Novel CLK1 inhibitors based on N-aryloxazol-2-amine skeleton - A possible way to dual VEGFR2 TK/CLK ligands

Article abstract of DOI:10.1016/j.ejmech.2016.11.003

Background Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing in cancer. CLK family kinases are also involved in alternative splicing and RNA processing in Duchenne muscular dystrophy, Alzheimer's disease, HIV-1, and influenza virus. Small inhibitors are valuable tools for better understanding the molecular mechanisms of splicing and may serve as seeds for a novel class of therapeutics. Achievements Here we describe a discovery of four novel CLK1 inhibitors possessing N-aryloxazol-2-amine skeleton. Their activity against CLK1 (IC₅₀: 20, 30, 40 and 80 nM) and some other CMGC kinases, predicted CLK binding poses, synthesis and physico-chemical characteristics are also stated. Additionally analysis of all PDB available CLK structures and interactions of their ligands was performed. There are only few powerful dual CLK/VEGFR inhibitors known in the literature. We proposed that our inhibitors have similar binding places and interactions in CLK1, 3 and VEGFR2 TK mostly due to the joint N-aryloxazol-2-amine pharmacophoric fragment. One of our N-aryloxazol-2-amines already proved a good activity against both VEGFR2 and CLK1 enzymes (23/80 nM, resp). We proposed that the presented class of compounds has a potential to be developed in dual VEGFR2/CLK clinical compounds with prospective synergy to treat cancer.

Full text of DOI:10.1016/j.ejmech.2016.11.003

Accepted Manuscript
Novel CLK1 inhibitors based on N-aryloxazol-2-amine skeleton - A possible way to
dual VEGFR2 TK / CLK ligands
Miroslav Murár, Juraj Dobiaš, Peter Šramel, Gabriela Addová, Gilles Hanquet, Andrej
Boháč
PII:
S0223-5234(16)30941-2
10.1016/j.ejmech.2016.11.003
EJMECH 9036
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 July 2016
Revised Date: 29 October 2016
Accepted Date: 2 November 2016
Please cite this article as: M. Murár, J. Dobiaš, P. Šramel, G. Addová, G. Hanquet, A. Boháč, Novel
CLK1 inhibitors based on N-aryloxazol-2-amine skeleton - A possible way to dual VEGFR2 TK / CLK
ligands, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.003.
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ACCEPTED MANUSCRIPT
N-aryloxazol-2-amine CLK1 inhibitors
O
S O
Z
O
N
N
H
O
Z:_(IC50, CLK1)
6-methoxynaphthalen-2-yl_(20 nM)
benzo[b]thiophen-2-yl_(30 nM)
(E)-2-phenylethen-1-yl_(40 nM)
4-(cyanomethyl)-3-(pyridin-2-yl)benzene-1-yl_(80 nM)
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Novel CLK1 Inhibitors Based on N-aryloxazol-2-amine Skeleton - a Possible Way to
Dual VEGFR2 TK / CLK Ligands
Miroslav Murára, Juraj Dobiaša, Peter Šramela,c, Gabriela Addováb, Gilles Hanquetc, Andrej
Boháča,d,*
a Department of Organic Chemistry and b Institute of Chemistry, Faculty of Natural Sciences,
Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia
c
Université de Strasbourg, Ecole européenne de Chimie, Polyméres et Matériaux (ECPM)
Laboratoire de Synthése et Catalyse (UMR CNRS 7509), 25, rue Becquerel, F-67087
Strasbourg, France
d Biomagi, Ltd., Mamateyova 26, 851 04 Bratislava, Slovakia
* Corresponding author
Email: Andrej Boháč* - andrej.bohac@fns.uniba.sk
Abstract
Background: Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by
modulating pre-mRNA splicing in cancer. CLK family kinases are also involved in alternative
splicing and RNA processing in Duchenne muscular dystrophy, Alzheimer’s disease, HIV-1,
and influenza virus. Small inhibitors are valuable tools for better understanding the molecular
mechanisms of splicing and may serve as seeds for a novel class of therapeutics.
Achievements: Here we describe a discovery of four novel CLK1 inhibitors possessing N-
aryloxazol-2-amine skeleton. Their activity against CLK1 (IC50: 20, 30, 40 and 80 nM) and
some other CMGC kinases, predicted CLK binding poses, synthesis and physico-chemical
characteristics are also stated. Additionally analysis of all PDB available CLK structures and
interactions of their ligands was performed. There are only few powerful dual CLK / VEGFR
inhibitors known in the literature. We proposed that our inhibitors have similar binding places
and interactions in CLK1, 3 and VEGFR2 TK mostly due to the joint N-aryloxazol-2-amine
pharmacophoric fragment. One of our N-aryloxazol-2-amines already proved a good activity
against both VEGFR2 and CLK1 enzymes (23 / 80 nM, resp). We proposed that the presented
class of compounds has a potential to be developed in dual VEGFR2 / CLK clinical
compounds with prospective synergy to treat cancer.
