residue names for CLK1/CLK3 are indicated if there
are residue differences. The coordinates for CLK1, CLK3 complexed with Cpd-
2 have been deposited in the PDB databank (PDB access codes = 6FYO,
6FYR).
Figure 3. First X-ray structure for CLK4. Comparison of interactions
made by CX-4945 in the ATP-binding sites of CLK4 and CLK3: Structural
basis for selectivity against CLK3. A, X-ray structure at 2.46 Å resolution of
the kinase domain of CLK4 (carbons in yellow, nitrogens in blue, oxygens in
red) complexed with CX-4945 (carbons in cyan, computed hydrogen positions
in white). Water molecules are shown as white spheres. Selected interactions
are shown as green dotted lines. B, Superposed on the X-ray structure of
CLK4/CX-4945 (coloring as in A) is the X-ray structure at 2.29 Å resolution of
CLK3 (carbons in white) complexed with CX-4945 (carbons in magenta). The
coordinates for CLK2, CLK3, CLK4 complexed with CX-4945 have been
deposited in the PDB databank (PDB access codes = 6FYL, 6FYP, 6FYV).
X-ray structures reveal the binding mode of Cpd-2
complexed with CLK1 and CLK3 kinase domains
The imidazopyridine core is sandwiched between A189, V175 of
the N-terminal lobe and L295, V324 of the C-terminal lobe. In
the hinge region, the imidazo-N accepts a hydrogen bond (3.0
Å) from NH-L244. In addition, the amide-NH donates a hydrogen
bond (3.0 Å) with non-optimal geometry to CO-L244. The
pyrazole makes an aromatic interaction with the gk residue F241
and also important vdW-interactions with V324. In addition, for
CLK1/Cpd-2 the pyrazole makes strong direct hydrogen bond
interactions with OE2-E206 and the catalytic lysine NZ-K193.
Interestingly, for CLK3/Cpd-2 the pyrazole makes only indirect
water mediated interactions with the corresponding E201 and
K186 (Fig. 4B and supporting information Fig. S2). Correlating
with this finding, for CLK3/Cpd-2 the pyrazole has a slightly
different orientation than for CLK1, possibly caused by the
[7]
As a further tool compound, Cpd-2 published by Araki et al.
was selected to determine the binding mode, and details of
interactions for its pyrazole and tert-butyl-nicotinamide groups.
The X-ray structures for Cpd-2 bound to CLK1 and CLK3 (at
2.32 Å, resp. 1.42 Å resolution) were obtained, shown in Fig. 4.
Interestingly, the overall electron density for CLK1/ Cpd-2
showed no phosphorylation, in particular not at S341 or T341,
and the latter loop conformation is consistent with no
phosphorylation (despite the co-presence of a phosphorylated
form after purification; i.e. crystallization selected the un-
phosphorylated form).
3
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difference of V324 vs. A319 for CLK1 vs. CLK3. The X-ray
structures thus predict that Cpd-2 should be slightly less potent
for CLK3, compared to CLK1, CLK2, CLK4. Importantly, the tert-
butyl group attached to the nicotinamide of Cpd-2 reaches
towards a region (Fig. 10), where DYRK1A showed a significant
difference to CLKs (Y243 for DYRK1A vs. L246 for CLK1, L248
for CLK2, K241 for CLK3, L246 for CLK4). In addition,
derivatives in the tert-butyl region could be envisaged with
further interactions, e.g. a hydrogen bond with OG-S299 of
CLK1 or with OE1-E254 of CLK1. Interestingly, a piperazinylone
derivative of Cpd-2 has been reported by the Takeda group to
approach to hit list generation was followed. An internally
developed machine learning technique (pQSAR [13]) based on
random forests and partial-least squares was employed to build
a virtual screening model. To train the model, activity data of
roughly three thousand compounds against a closely related
kinase was used as a surrogate for CLK2, since it was known
from few compounds that selectivity between the kinases was
low. Model quality for the data set was reasonable for virtual
screening with an R2 of 0.75, a Q2 of 0.75 and an external R2
Fit
of 0.36. For this model, the hit list was filtered by a minimum
predicted pIC50 of 5.
have high potency for CLK1 [8]
.
To reduce the size of the initial hit lists and remove obviously
uninteresting compounds, duplicates were removed and the
output of all methods was filtered by some simple properties (for
details see supporting information) resulting in a final list of
roughly 30K compounds.
