Hydroxybenzothiophene ketones are efficient pre-mRNA splicing modulators due to dual inhibition of Dyrk1A and Clk1/4

Article abstract of DOI:10.1021/ml500059y

Dysregulated usage of pre-mRNA splicing sites contributes to the progression of cancer, neurodegenerative diseases, and viral infections. Serine/arginine-rich (SR) proteins play major roles in the splice site recognition and are largely regulated by phosp

Full text of DOI:10.1021/ml500059y

Letter
pubs.acs.org/acsmedchemlett
Hydroxybenzothiophene Ketones Are Efficient Pre-mRNA Splicing
Modulators Due to Dual Inhibition of Dyrk1A and Clk1/4
Christian Schmitt,† Parisa Miralinaghi,† Marica Mariano,† Rolf W. Hartmann,†,‡ and Matthias Engel*,†
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123 Saarbrucken, Germany
̈
‡Department of Drug Design and Optimization, Helmholtz-Institut fur Pharmazeutische Forschung Saarland, Campus C2.3, D-66123
̈
Saarbrucken, Germany
̈
S
* Supporting Information
ABSTRACT: Dysregulated usage of pre-mRNA splicing sites contributes
to the progression of cancer, neurodegenerative diseases, and viral
infections. Serine/arginine-rich (SR) proteins play major roles in the
splice site recognition and are largely regulated by phosphorylation. This
provides an option for the pharmacological correction of aberrant splicing
by inhibiting the relevant kinases. Cdc2-like kinases (Clks) and dual
specificity tyrosine phosphorylation-regulated kinases (Dyrks) were both
reported to phosphorylate numerous SR proteins in vitro and in vivo. In
this study, we describe the discovery of new selective dual Clk/Dyrk1A/1B
inhibitors, which are able to modulate alternative pre-mRNA splicing of
model gene transcripts in cells with submicromolar potencies. The optimization process yielded a dual Clk and Dyrk inhibitor
with exceptionally high ligand efficiency. Our results suggested that dual inhibition of both Clk1 and Dyrk1A increased the
efficacy of pre-mRNA splicing modulation.
KEYWORDS: Dual kinase inhibitor, cdc2-like kinases, dual specificity tyrosine (Y) phosphorylation regulated kinases,
alternative splicing modifiers
lternative splicing of pre-mRNAs creates several mRNA
inhibition may have a therapeutic utility, e.g., for the treatment
Aisoforms that code for proteins having distinct functions. of neurodegenerative tauopathies, which are partly caused by an
imbalanced 3R/4R tau isoform ratio resulting from dysregu-
lated alternative splicing;10−12 both Dyrk1A and Clk1 were
reported to be causally involved due to hyperphosphorylation
of SR proteins.10−14
This versatile mechanism is involved in the control of
embryonic development, cell growth, and apoptosis.1 However,
dysregulated or nonfunctional splicing contributes to the
development and progression of neurodegenerative disorders,2
cancer, and viral infections, e.g., by HIV-1.3,4 The splice site
recognition is mainly determined by the interactions of serine-
arginine-rich (SR) proteins and heterogeneous nuclear
ribonucleoproteins with specific exonic and intronic pre-
mRNA sequences.5 SR proteins mostly promote the inclusion
of exons via binding to exonic splicing enhancers.5,6 Their
activity is largely regulated by multisite phosphorylations.5,7 To
date, several kinases have been reported to phosphorylate SR
proteins, including Clk1, SRPK1, topoisomerase I, and
Dyrk1A.5 For instance, Clk1 activity promotes the expression
of HIV-1 Gag proteins by altering the splicing of the viral RNA8
and mediates rephosphorylation of SR proteins after initial heat
shock response to viral infections, enabling the resumption of
viral RNA processing.9 Given the multisite phosphorylation of
SR proteins, co-operative effects of the relevant protein kinases
on the splicing activity can be expected. Several SR proteins
were reported to be substrates of both Clk1 and Dyrk1A,
including AF2/ASF, SC35, and SRp55,10−12 suggesting that it
might be possible to co-operatively enhance the modulation of
alternative splicing by coinhibition of the structurally related
kinases (both belonging to the CMGC family of the kinome);
however, this was not investigated so far. Such a simultaneous
Many natural and synthetic compounds were described to
interfere with splicing, some of which are general splicing
inhibitors, such as spliceostatin (reviewed in15). Compounds
that were reported to modulate alternative splicing of particular
genes are mostly pleiotropic effectors and/or act via poorly
defined mechanisms; they include cytostatic agents such as
camptothecin, cardiac glycosides, flavonoids, ethanol, dietary
supplements and some indole derivatives.15 In contrast, very
few compounds originated from a target−oriented develop-
ment of alternative splicing modulators.15 This is also true for
kinase inhibitors, in spite of the known importance of SR
protein phosphorylations in the regulation of alternative
splicing; some of the few examples are TG003,16,17 KH-
CB19,18 and NB-506.19 TG003, which is the best studied of
these compounds, is a dual inhibitor of Clk’s and Dyrk1A (cf.