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Keywords
dual CLK1 / VEGFR2 kinase ligands; CDK9, GSK3, DYRK1A, GSK3α/β; multitarget
kinases inhibitors
1. Introduction
Alternative splicing is a regulated process during the gene expression that greatly
increases the biodiversity of proteins and allows the human genome to direct the synthesis of
many more proteins (50 - 100 000) than would be expected from human 20,000 protein-
coding genes [1]. There is a growing recognition of the importance of protein kinases in the
control of alternative splicing. Splicing requires reversible phosphorylation of serine /
arginine-rich (SR) proteins in eukaryotic mRNA. These phosphorylation events are dependent
on SR protein and cdc2-like kinase (CLK) families [2]. The CLKs are an evolutionarily
conserved group belonging to the CMGC Ser / Thr protein kinase family from Human
Kinome [3-4] (for CMGC part see supplementary material (chapter: CLKs in the Human
Kinome). CLKs are primarily localized to the cytoplasm and nucleus [5]. CLKs are part of
LAMMER PK family possessing identity of the motif “EHLAMMERILG” in their kinase
subdomain (see supplementary material: chapter Superimposed CLKs with marked LAMMER
subdomain). This motif was reported to be essential for kinase activity [6]. CLKs (CDC2-like
or LAMMER kinases) are dual specific kinases that have been shown to autophosphorylate on
serine, threonine and tyrosine residues and phosphorylate exogenous substrates on serine and
threonine residues. The CLK family kinases are found in diverse species, from yeast to
human. A critical role of the CLK family kinases is the regulation of mRNA splicing. CLK
have shown to interact with, and phosphorylate, serine- and arginine-rich (SR) proteins [7-8].
SR proteins are splicing factors that regulate the assembly of the spliceosome, a
macromolecular complex where RNA splicing occurs in nucleus [9]. CLKs can co-exist as
full-length catalytically active and alternatively-spliced truncated inactive forms [10]. The
CLK family consists of four isoforms CLK1-4 exhibiting the typical protein kinase fold (see
supplementary material: chapter Graphical abstract). CLK1-4 isoforms possess different
length of amino acids (AAs) chain: Clk1 (484 AAs), Clk2 (499 AAs), Clk3 (638 AAs), Clk4
(481 AAs) [11]. CLK1 regulates its own splicing [12]. CLKs inhibitors suppress cell growth
and induce apoptosis by modulating pre-mRNA splicing in cancer [13]. CLK family kinases
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are also involved in alternative splicing and RNA processing in Duchenne muscular
dystrophy, Alzheimer’s disease, HIV-1, and influenza virus [14]. CLK1 is involved in the
pathophysiology of Alzheimer's disease, hence the inhibition of CLK1 can be used as a
therapeutic strategy for it [9]. CLK1 in the host cells is responsible for alternative splicing of
the M2 gene of influenza virus during influenza infection and replication. Therefore CLK1
inhibitors may have potential in anti-influenza drug screening [15]. CLK1 has been shown to
interact and phosphorylate other protein kinases as well as protein phosphatases [12]. Human
CLK2 links cell cycle progression, apoptosis and telomere length regulation [16]. CLK2 acts
as a suppressor of hepatic gluconeogenesis and glucose output [17]. CLK2 is overexpressed in
breast tumours. Downregulation of CLK2 inhibits breast cancer growth [18]. CLK3 is a
protein kinase with a non-conserved N-terminal domain.
CLK small molecule inhibitors are valuable tools for better understanding the molecular
mechanisms of splicing and may serve as seeds for a novel class of therapeutics [2, 14].
The limitations of many mono-kinase inhibitors can be overcome by agents with
multi-target action by increasing their potency, due to the synergistic effect. A review was
published in 2015 about the most recent examples of multi-kinase inhibitors. [19] Some dual
inhibitors for distanced kinases CLK1 and CK1 were developed recently based on pyrido[3',
2':4,5]thieno[3,2-d]pyrimidin-4-amine skeleton. [20]
2. Results and discussion
2.1. Analysis of hu-CLKs structures in PDB
With the aim to perform docking experiments we analysed CLKs structures in the
PDB database [21]. Currently, there are 8 X-ray structures of human CLK1-3 in PDB DB:
two hu-CLK3 proteins and six CLK1-3 / ligand complexes. No structure of hu-CLK4 was
published. According to our analysis, all CLK structures in PDB are in an active DFG-IN
kinase conformation. (Table 1)
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Table 1. hu-CLK structures published in PDB database.