Taken together, our X-ray structures for the tool compounds
TG003, CX-4945 and Cpd-2 showed the structural basis for
possibilities to optimize selectivity and affinity in the ATP binding
pocket. In parallel with the successful efforts to obtain CLK X-ray
structures of various tool compounds from the literature, we had
already initiated efforts to find novel highly potent CLK inhibitors
that would be more selective over other kinase families than
existing tool compounds.
Assay development of CLK2 mobility shift assay
Kinases are classical disease targets being addressed by a
[14]
broad spectrum of assay technologies
enabling hit finding
Identification of a novel CLK2 inhibitor class using an in
silico driven focused screen
campaigns. In the case of CLK2, Caliper microfluidic technique
was chosen due to its (i) high reliability, (ii) data quality, (iii) low
compound interference effects and (iv) medium throughput
capability. The assay principle is based on the different
electrophoretic mobility of a fluorescent-label peptide after
phosphorylation by CLK2 to follow the conversion of an un-
phosphorylated substrate to phosphorylated product in real time.
During the initial assay development phase, CLK2 kinase activity
was characterized in regard to enzyme kinetic parameters as
ATP Km, enzyme time kinetic and DMSO sensitivity
(experimental details shown in supporting information). CLK2
enzyme concentrations of 4 to 7 nM converted 15 to 25% of
substrate after 60 minutes reaction time. The enzymatic reaction
displayed a high linearity between 4 to 90 min reaction time
(R2=0.99). A substrate conversion rate of 15% ensured a reliable
assay window being required for a robust screening assay with
high data quality. The apparent ATP Km values were determined
in a time interval of 4 to 90 min generating ATP Km values
between 34 and 54 µM. The screening assay was performed at
ATP Km (50 µM) to guarantee a high sensitivity for CLK2
inhibitor identification. Since chemical screening libraries are
dissolved in DMSO, its influence on CLK2 activity was analyzed.
CLK2 activity was not significantly affected by DMSO
concentrations up to 1%. Higher DMSO concentrations slightly
decreased CLK2 activity, whereas the linearity of enzymatic
reaction was not changed. In a last step, the CLK2 kinase assay
was validated by analyzing the three CLK kinase inhibitors Cpd-
2, CX-4945 and TG003 (Fig. 5). The assay generated robust
data quality by showing highly reliable IC50 fitting curves. The
determined IC50 values were in good agreement with the
published IC50 data, confirming the validity of the CLK2 kinase
activity assay (Fig. 5).
All four CLK isoforms were successfully expressed in E. coli and
purified by a standard 2-step method. The crucial factor was the
co-expression of λ-phosphatase which resulted in quite
homogeneous protein preparations with no, or just a low degree
of, λ-phosphorylation (supporting information Table S1). Without
co-expression of the λ-phosphatase, the recombinant proteins
were extensively phosphorylated
(6 to > 10 phosphate
residues) causing inhomogeneous elution during purification and
a tendency to form high molecular weight aggregates. In
addition, the closely related CLK2 and CLK3 were completely
dephosphorylated in the presence of λ-phosphatase during
expression, whereas CLK1 and
4
still showed some
phosphorylation. More in-depth analysis performed by peptide
mapping with tryptic digests of the purified proteins revealed that
at least one of the two phosphate groups is attached to either
S341 and/or T342 as shown in supporting information Fig. S6.
Similarly CLK4 was phosphorylated at either S340 or T341.
These two adjacent residues are located within the activation
loop of the kinase. Further details are shown in supporting
information.
To create an initial set for compound screening, two different
virtual screening approaches were applied. (a) Structural
information was leveraged by docking the internally available
compound set into the binding site of CLK2. Since at that time
point no internal structures were yet available, the structure of
CLK2 (access code 3NR9 [9]) was downloaded from the PDB [10]
,
and chain A prepared for docking experiments using the protein
preparation workflow in MAESTRO. The obtained structure was
[11]
used as input to two different docking engines – ICM
and
GLIDE [12]. To obtain more diverse results from both methods,
the settings for both were varied. Detailed information can be
found in the supporting information. (b) Even though no activity
information was available in house for CLK2, a ligand-based
4
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1 2 0
1 0 0
8 0
T G 0 0 3
I
T G 0 0 3 I I
T G 0 0 3 I I I
C X - 4 9 4 5
I
6 0
C X - 4 9 4 5 I I
C X - 4 9 4 5 I I I
4 0
C p d - 2
I
2 0
C p d - 2 I I
C p d - 2 I I I
0
- 2 0
- 1 0
- 9
- 8
- 7
- 6
- 5
l o g [ i n h i b i t o r ] ,
M
TG003
260 +/- 2 nM 17.5 +/- 0.5 nM 8.5 +/- 0.1 nM
200 nM 3.8 nM 2.4 nM
CX-4945
Cpd-2
in house IC50
published IC50
Figure 6. Enrichment of CLK2 inhibitors in primary screen. Activity
distribution of hits in 30K screen. The different inhibitor potency ranges are
colored from light blue (-30 to -40% activity) till dark blue (full inhibition -90 to
100%). On top of the diagram, the number of hits for the different potency
ranges are displayed. Inactive hits are marked in yellow.