Table S1),20 like harmine (Figure 1) and some other
compounds.14 However, the majority of these compounds
was not analyzed for the potential effect on alternative splicing,
Received: February 7, 2014
Accepted: July 14, 2014
Published: July 14, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Supporting Information). Therefore, we replaced the central
aromatic core by bioisosteric benzothiophene, benzofuran, and
indole heterocycles, considering in each case the two possible
hydroxyl positions (Tables 1 and S3, Supporting Information).
The 6-hydroxyl-substituted compounds 4 to 9 (Table S3,
Supporting Information) exhibited no improvement of potency
compared to the naphthalene-based inhibitors. However,
swapping the hydroxyl group from the 6- to the 5-position
on the benzothiophene core led to a 10-fold increase of the
activity toward the target kinases (compound 10, Table 1).
Inhibition of Clk1 was slightly preferred, displaying an IC50 of
about 100 nM. Because such a beneficial effect was not
observed for any of the benzofuran- and indole-based analogues
(11, 12, 13, and 15), further optimization efforts focused on
the 5-hydroxybenzothiophene ketones. To explore the effect of
the phenyl ring substitution pattern, the 3-hydroxyl group was
moved to the para-position in 14, which significantly decreased
the Dyrk1A inhibition by about 10-fold; however, the Clk1
inhibitory potency was less affected. Introduction of an
additional para-methyl group (16) doubled the potency toward
Dyrk1A and Clk1 compared with 10. While 16 was the most
potent compound of this series, the complete loss of activity
with 17 suggested a steric clash of the meta-methyl group with
the ATP binding pocket. Interestingly, the introduction of
fluorine in the phenyl moiety (18) dramatically increased the
selectivity for Clk1 (IC50 = 50 nM) over Dyrk1A (IC50 = 2.0
μM). Most likely, the conformational constraints effected by
the ortho-substitution were better tolerated by the Clk1
binding site. A comparison of 21 and 22 with 10 revealed
that the hydroxyl group at the benzothiophene but not at the
phenyl moiety was essential to the inhibitory activity. We
wanted to investigate further if the phenyl ring was at all
necessary for the biological activity by synthesizing compound
23, in which it was replaced by methyl. Strikingly, 23 exhibited
Figure 1. Reported Clk1 and Dyrk1A inhibitors and the newly
identified dual Clk/Dyrk inhibitor 1.