Protein structure a
Kinase / ligand complex structure a
hu-CLK1 b
none
PDB: 2VAG (V25; IC50 = 19.7 nM;
2007, 1.80 Å, diphosphorylated kinase) [22]
PDB: 1Z57 (DBQ;c 2005, 1.70 Å) [2]
PDB: 3NR9 (NR9; 2010, 2.89 Å) [23]
hu-CLK2 b
hu-CLK3 b
none
PDB: 2EU9 (2005, 1.53 Å) [2] PDB: 3RAW (3RA;d 2011, 2.09 Å) [25]
PDB: 2EXE (2005, 2.35 Å,
phosphorylated) [24] e
none
PDB: 2WU6 (DKI; IC50 = 29.2 nM; 2009, 1.92 Å) [22]
PDB: 2WU7 (V25; IC50 = 530 nM; 2009, 2.25 Å) [22]
none
hu-CLK4
a/ The data are described in the following order: PDB: code (ligand code, it´s activity; year of deposition in PDB
DB; X-ray resolution; notes, if any).
b/ All CLK kinase structures in PDB are in an active DFG-IN conformation: DFG fragment (Asp-Phe-Gly triade
and their isoform specific primary sequence numbers: 325-327 (hu-CLK1), 327-329 (hu-CLK2), 320-322 (hu-
CLK3).
c/ also known as 10Z-Hymenialdisine
d/ also known as leucettine (L41)
e/ this is an incomplete structure of CLK3
2.2. Analysis of hu-CLKs PDB ligand interactions
To understand the ligand / CLK bindings, interactions diagrams of all inhibitors (2 x
V25, DBQ, DKI, NR9 and 3RAW) from PDB hu-CLK complexes were composed. The
ligand structures on diagrams are drawn uniformly according their positions in an Africa-like
kinase perspective [26]. Some biological activities of CLK PDB inhibitors are listed too [27].
The data for all CLKs PDB ligands can be found in supplementary material (chapter: Analysis
of hu-CLKs PDB ligand interactions). Here we present an example of analysis for ligand V25
from both complexes CLK1 (PDB: 2VAG) and CLK3 (PDB: 2WU7). (Fig. 1)
2.2.1. (E)-Ethyl 3-(2-amino-1-cyanovinyl)-6,7-dichloro-1-methyl-1H-indole-2-
carboxylate (V25) in complex with hu-CLK1 and 3
For compound V25 [SciFinder CAS: 1354037-26-5; Reaxys RRN: 21739116] the
following IC50 activities were described in the literature: hu-CLK1 (19.7 nM), hu-CLK3 (530
nM) and hu-DYRK1A (55.2 nM) [27].
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2.3. Interaction diagrams
Figure 1. The structure and intermolecular interactions of V25 determined in hu-CLK1 and 3
complexes.
2.4. CLKs inhibitors
In order to know how many inhibitors exist, CLKs abbreviations and synonyms [28] were
collected and used to search biological active CLK compounds in the Reaxys DB [27]. The
search resulted in the amount of inhibitors depicted for each kinase in parenthesis: CLK1
(176), CLK2 (108), CLK3 (84), CLK4 (261). By combining the above hit sets, we found 388
known compounds connected with CLKs activities.
2.5. Clinical drugs as CLKs inhibitors
There are only few clinical drugs able to inhibit CLK kinases. The drugs CLK activities
were mostly determined by searching their side effects [29] by screening pharmaceutical
compounds against 456 human kinases (Kinome scan). The determined drug CLK affinities
are as follows: CLK1 (sunitinib KD = 22 nM, bosutinib KD = 600 nM, nilotinib KD = 2100
nM); CLK2 (sunitinib KD = 20 nM, ruxolitinib KD = 460 nM, bosutinib KD = 1700 nM);
CLK3 (bosutinib KD = 300 nM); CLK4 (sunitinib KD = 29 nM, ruxolitinib KD = 1700 nM,
imatinib KD = 2100 nM). The above drugs were developed for different primary target(s) as
CLKs. From them only sunitinib was developed against VEGFR2 TK. The dual CLK and
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VEGFR2 activities of sunitnib are interesting because both CLKs and VEGFR2 inhibitors are
important as potential anticancer drugs.
2.6. VEGFR2 TK drugs
VEGFR2 TK (also named as KDR kinase) is an important receptor for VEGF signalling.
VEGF is a key mediator of angiogenesis in cancer [30]. Similarly as for CLKs above we
identified 6299 active compounds against VEGFR1-3 in Reaxys DB. Within recent ten years
pharmaceutical companies produced nine approved anticancer drugs inhibiting VEGFR2 TK
[31]. Their structures, year of approval and name of owning pharma company are published in
supplementary material (chapter: A list of VEGFR2 approved drugs).
2.7. Synthesis of CLK1 inhibitors 6, 10, 21 and 29
CLK1 inhibitors 6, 10, 21 and 29 and their intermediates were prepared according the
procedures depicted on Schemes 1-4. Synthetic details and physico-chemical characteristics
for newly prepared compounds are given in the Experimental part. Synthetic details about
known compounds and figures of spectra of all compounds are stated in supplementary
material (chapter: Supplementary Experimental).
Scheme 1. Synthetic pathway to final naphthalenyloxazolamine 6.
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Scheme 2. Synthetic pathway to required benzothiophenyloxazolamine 10.
Scheme 3. Synthesis of final styryloxazolamine 21.
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Scheme 4. Synthetic pathway to aminooxazolephenylacetonitrile 29.