Figure 5: CLK2 assay validation. IC50 fit curves of three reference inhibitors.
Inhibitors tested at concentrations between 300 pM and 10 µM in three dilution
series with duplicates. Green: Cpd-2, blue: CX-4945, red: TG003; circle: first
dilution series, triangle: second dilution series, and square: third dilution series
High resolution X-ray structure reveals binding mode of
new CLK2 inhibitor class exemplified by Indazole1
CLK2 30K screen and hit validation
A focused library of 30K compounds was assembled from the
Novartis compound archive using the aforementioned in silico
methods. The library was screened at 10 µM compound
concentration. The screen displayed an excellent assay quality
mirrored by (i) Z’ > 0.5 for all 86 tested plates (average Z’: 0.8)
and (ii) a high reproducibility of IC50 values of 3 published CLK
inhibitors, which were included in the beginning and the end of
each screening run (assay quality data included in supporting
information). The successful in silico compound selection of the
focused screening set was underlined by (i) a high hit rate of
25.5% for compounds reducing CLK2 kinase activity by more
than 30% and (ii) the enrichment of a potent inhibitor population
around 100% inhibition (Fig. 6).
In order to elucidate the binding mode and interactions of the
new indazole inhibitor class (as a basis for possible structure
guided optimization) we embarked on an X-ray structure
determination for the most potent inhibitor Indazole1. We could
obtain co-crystals of CLK2(S136–D496)/Indazole1 which
diffracted to 2.39 Å. The refined structure (supporting
information Table S2) revealed the details of the interactions
(Fig. 7A). The indazole core is sandwiched between A191 of the
N-terminal lobe and L297, V326 of the C-terminal lobe. The
benzyl part of the indazole also makes an aromatic interaction
with the gk residue F243. In the hinge region, the 1-NH of the
indazole donates a hydrogen bond (2.8 Å) to CO-E244 and the
2-N accepts a hydrogen bond (3.0 Å) from NH-L246. The 3-
methyl extension on the indazole makes vdW-interactions with
L169. Importantly, the pyridine-N of Indazole1 accepts
a
Initial hits were subjected to different filters e.g. chemical
clustering, comparison with in house kinase selectivity data, and
physicochemical properties to validate the most promising
compounds (filter criteria, supporting information). Finally, 773
potential CLK2 inhibitors were selected for validation in 8-point
concentration series. The predictive strength of the CLK2 assay
was underlined by (i) the high confirmation rate of 81% and (ii)
the good correlation between the potency (%inhibition) in the
primary screen and the corresponding IC50 values obtained in
validation. Almost half of all tested compounds displayed IC50
values < 1 µM. A deeper analysis of the validated hits and a
SAR by archive led to the identification of several new CLK2
inhibitor classes. The indazole cluster was selected for detailed
characterization by X-ray, biophysical, biochemical and cellular
approaches to verify the binding mode and specificity of
inhibitors.
hydrogen bond (3.0 Å) from the catalytic lysine NZ-K193 (the
position of K193 is further stabilized by a saltbridge with E208
and a water mediated hydrogen bond with D327). Interestingly,
the 3-oxy ether oxygen, which is attached to the pyridine,
accepts an intramolecular hydrogen bond (2.8 Å) from the amine
NH3+, thus stabilizing the conformation of the 3-extension.
Importantly, in this 3-extension the amine NH3+ donates a
strong hydrogen bond (2.6 Å) to OD1-N295.
Indazole1 was submitted for testing in CLK1-4/DYRK1A
radiometric protein kinase assays by ProQinase (see supporting
information), with IC50 values of 12 nM (CLK1), 10 nM (CLK2),
2250 nM (CLK3), 12 nM (CLK4), and 73 nM (DYRK1A), showing
the highest selectivity in inhibition of CLK2 vs CLK3 (225 fold) so
far reported in literature. In order to explain the high selectivity
over CLK3, it is important to consider that the small sidechain of
residue A319 underneath provides less vdW-interactions with
the aromatic core of the inhibitor than the larger V324 for CLK4
(V324 for CLK1, V326 for CLK2). Therefore, the interactions of
5
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described in the supporting information. As demonstrated by
modelling (Fig. 8B), various indazole functional side chains can
form interactions with asparagine N295 or glutamic acid E171,
and the potencies of Indazole1-5 were found to be in a similar
range.