2.8. Biological screening
Compounds 6, 10, 21 and 29 previously developed to inhibit VEGFR2 TK were
screened on eleventh protein kinases by radiometric protein kinase assay in 10 semi-
logarithmic concentrations on a panel of eleventh protein kinases: CDK2/CyclinA (cyclin-
dependant kinase); CDK5/p25; CDK9/CyclinT; PIM1 (proto-oncogene serine/threonine-
protein kinase); DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase);
GSK3α/β (glycogen synthase kinase); GSK3; CLK1 (CDC2-like or LAMMER family dual
specificity protein kinase); CK1δ/ε (caseine kinase 1); CK1; HASPIN (haploid germ cell-
specific nuclear protein kinase); AURKB (Aurora kinase B); RIPK3 (receptor-interacting
serine/threonine-protein kinase) and TLK (tousled-like kinase). The IC50 activities were
obtained by Dr. Stéphane Bach and Thomas Robert (Station Biologique de Roscoff, Place
Georges Teissier, CS 90074, 29688 Roscoff cedex, France). All compounds 6, 10, 21 and 29
were showed to be powerful CLK1 inhibitors. The results are given in Figure 2. (see also the
Supplementary material chapter Biological activity assay) Compound 29 was screened also on
VEGFR2 TK activity. Surprisingly, this compound performed dual VEGFR2 / CLK1 kinase
activity even though VEGFR2 is not relative kinase to CLKs and belongs to distant TK
subgroup in the Human Kinome [4].
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O
O
O
S
S O
S O
O
O
N
N
N
N
H
H
O
O
6
10
BM09414
BM09915
IC50 = 20 nM (hu-CLK1)
1 000 nM (DYRK1A)
IC50 = 30 nM (hu-CLK1)
500 nM (DYRK1A)
N
O
N
S O
O
S O
O
O
N
N
H
O
N
N
H
21
O
29
BM10015
IC50 = 40 nM (hu-CLK1)
200 nM (CDK9)
BM07114
IC50 = 80 nM (hu-CLK1)
23 nM (VEGFR2 TK)
4 000 nM (GSK3)
7 000 nM (DYRK1A)
7 500 nM (GSK3 / )
Figure 2. Novel hu-CLK1 inhibitors 6, 10, 21, 29 and their biological activities determined
against some CMGC protein kinases together with VEGFR2 TK activity for 29.
2.9. Predicted CLK1 inhibitors binding poses
In order to find binding poses for CLK1 inhibitors (6, 10, 21 and 29), docking
experiments [32] on seven CLK proteins from PDB DB were performed. An incomplete
2EXE structure of CLK3 was excluded. (Table 1) Most of the PDB CLK proteins showed to
be unfavourable for docking experiments due to their inappropriate kinase conformation.
Only the kinase variant from hu-CLK3 (2WU6) significantly outperformed the other models
by docking scores and predicted poses for all ligands (6, 10, 21 and 29). As can be seen from
ligands superimpositions especially the poses of their joint N-aryloxazole-2-amine parts were
almost identical. (Fig. 3)
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Firgure 3. The superimposed poses of inhibitors 6, 10, 21 and 29 from aligned complexes
obtained after ligands docking in CLK3 (2WU6) protein conformer.
The most active inhibitor 6 (IC50 = 20 nM) was predicted to form three hydrogen bonds (HBs)
with two amino acids Leu239 and Asn242 (both from a kinase hinge region) and one ligand
specific HB between -OMe group and Lys186. Moreover, N-aryloxazole-2-amine ligands
showed their poses and binding interactions to be similar in both hu-CLK3 (2WU6) and
VEGFR-2 TK (1Y6A). (Fig. 4 and 7)
Figure 4. Interactions of AAZ in VEGFR2 TK (1Y6A) and 6 in CLK3 (2WU6) to
demonstrate similar binding interactions of N-aryloxazol-2-amine in both kinases.
As mentioned above, both available CLK1 (1Z57, 2VAG) were not suitable for docking of
our CLK1 inhibitors. In order to investigate problematic amino acid residues predicted CLK3
(2WU6) / (6, 10, 21 and 29) complexes were aligned with both CLK1 protein conformers.
Then we could recognise that the most important unfavourable ligand interactions in CLK1
(2VAG) were between an amino group of N-aryloxazol-2-amines and too closely located
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carbonyl group of Leu244 from a hinge region. Due to the above repulsion the structures (6,
10, 21 and 29) could not be stabilized in good position in CLK1 (2VAG). (Fig. 5) In cases of
6 and 29 also some steric clashes with Phe172 were seen but they disappeared after a
problematic ligand group rotation.
Figure 5. In the left: the predicted pose of inhibitor 6 in CLK3 (2WU6) showing two
hydrogen bonds (1.7 and 1.8 Å, in blue) with a hinge Leu239. In the right: Inhibitor 6
positioned in CLK-1 (2VAG) possessing unfavourable interactions with Leu244 (2.06 Å
electrostatic repulsion, 1.4 Å steric clash, both in red).