Indazole1 with NH-L246 from the hinge area and lysine NZ-
K193 are most ideal in the case of CLK2 (and in analogy, CLK1
and CLK4), but not in case of CLK3. In the internal caliper
kinase assay panel, Indazole1 had a CLK2 IC50 of 2.7 nM and
showed high selectivity of at least 600-fold when tested against
34 kinases, apart from protein kinase A, where selectivity was
only 70-fold (supporting information, Table S5).
Figure 8. SAR for the CLK2 inhibitors Indazole 1-5. A, Table with IC50
values in Caliper assay and thermal shift assay data. B, Docking model of
compounds Indazole1-5. Depending on the compound, interactions in different
regions below the p-loop can be identified.
CLK2 inhibitors Indazole1 and Indazole2 rescue spine
density in mouse brain slices
The tool compound TG003 was shown to rescue spine density
in Shank3 deficient neurons at 10 μM concentration [1]. We used
the same assay to evaluate whether Indazole1 and Indazole2
were able to rescue the loss of spine density at lower
concentrations. Remarkably, Indazole2 significantly improved
spine density at 1 µM concentration, whereas the most potent
CLK2 inhibitor Indazole1 already showed efficacy at
a
concentration of 300 nM (Fig. 9). These results indicate a 10-fold
and 30-fold increase of potency for Indazole2 and Indazole1,
respectively, compared to TG003.
We next focused on safety related questions regarding CLK
inhibition, in particular due to a publication which has linked
CLK1, CLK2, and CLK4 inhibition with genotoxicity via a
mechanism of action on chromosomal abscission [15]
.
Figure 7. X-ray structure for CLK2/Indazole1 reveals binding mode and
interactions, which explain the high potency of Indazole1 for CLK2 and
why it is 96x more potent than TG003 in the CLK2 Caliper assay. A, X-
ray structure at 2.39 Å resolution of the kinase domain of CLK2 (carbons in
yellow, nitrogens in blue, oxygens in red) complexed with Indazole1
(carbons in cyan, computed hydrogen positions in white). Water molecules
are shown as white spheres. Selected interactions are shown as green
dotted lines. B, Superposed X-ray structures of TG003 (carbons in magenta)
and Indazole1 (carbons in cyan), when complexed with CLK2. The
coordinates for CLK2 complexed with TG003, Indazole1 have been
deposited in the PDB databank (PDB access codes = 6FYI, 6FYK).
Indazole analogs of Indazole1 and initial SAR
All 4 analogs of Indazole1 from the indazole series identified via
SAR by archive also showed a CLK2 IC50 in the Caliper assay
below 50 nM, and stabilized CLK2 > 4 °C in a thermal shift
assay (see table in Fig. 8A). All syntheses of Indazole2-5 can be
performed in analogy to the synthesis of Indazole1, which is
Figure 9. Indazole1 and Indazole2 rescue spine density in Shank3
knockdown neurons. Left, representative images of CA1 apical secondary
6
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10.1002/cmdc.201800344
ChemMedChem
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dendrites treated on DIV 14 for 24 hrs with Indazole1 or Indazole2 prior to
immunostaining for GFP. Right, quantitative analysis with plots indicating
(e.g. Cl edge interactions), L248 (vdW-interactions) are
candidates for obtaining high affinity.
group means
± SEM (one-way ANOVA, p<0.0001, Dunn’s multiple
comparisons test).
MNT measurements of TG003 and Indazole1 in primary
human lymphocytes to assess in vitro genotoxicity of CLK
inhibitors
To assess potential clastogenic and/or aneugenic effects of the
CLK inhibition, two potent CLK inhibitors from two different
chemical series were tested in the cytokinesis-blocked
micronucleus (MN) assay in primary human lymphocytes. The
treatment duration was 28 hours continuous incubation in the
absence of S9 metabolic activation. Both compounds were
tested up to clearly cytotoxic concentrations. In the experiment
with Indazole1, a significant concentration-dependent increase
in the frequencies of micronucleated binucleates (MNBN) was
seen at all analyzed concentrations ranging from moderate to
high cytotoxicity (supporting information, MNBN Table S6).