In the other CLK1 (1Z57) conformer, the most important unfavourable interactions were
identified between the oxygen from -SO2Et group of the ligand and too close positioned
carboxylate oxygen from a hinge Asp250. This interaction could not be eliminated by simple
changing the conformation of -SO2Et group. The problematic Asp250 in CLK1 (1Z57) is
replaced by Glu245 in CLK3 (2WU6). Even though positions of Asp250 and Glu245 are
similar, the problematic carboxylate in larger Glu245 is folded out of the -SO2Et ligand group
excluding so unfavourable interactions in CLK3 compared to CLK1. (Fig. 6) Similarly as in
CLK1 (2VAG) also in CLK1 (1Z57) the inhibitors 6 and 29 possess some clashes also with
Phe172.
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Figure 6. A complex 6 / CLK3 (2WU6), obtained after docking, was aligned with CLK1
(1Z57) kinase. A hinge Glu245 from CLK3 (2WU6) is replaced by Asp250 (yellow) in CLK1
(1Z57) and forms unfavourable interaction with oxygen from -SO2Et group of 6. Observed
strong electrostatic repulsion (1.7 Å, red) is hindering ligand 6 to be docked in CLK1 (1Z57)
in an appropriate position.
Table 2. A summary for N-aryloxazole2-amine 6 interactions favourable for CLK3 (2WU6)
and problematic for both CLK1s (2VAG, 1Z57).
oxazole-2-amine of 6
-SO2Et of 6
hinge-Leu239 (CO, NH: 1.7, 1.8 Å)
hinge-Asn242 (CONH2: 2.1 Å)
CLK3 (2WU6)
hinge-Leu244 (NH: 1.7 Å)
hinge-Ser247 (NH: 2.5 Å)
CLK1 (2VAG)
CLK1 (1Z57)
UI: Leu244 (CO) (1.4, 2.1 Å)
hinge-Leu244 (CO, NH) (2 x 1.8 Å)
hinge-Ser247 (NH: 2.5 Å)
UI Asp250 (COO-: 1.7 Å)
UI: unfavourable interactions
in CLK3, CLK1 isoforms there are some replaced AAs residues (Leu239 to Leu244 and Asn242 to Ser 247,
resp.)
To conclude the above observation, a different conformation of CLK1 kinase will be required
to find the correct binding poses for N-aryloxazole-2-amine ligands with respect to their
CLK1 activity. Therefore we aligned CLK1 proteins (1Z57, 2VAG) with 6 / CLK3 (2WU6)
complex. In order to enable improved ligand docking to CLK1 the geometry minimization
was carried out on both CLK1 (1Z57, 2VAG) conformers with ligand 6 from CLK3 (2WU6)
complex. This approach resulted in CLK1 optimized protein models (1Z57opt, 2VAGopt)
with significantly improved performance to give docked CLK1 inhibitors (6, 10, 21 and 29)
positioned to keep essential hinge hydrogen bonds and docking scores that followed their IC50
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values. These models will be used for further CLK1 ligands development. (see supplementary
material, chapter: Predicted inhibitors binding poses in CLK1 optimised protein)
2.10. N-aryloxazole-2-amines in VEGFR2 and CLK
Compound 29 inhibits both kinases VEGFR2 (IC50 = 23 nM) and CLK1 (IC50 = 80
nM). The reason for a dual kinase activity of 29 can be explained by its similar binding pose
and interactions in each kinase where the pair of hinge amino acid residues (Cys917 / Asn921
in VEGFR2; Leu239 / Asn242 in CLK3 or Leu244 / Ser247 in CLK1) fixes a pharmacophoric
N-arylooxazole-2-amine group in an ATP binding pocket. The interaction diagrams for
ligands AAZ / VEGFR2 and 6 / CLK3 were composed. (Fig. 7).
Figure 7. The interactions of AAZ ligand with VEGFR2 TK and 6 in CLK3 are very similar
especially for their joint N-aryloxazole-2-amine part.
2.11. Dual VEGFR / CLK active compounds
With aim to determine the amount of dual VEGFR / CLK inhibitors an intersection
between previously found 388 CLKs inhibitors and 6 299 VEGFRs TKIs in Reaxys DB was
performed. This experiment resulted in 85 dual modulators with activities mostly determined
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between 1 - 10 ꢀM concentrations. Many of duals were uncovered by searching target-off
activities through Kinome scan for another biomacromolecule against that compounds were
primarily developed. As we have seen, the activities of many of them were not very important
or they were unbalanced for both kinases. A list of Reaxys codes (RRN) for 85 dual inhibitors
are given in supplementary material (chapter: Reaxys RRN codes of 85 compounds possessing
dual VEGFR / CLK activities). Among 85 structures there are about 7 with more balanced
CLKs / VEGFRs activities (RRN: 18647647, 20385666, 20278959, 22546881, 20385602,
21237166 and 22449988.). One structure, a triazole-staurosporine like compound (RRN:
18647647, CAS: [1359754-81-6]) possess submicromolar bivalent activities to VEGFR2 and
CLK2 (200 / 350 nM, resp.) [33]. We found also that sunitinib (RRN: 15426924, [CAS:
341031-54-7]) a multi-targeted tyrosine kinase ligand (PDGF, VEGFR2 TK) has strong
ability to inhibit CLKs. KD affinities for sunitinib are listed here: CLK1 (22 nM) CLK2 (20
nM), CLK4 (29 nM) [29]. There are only few powerful and activity balanced dual VEGFR2 /
CLKs inhibitors described in the literature. Such compounds could be advantageous for the
development of clinical drugs treating tumours by two VEGFR2 and CLKs independent
pathways. From this point of view N-aryloxazol-2-amine CLK1 inhibitors (6, 10, 21 and 29)
previously designed as VEGFR2 TKIs seem to be a good starting point for such development.
3. Conclusions
Four novel N-aryloxazol-2-amine CLK1 inhibitors (6, 10, 21 and 29) were identified
and their ATP binding poses in CLK kinase were predicted. Compound 29 has comparable
activity for CLK1 and VEGFR2 TK. The observed dual inhibition of 29 was explained by
similar
binding
interactions
of
its
pharmacophoric
N-(5-(ethylsulfonyl)-2-
methoxyphenyl)oxazol-2-amine fragment that has joint affinities to CLK1 and VEGFR2
kinases via hinge amino acid residues HBs. We found that there are only few dual VEGFR /
CLK inhibitors with comparable and strong activities on both targets. Such dual kinase
compounds could be advantageous for development of clinical compounds targeting tumours
by two independent and synergy pathways. Therefore presented N-aryloxazole-2-amine
CLK1 inhibitors (6, 10, 21 and 29) can be used in the further study of the molecular
mechanisms of splicing and seem to be also good seeds for development a novel class of
VEGFR2 / CLKs dual kinase therapeutics.
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4. Experimental
The syntheses of biologically active compounds 6, 10, 21 and 29 and their intermediates are
described in the Supplementary material (chapter: Supplementary Experimental).
Acknowledgements
The research was supported by Biomagi, Ltd. (development of kinase research, compounds
design and selection), VEGA 1/0634/13 (organic synthesis), bilateral project APV SK-FR-
2015-0014 (researchers and student exchange), program Cotutelle and Collège Doctoral
Européen - Université de Strasbourg (support for joint PhD student PŠ), ITMS 26240220007
(HPLC MS). Our big thanks belong to Dr. Nadine Martinet (Consultant in Cosmetics and
Medicinal Chemistry, Nice, France, nadine.martinet1@free.fr) for chemistry / biology
networking and Dr. Stéphane Bach (bach@sb-roscoff.fr) from Station Biologique de Roscoff,
France for biological screening. Our acknowledgement is addressed also to European
cooperation in science and technology COST Actions: StemChem CM1106 and MuTaLig
CA15135 for research networking. This paper is dedicated to our teachers and enthusiastic
colleagues prof. Marta Sališová and prof. Margita Lácová from Comenius University in
Bratislava, Slovakia. We appreciate their research knowledge, management and personal
behaviour that we and our students could and still can learn from them.
References
[1] Black D.L. Mechanisms of alternative pre-messenger RNA splicing, Annual Review of
Biochemistry 2003, 72, 291-336, DOI:10.1146/annurev.biochem.72.121801.161720.
[2] Bullock A.N., Das S., Debreczeni J.E., Rellos P., Fedorov O., Niesen F.H., Guo K.,
Papagrigoriou E., Amos A.L., Cho S., Turk B.E., Ghosh G., Knapp S. Kinase domain
insertions define distinct roles of CLK kinases in SR protein phosphorylation, Structure
2009, 17, 352-362. DOI: 10.1016/j.str.2008.12.023.
[3] Manning G., Whyte D.B., Martinez R., Hunter T., Sudarsanam S. The Protein Kinase
Complement of the Human Genome Science 2002, 298, 1912-1934, DOI:
10.1126/science.1075762.
[4] Cell Signaling Technology interactive human kinome tree.
http://www.cellsignal.com/contents/science-protein-kinases/protein-kinases-interactive-
human-kinome/kinases-human-kinome (accessed 22.06.0216).
[5] Sessa G., Raz V., Savaldi S., Fluhr R., PK12, a plant dual specificity protein kinase of the
LAMMER family, is regulated by the hormone ethylene Plant Cell 1996, 8, 2223-2234.
15

ACCEPTED MANUSCRIPT
[6] Savaldi-Goldstein S., Sessa G., Fluhr R. The ethylene inducible PK12 kinase mediates the
phosphorylation of SR splicing factors Plant J. 2000, 21, 91-96.
[7] Nayler O., Stamm S; Ullrich A. Characterization and comparison of four serine- and
arginine-rich (SR) protein kinases Biochem J. 1997, 326, 693-700.
[8] a/ Colwill K., Pawson T., Andrews B., Prasad J., Manley J.L., Bell J.C., Duncan P.I. The
Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear
distribution. EMBO J. 1996, 15, 265-275. b/ Prasad J., Manley J.L. Mol. Cell Biol. 2003,
23, 4139-4149. c/ Muraki M., Ohkawara B., Hosoya T., Onogi H., Koizumi J., Koizumi
T., Sumi K., Yomoda J.-I., Murray M.V., Kimura H., Furuichi K., Shibuya H., Krainer
A.R., Suzuki M., Hagiwara M. Manipulation of Alternative Splicing by a Newly
Developed Inhibitor of Clks J. Biol. Chem. 2004, 279, 24246-24254, DOI:
10.1074/jbc.M314298200.