TG003 also induced
a significant concentration-dependent
increase in the frequencies of MNBN (supporting information,
MNBN Table S7). The increases reached statistically significant
levels at the two highest analyzed concentrations that showed
moderate to high cytotoxicity. Our data show that both
compounds are genotoxic in the MN test. These results are in
alignment with data already published by Petsalaki et al.[15]
,
where TG003 or depletion of CLK1, CLK2 or CLK4 increased
the frequency of micronucleated cells. Indazole1, a more potent
inhibitor of CLKs than TG003, was also more potent in inducing
micronuclei in reaching higher frequencies of MNBN at relatively
low concentrations, correlating with IC50 values for CLKs.
Therefore our data confirm the hypothesis that inhibition of CLKs
1, 2 and 4 confers a genotoxic potential.
Figure 10. Comparison of CLK1, CLK2, CLK3, CLK4, DYRK1a kinase
domain X-ray structures in ATP-sites. A, CLK1 (from complex with Cpd-2).
B, CLK2 (from complex with CX-4945). C, CLK3 (from complex with CX-4945).
D, CLK4 (from complex with CX-4945). E, Dyrk1a, published by Falke et al.
[16]. The red star indicates the position where CLK1, CLK2 and CLK4 have a
valine (V326 for CLK2), whereas CLK3 has the smaller alanine A319. The
coordinates for CLK1/Cpd-2, CLK2/CX-4945, CLK3/CX-4945, CLK4/CX-4945
have been deposited in the PDB databank (PDB access codes = 6FYO, 6FYL,
6FYP, 6FYV).
Conclusions
One of our initial questions was whether CLK inhibition would be
a feasible approach to treat PMDS. By leveraging a combination
of orthogonal in silico screening methods based on structural
information as well as machine learning, we were able to quickly
identify a large set of different target scaffolds to answer this
question. The high screening hit rate substantiates that this
approach is not only feasible but can replace full deck screening.
The resulting CLK inhibitors Indazole1 and Indazole2 showed
efficacy on spine density restoration in Shank3 deficient
neurons. However, the data on the induction of micronuclei in
human lymphocytes, obtained with two compounds from
different chemical series, TG003 and Indazole1, confirm the
hypothesis that CLK 1, 2 or 4 inhibition per se will induce a
Besides our own focus on CLK2 as a molecular target for the
treatment of autistic features in PMDS patients with SHANK3
[1]
deletions
,
various groups have suggested functional
modulation of the closely related kinase DYRK1A as a potential
therapeutic modality in addressing the cognitive and behavioral
deficits in Down syndrome [6]. We hope this work may prove
helpful in the development of ATP-competitive DYRK1A
inhibitors with the likely required selectivity over CLK1, CLK2,
and CLK4, which are associated with adverse genotoxic
potential.
genotoxic effect, which is very plausible given the mechanistic
Possible applications of CLK2 inhibitors in the treatment of
certain cancer indications (e.g. breast cancer) are also still of
high interest. Our new X-ray structures should prove to be useful
for the design and/or optimization of further CLK-family inhibitor
series for cancer indications, with respect to affinity, selectivity
and detailed molecular mechanism.
[15]
involvement of the pathway in chromosome integrity
.
Inhibition of CLK1, CLK2, and CLK4 is therefore concluded not
to be a safe strategy to treat PMDS.
Concerning the potential to reach selectivity, a comparison of the
various in house X-ray structures for CLK-family members (Fig.
10) showed us that L248, S249, S301, V326 in the ATP binding
site of CLK2 are possible candidates for compound interactions
to obtain a certain degree of selectivity (e.g. vs. CLK3 or
DYRK1A). CLK1, CLK2 and CLK4 have a valine at the V326
position of CLK2, whereas CLK3 has the smaller alanine A319 at
the corresponding position. On the other hand, we concluded
that selectivity for CLK2 vs. CLK1 or CLK4 would be challenging
to obtain. In addition to hinge binding interactions, interactions
with the CLK2 residues K193 (e.g. carboxylate, pyrazole), E208
(e.g. carboxylate, pyrazole), F243 (aromatic interaction), F174
Experimental Section
Expression and purification of human CLK1-4.
Cloning: The cDNAs of the four CLK kinase domains were
codon-optimized in-house and synthesized by GeneArt AG
(Regensburg, Germany). The resulting products (Invitrogen™
GeneArt™ Strings™ DNA Fragments) were cloned into a
7
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10.1002/cmdc.201800344
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proprietary E. coli/ T7 expression vector featuring an amino
terminal hexahistidine tag (His6-tagged) followed by human
rhinovirus 3C protease cleavage site (LEVLFQ’GP). The
boundaries of the kinase domains were set as follows: CLK1
(Uniprot P49759-1) H148-I484, CLK2 (Uniprot P49760-1) R130-
D496 or S136-D496 respectively, CLK3 (Uniprot P49761-4),
Q275-T632, and CLK4 (Uniprot (Q9HAZ1-1) H146-K480.
The gene encoding full-length λ-phosphatase from lambda
phage was cloned either into a pET derived expression vector or
into an auxiliary plasmid for co-expression. The latter is based
on the plasmid pACY184 featuring an alternative origin of
After 60 minutes, the kinase reaction was stopped by EDTA
addition. Then, the plates were analyzed on a Caliper system.
Data analysis and visualization
Km and IC50 values were determined using Prism software 7.03
(Graphpad Software Inc. San Diego, CA). Larger primary
screening and IC50 validation raw data files were analyzed using
an in house software. The data was visualized using Spotfire
Decision Site 8.1 (Spotfire). The data of each screening plate
was normalized by plate specific controls, (i) active control (AC,
n=16), 100% inhibition and (ii) neutral control (NC, n=16), 0%
inhibition using following equation: %inhibition = -100x(1-(x-
AC)/(NC-AC)). Furthermore, the plate controls were used to
evaluate the assay quality by calculating the Z’-factor: Z’= 1-
replication,
a different resistance marker (chloramphenicol
instead of kanamycin) and the arabinose controlled BAD
promoter.
Expression: BL21(DE3) Tuner E. coli cells (Novagen) were
transformed with the individual plasmids coding for CLK isoform
1 to 4 and additionally the pACY184 vector containing the gene
for λ-phosphatase in order to achieve an in vivo de-
phosphorylation of the expressed kinases. The bacterial cells
were cultivated in Terrific Broth (TB) culture media
(3xSDNC + 3xSDAC)/(meanNC –meanAC
)
Differential scanning fluorimetry (DSF) assays to determine
thermal stability of CLK proteins
complemented with 0.1
M
morpholino-ethanesulfonic acid
Conformational stability reflects a common property of proteins
which is influenced by the interaction with low molecular weight
ligands. Thermal shift assays formats like differential scanning
fluorimetry (DSF) aka ThermoFluor™ analyzing thermal stability
of proteins enable the identification and characterization of
ligand binding events. 384well microtiter well plate DSF assays
for CLK proteins were developed using a CFX384™ real-time
PCR instrument (Bio-Rad, Hercules, CA). Heat induced
unfolding of CLK proteins was followed by measuring the
(MOPS), 30 mg/l kanamycin, 30 mg/ml chloramphenicol and 2
mM arabinose (for inducing the expression of the for λ-
phosphatase). Cells were grown at 37o C until an optical density
of 0.8 and induced with 0.1 mM isopropyl-thiogalactoside (IPTG)
for induction for expression; the temperature was lowered to 20o
C and cells further incubated overnight.
Purification: E. coli cells were harvested by centrifugation and
lysed with a high pressure homogenizer device. The His6-
tagged CLK isoforms were all purified in a first step with
immobilized metal affinity chromatography (IMAC) by using a 5
ml HisTrap column (GE Healthcare). The column was washed
with loading buffer (50 mM Na-phosphate pH 8.0, 300 mM NaCl,
20 mM imidazole, 10 % glycerol and 0.5 mM TCEP) for several
column volumes, once the sample was loaded. The His6-tagged
kinases were eluted in an imidazole gradient ranging to 300 mM
within 5 column volumes. For cleavage of the N-terminal His6-
tag fractions containing sufficiently pure protein were pooled,
and in-house produced HRV-3C protease (PreScission
protease) was added in molar ratio of 1:100. The mixture was
dialyzed against size exclusion chromatography (SEC) buffer
(50 mM HEPES pH 7.4, 300 mM NaCl, 10 % glycerol and 0.5
mM TCEP) overnight at 4o C. For separation of the cleaved
fusion tag and as a final purification step the volume of the
dialysis mixture was concentrated to approximately 5 ml and
loaded onto a Superdex 75 (16/60 or 26/60) size exclusion
column using the SEC buffer. Typically, the cleaved kinases
eluted in a symmetric major peak around 0.5 column volumes,
preceded by a smaller peak containing aggregated protein.
Fractions from the large peak were pooled and deep frozen at -
80°C in aliquots until further use.
fluorescence increase caused by
a
solvatochromic dye
SYPRO®Orange (ThermoFisher Scientific™, Waltham, MA).