[9] Jain P., Karthikeyan C., Moorthy N.S., Waiker D.K., Jain A.K., Trivedi P. Human CDC2-
like kinase 1 (CLK1): a novel target for Alzheimer's disease Curr Drug Targets. 2014, 15,
539-50.
[10] Hanes J., von der Kammer H., Klaudiny J., Scheit K.H. Characterization by cDNA
Cloning of Two New Human Protein Kinases: Evidence by Sequence Comparison of a
New Family of Mammalian Protein Kinases J. Mol. Biol. 1994, 244, 665-672, DOI:
10.1006/jmbi.1994.1763.
[11] The UniProt database. http://www.uniprot.org/ (assessed 03.04.2016).
[12] García-Sacristán A., Fernández-Nestosa M.J., Hernández P., Schvartzman J.B., Krimer
D.B. Protein kinase clk/STY is differentially regulated during erythroleukemia cell
differentiation: a bias toward the skipped splice variant characterizes postcommitment
stages Cell Res. 2005, 15, 495–503, DOI:10.1038/sj.cr.7290319.
[13] Araki S., Dairiki R., Nakayama Y., Murai A., Miyashita R., Iwatani M., Nomura T.,
Nakanishi O. Inhibitors of CLK protein kinases suppress cell growth and induce
apoptosis by modulating pre-mRNA splicing PLoS One 2015, 10, e0116929, DOI:
10.1371/journal.pone.0116929. eCollection 2015.
[14] Shinsuke Araki, Ryo Dairiki, Yusuke Nakayama, Aiko Murai, Risa Miyashita, Misa
Iwatani, Toshiyuki Nomura, Osamu Nakanishi Inhibitors of CLK Protein Kinases
Suppress Cell Growth and Induce Apoptosis by Modulating Pre-mRNA Splicing PLoS
One. 2015, 10, e0116929, DOI: 10.1371/journal.pone.0116929.
[15] Mian B.Z., Chao L., Jian-Song F., Wen-Wen L., Ai-Lin L., Li-Shu Z.,; Guan-Hua D.
Drug discovery of host CLK1 inhibitors for influenza treatment Molecules 2015, 20,
19735-19747.
[16] Jiang N., Bénard C.Y., Kébir H., Shoubridge E.A., Hekimi S. CLK2 may serve as a link
between cell cycle progression, apoptosis, and telomere length regulation. J. Biol. Chem.
2003, 278, 21678-84.
[17] PhosphoSitePlus Cell Signaling Technology.
http://www.phosphosite.org/proteinAction.do?id=810&showAllSites=true (assessed
26.06.2016).
[18] Yoshida T., Kim J.H., Carver K., Su Y., Weremowicz S., Mulvey L., Yamamoto S.,
Brennan C., Mei S., Long H., Yao J., Polyak K. CLK2 Is an Oncogenic Kinase and
Splicing Regulator in Breast Cancer Cancer Res. 2015, 75, 1516-26, DOI: 10.1158/0008-
5472.CAN-14-2443.
[19] Garuti L., Roberti M., Bottegoni G. Multi-Kinase Inhibitors Curr. Med. Chem. 2015, 22,
695-712, DOI:10.2174/0929867321666141216125528.
[20] Loidreau Y., Deau E., Marchand P., Nourrisson M.-R., Logé C., Coadou G., Loaëc N.,
Meijer L., Besson T. Synthesis and molecular modelling studies of 8-
16

ACCEPTED MANUSCRIPT
arylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amines as multitarget Ser/Thr kinases
inhibitors Eur. J. Med. Chem. 2015, 92, 124-134, DOI: 10.1016/j.ejmech.2014.12.038.
[21] RCSB Protein Data Bank. http://www.rcsb.org (accessed 22.06.2016).
[22] Fedorov O., Huber K., Eisenreich A., Filippakopoulos P., King O., Bullock A.N.,
Szklarczyk D., Jensen L.J., Fabbro D., Trappe J., Rauch U., Bracher F., Knapp S.
Specific Clk Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing
Chem. Biol. 2011, 18, 67-76, DOI: 10.1016/j.chembiol.2010.11.009.
[23] Chaikuad A., Savitsky P., Krojer T., Muniz J., Filippakopoulos P., Rellos P., Keates T.,
Fedorov O., Pike A., Eswaran J., Berridge G., Phillips C., Zhang Y., von Delft F.,
Weigelt J., Arrowsmith C., Edwards A., Bountra C., Knapp S (Structural Genomics
Consortium) S to be published, DOI: 10.2210/pdb3nr9/pdb.
[24] Papagrigoriou E., Rellos P., Das S., Bullock A., Ball L.J., Turnbull A., Fedorov O.,
Johansson C., Ugochukwu E., Sobott F., von Delft F., Edwards A., Sundstrom M.,
Weigelt J., Arrowsmith C., Knapp S. Crystal structure of the phosphorylated CLK3 to be
published, DOI: 10.2210/pdb2exe/pdb.
[25] Filippakopoulos P., Fedorov O., King O., Bullock A., Muniz J.R.C., von Delft F.,
Arrowsmith C.H., Edwards A.M., Weigelt J., Bountra C., Knapp S., Crystal Structure of
human CDC-like kinase 3 isoform with a benzo-dioxol ligand (Structural Genomics
Consortium) to be published, DOI: 10.2210/pdb3raw/pdb.
[26] Lintnerová L., García-Caballero M., Gregáň F., Melicherčík M., Quesada A.R., Dobiaš
J., Lác J., Sališová M., Boháč A. A development of chimeric VEGFR2 TK inhibitor
based on two ligand conformers from PDB: 1Y6A complex - Medicinal chemistry
consequences of a TKs analysis Eur. J. Med. Chem. 2014, 72, 146-159,
DOI:10.1016/j.ejmech.2013.11.023.
[27] Reaxys database https://www.reaxys.com (accessed 22.06.2016)
[28] The UniProt database. http://www.uniprot.org/ (accessed 26.06.2016).
[29] The targets and their inhibitors.
http://www.guidetopharmacology.org/GRAC/searchPage.jsp (accessed 26.06.2016).
[30] Carmeliet P. VEGF as a key mediator of angiogenesis in cancer Oncology 2005, 69
(Suppl. 3), 4-10, DOI:10.1159/000088478.
[31] IUPHAR/BPS Guide to PHARMACOLOGY.
http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1813
(accessed 31.1.2016).
[32] Irwin J.J., Shoichet B.K., Mysinger M.M., Huang N., Colizzi F., Wassam P., Cao Y.
Automated docking screens: a feasibility study, J. Med. Chem. 2009, 52, 5712-5720,
DOI: 10.1021/jm9006966. Software Dock Blaster: http://blaster.docking.org/ (assessed
26.06.2016).
[33] Gu G., Wang H., Liu P., Fu C., Li Z., Cao X., Li Y., Fang Q., Xu F., Shen J., Wang P.G.
Discovery and structural insight of a highly selective protein kinase inhibitor hit through
click chemistry Chem. Commun. 2012, 48, 2788-90, DOI: 10.1039/c1cc15851a.
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List of captions (for review only)
Scheme 1. Synthetic pathway to final naphthalenyloxazolamine 6.
Scheme 2. Synthetic pathway to required benzothiophenyloxazolamine 10.
Scheme 3. Synthesis of final styryloxazolamine 21.
Scheme 4. Synthetic pathway to aminooxazolephenylacetonitrile 29.
Figure 1. The structure and intermolecular interactions of V25 determined in hu-CLK1 and 3
complexes.
Figure 2. Novel hu-CLK1 inhibitors 6, 10, 21, 29 and their biological activities determined
against some CMGC protein kinases together with VEGFR2 TK activity for 29.
Firgure 3. The superimposed poses of inhibitors 6, 10, 21 and 29 from aligned complexes
obtained after ligands docking in CLK3 (2WU6) protein conformer.
Figure 4. Interactions of AAZ in VEGFR2 TK (1Y6A) and 6 in CLK3 (2WU6) to
demonstrate similar binding interactions of N-aryloxazol-2-amine in both kinases.
Figure 5. In the left: the predicted pose of inhibitor 6 in CLK3 (2WU6) showing two
hydrogen bonds (1.7 and 1.8 Å, in blue) with a hinge Leu239. In the right: Inhibitor 6
positioned in CLK-1 (2VAG) possessing unfavourable interactions with Leu244 (2.06 Å
electrostatic repulsion, 1.4 Å steric clash, both in red).
Figure 6. A complex 6 / CLK3 (2WU6), obtained after docking, was aligned with CLK1
(1Z57) kinase. A hinge Glu245 from CLK3 (2WU6) is replaced by Asp250 (yellow) in CLK1
(1Z57) and forms unfavourable interaction with oxygen from -SO2Et group of 6. Observed
strong electrostatic repulsion (1.7 Å, red) is hindering ligand 6 to be docked in CLK1 (1Z57)
in an appropriate position.
Figure 7. The interactions of AAZ ligand with VEGFR2 TK and 6 in CLK3 are very similar
especially for their joint N-aryloxazole-2-amine part.
Table 1. hu-CLK structures published in PDB database.
Table 2. A summary for N-aryloxazole2-amine 6 interactions favourable for CLK3 (2WU6)
and problematic for both CLK1s (2VAG, 1Z57).
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HIGHLIGHTS
•
•
•
•
four novel N-aryloxazol-2-amine CLK1 inhibitors (20,30,40,80 nM) were discovered
their binding poses in CLK1 and 3 were predicted
analysis of all CLK PDB structures and interactions of their ligands were performed
an advantageous dual VEGFR2 / CLK activity of N-aryloxazol-2-amines was
proposed