The standard assay conditions were as follows: 50 mM HEPES
pH 7.4, 150 mM NaCl, 0.5 mM TCEP4, 1% DMSO, 1.9 µM
CLK1, CLK2, CLK3 or CLK4 and 5 µM SYPRO®Orange. All
DSF assays were performed in 10 µl volume using sealed PCR
plates. The CLK samples were heated from 20 to 60oC with a
heating rate of 1oC/min and every 0.5oC the fluorescence was
detected by using the FRET channel (excitation: 450 - 490 nm;
emission: 560 - 580 nm). To determine the stabilization of CLK
proteins by ligands, control wells containing the proteins without
ligand and 1% DMSO were included in each experiment. The
melting temperature (Tm) was determined by plotting the
fluorescence values against temperature and fitting the data to
the Boltzmann equation using an in-house software program.
The inflection point of the first derivative of the melting curve
represented the Tm. Melting temperature shifts (ΔTm), a measure
to quantify the protein stabilization, was calculated by ΔTm
=
Tm(CLKligand) – Tm(CLKDMSO). A ΔTm > 3-fold standard deviation
of Tm (CLKDMSO) was classified as a significant stabilization. The
standard deviation of CLK Tm was in the range of 0.1 to 0.2oC for
all experiments enabling the identification of hits causing a ΔTm
> 0.5oC. For the compound tests, 40 nl of 2 mM and 10 mM
compound solution was spotted into PCR plates (final
concentrations of 8 and 40 µM) and mixed with 10 µl of CLK/dye
solution. The assay reaction was pre-incubated for 30 min at
room temperature before analysis in the PCR instrument.
CLK2 kinase activity assay
The in vitro CLK2 kinase assay was established using Caliper
mobility shift assay technology (Perkin-Elmer, Waltham, MA). In
house recombinant CLK2 kinase domain (130-496) was used to
establish Caliper kinase assay. The kinase assay buffer mix
contained 50 mM HEPES pH 7.5, 1 mM DTT, 0.02% Tween 20,
0.02% BSA, 10 mM beta-glycerophosphate, 10 µM sodium
orthovanadate, 6 nM CLK2, 3 mM MgCl2, 2 µM peptide (FL-26,
5-FAM-ARKRERTYSFGHHA-COOH, PerkinElmer, Waltham,
MA). The assay buffer mix was added with a Thermo Multidrop
Combi dispenser to 384well microtiter well assay plates,
containing 40 nl 5 mM pre-spotted compound solution. The
assay was run at 10 µM compound screening concentration
(total number of compounds: 30129). CLK2 was pre-incubated
with the test compounds for 45 minutes and then the enzymatic
reaction was started by adding ATP (final concentration 50 µM).
Crystallization
Co-crystals of CLK1(148-484) (numbering according to
P49759) in complex with Cpd-2 were obtained by adding a 3-
fold molar excess of compound (all complexes were incubated
ca. 2-3 hours at 4°C and centrifuged before crystallization).
The reservoir solution consisted of 200 mM AS, 100 mM Bis-
Tris pH5.5, 25% (w/v) PEG3350. Co-crystals of CLK2(130-
496) (numbering according to P49760) in complex with TG003
were obtained by adding a 3-fold molar excess of compound.
The reservoir solution consisted of 100 mM HEPES pH7.5,
8
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10.1002/cmdc.201800344
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FULL PAPER
25% (w/v) PEG3350. With the aim to increase the resolution
for CLK2, we redesigned the CLK2 construct based on our
CLK2/TG003 crystal structure by removing a flexible part at
the NH2-terminus to possibly obtain a different crystal form.
Co-crystals of CLK2(136-496) (numbering according to
P49760) in complex with compounds Indazole1, CX-4945
were obtained by adding a 3-fold molar excess of compound.
For Indazole1, the reservoir solution consisted of 200 mM
trimethylamine n-oxide, 100 mM Tris pH8.5, 20% (w/v)
PEG2000 MME. For CX-4945, the reservoir solution consisted
of 100 mM HEPES pH7.0, 30% (v/v) Jeffamine ED-2001. Co-
crystals of CLK3(275-632) (numbering according to P49761) in
complex with compounds CX-4945, Cpd-2 were obtained by
adding a 3-fold molar excess of compound. For CX-4945, the
reservoir solution consisted of 200 mM AS, 100 mM Bis-Tris
pH5.5, 25% (w/v) PEG3350. For Cpd-2, the reservoir solution
consisted of 100 mM Hepes pH7.5, 25% (w/v) PEG4000. Co-
crystals of CLK4(146-480) (numbering according to Q9HAZ1)
in complex with compound CX-4945 were obtained by adding
a 3-fold molar excess of compound. The reservoir solution
consisted of 50 mM AS, 50 mM Bis-Tris pH6.5, 30%(v/v)
Pentaerythritol ethoxylate (15/4 EO/OH). Co-crystals were
obtained at 20 °C and by sitting drop vapor diffusion. The
drops were made up of 200 nL of protein solution and 200 nL
of well solution.
toxicity/cell cycle delay. Indeed cytochalasin B, an inhibitor of
actin polymerization, blocks cytokinesis without interfering with
[21]
nuclear division
producing two daughter nuclei in the same
cytoplasm. Therefore, in the presence of the inhibitor, cells that
have completed one cell cycle after treatment will appear as a
binucleated cell, cells that divided more than once will appear as
polynucleated cells (trinucleated and tetranucleated cells) and
finally the cells that did not divide at all, will remain
mononucleates. For each substance at least three
concentrations (2 cultures per concentration) and 2000 cells per
concentration were analyzed. Per culture 1000 binucleated
lymphocytes were analysed for the presence of one, two or
more micronuclei (MN) and expressed as per thousand
micronucleated binucleates (MNBN). Also the percentage of
binucleates,
polynucleated
cells,
mitotic
cells
and
mononucleated cells with or without MN were recorded.
Cytotoxicity parameter: As a measure of cytotoxicity and cell
cycle delay the relative division index was used. The criteria for
determining a positive result is based on the reproducible
increase in the number of MNBN and on statistical differences
between controls and treated samples as determined with the
Chi-square test (95% confidence limit is used).
Acknowledgements
We thank Ulrich Schopfer, Tewis Bouwmeester, Fabrizio
Gasparini and Ricardo Dolmetsch for their advice and support of
our project. We thank Neil Press for critically reading the
manuscript and helpful comments. We thank Eric Martin for his
virtual screening model and advice for our project. The
crystallographic experiments were performed on the X10SA
beamline at the Swiss Light Source, Paul Scherrer Institute,
Villigen, Switzerland.
Data collection, structure determination and refinement
All crystals were cryo-protected in well solution supplemented
with 20% ethylene glycol and flash frozen in liquid nitrogen.
Data sets were collected at the Swiss Light Source Facility
(SLS, Villigen, Switzerland) for all the crystals. The data were
[17]
processed with XDS
. The X-ray structures of CLK
complexes were determined by molecular replacement
(PHASER [18]) using various published and in house CLK X-ray
[18]
structures as the search model. Programs REFMAC
COOT
and
[19]
were used for refinement and model (re)building.
The final refined structures have R (Rfree) values ranging from
0.176 to 0.228 (0.189 to 0.267) and showed excellent
geometry in the Ramachandran plot. All residues were in the
allowed regions for all X-ray structures, except for ca. 1% of
the residues (for which the electron density confirms the local
structure). As an example, for the complex CLK2(136-496)
and Indazole1 excellent electron density around R162, T289,
D327, S343, S359 unequivocally confirmed the local structure.
See supporting information Tables S2, S3, S4 for details of the
data collection and structure refinement. The crystallographic
data for the seven X-ray structures have been deposited at the
RSCB Protein Data Bank (PDB, www.pdb.org) with the
respective access codes 6FYI, 6FYK, 6FYL, 6FYO, 6FYP,
6FYR, 6FYV. All of the figures showing crystal structures were
prepared using PyMOL (Schrödinger, LLC (2015) The PyMOL
Molecular Graphics System, Version 1.8, New York).
The authors declare that they have no conflict of interests with
the contents of this article.
Key words: CLK2 • CLK-family • DYRK1A • kinase inhibitor •
X-ray structure • virtual screening • mobility shift assay •
indazoles • Phelan-McDermid syndrome • genotoxicity •
alternative splicing
The atomic coordinates and structure factors (codes 6FYI,
6FYK, 6FYL, 6FYO, 6FYP, 6FYR, 6FYV) have been deposited
in the Protein Data Bank (http://wwpdb.org/).
Genotoxicity assay in primary human lymphocytes:
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Entry for the Table of Contents
CLK2 inhibition has been proposed as a potential mechanism to
improve autism and neuronal functions in Phelan-McDermid
syndrome (PMDS). Herein, we report the discovery of a very
potent indazole CLK inhibitor series, and the CLK2 X-ray
structure of its most potent analog. Novel high resolution X-ray
structures of all CLKs, including the first CLK4 X-ray structure,
bound to known CLK2 inhibitor tool compounds (e.g. TG003,
CX-4945), are also shown and yield insights into inhibitor
selectivity in the CLK family. Efficacy of new CLK2 inhibitors
from the indazole series was demonstrated in the mouse brain
slice assay, and potential safety concerns were investigated. We
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10.1002/cmdc.201800344
ChemMedChem
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This article is protected by copyright. All rights reserved.