and the coinhibition of the Dyrk2 isoform, which functions as a
negative regulator of cell proliferation in vitro and suppressor of
tumor progression in vivo was a further drawback.21 This was
also noted for TG003, limiting its usefulness.22
Herein we describe the discovery of a new class of potent and
selective dual Clk and Dyrk1A/1B inhibitors. Additionally, we
provide evidence that dual inhibition of Clk1 and Dyrk1A was
more effective in the modulation of alternative splicing of two
different model gene transcripts in cells than inhibition of Clk1
alone. Screening of our in-house collection of diverse enzyme
inhibitors revealed a 6-hydroxynaphthalene ketone 1 (Figure 1)
as a moderate Dyrk1A and Clk1 inhibitor. Importantly, 1 did
not affect the “untouchable” Dyrk2 isoform. Therefore, we
optimized 1 in terms of potency as well as selectivity. Our initial
modification, the introduction of small substituents with
distinct electronic effects at the phenyl ring did not have any
significant impact on the biological activities (2 and 3, bearing
para-methyl and para-fluorine, respectively; cf. Table S2,
a
Table 1. Inhibitory Potencies of 5-Hydroxybenzoheterocyclic Inhibitors
IC50 [μM]/% inhibition @ 5 μM
compd
X
R1
R2
R3
Dyrk1A
Dyrk1B
Dyrk2
Clk1
Ck2α
10
s
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
F
0.4
3.1
15
0.2
6.1
41%
2.7
0.8
1.3
0.2
7
49%
34%
24%
25%
25%
38%
50%
8%
0.1
1.9
2
12%
21%
22%
31%
33%
17%
10%
11
0
H
12
NH
H
13
NMe
H
3.4
3.4
9.7
0.2
11
2.1
0.3
1−5
0.05
25%
0.05
0.3
0.4
0.2
14
S
O
S
S
S
S
S
S
S
S
S
S
OH
OH
Me
OH
H
15
H
16
OH
Me
OH
OH
CN
H
17
18
2
0.4
0.4
2.3
1
30%
27%
6%
20%
15%
10%
13%
24%
9%
19
F
H
H
H
1.2
6
20
H
21
H
2
37%
15%
46%
−7%
41%
22
42
6%
0.1
40%
1.6
23
0.2
10%
0.9
0.83
0.06
47%
0.1
24
25
TG003
0.17
a
[ATP] = 100 μM.
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Figure 2. Modulation of the Clk1/Sty splicing pattern by the Dyrk1A/Clk1 inhibitors in STO cells. The test compounds were added to the cell
medium at the indicated concentrations for 4 h. DMSO was used as control (0.05%). Total RNA was purified, and the splicing pattern was analyzed
by RT-PCR. The splicing variants and their expected PCR products using the primers indicated by arrows are depicted on the right. For comparison,
IC50 values against the recombinant enzymes are also given; for a more detailed description, see Supporting Information, 1. Biology.
Figure 3. Modulation of SC35 pre-mRNA splicing by the Dyrk1A/Clk1 inhibitors in STO cells. The test compounds were added to the cell medium
at the indicated concentrations for 4 h. Total RNA was purified, and the splicing pattern was analyzed by RT-PCR. The splicing variants and their
expected PCR products using the primers indicated by arrows are depicted on the right (for a more detailed description, see Supporting Information,
1. Biology).
the highest inhibitory potency besides 16, indicating that the
phenyl ring might even impede an optimum protein−ligand
interaction. Considering its low molecular mass (192.2 Da), 23
probably represented an optimal ligand for the ATP binding
pockets of Dyrk1A and Clk1, as indicated by its exceptionally
high ligand efficiency of 0.74. Since the phenyl ring was not
needed for activity, it was straightforward to investigate whether
an amide function was also tolerated in place of the methyl
ketone. The Weinreb amide 25 turned out to be nearly as
potent against Clk1 as 23, but it was considerably less active
against Dyrk1A and Dyrk1B. Compound 25 was thus identified
as a potential new lead for the development of selective Clk1
inhibitors.
structurally similar benzothiophene−based inhibitors for this
potential off-target activity. Indeed, several compounds were
identified as 17β-HSD1 inhibitors, including 16 (IC50 = 195
nM); however, 23 and 25 were completely inactive toward this
enzyme (Table S4, Supporting Information), thus excluding
side effects on the steroidal biosynthesis by our most active
compound 23.
We expected good cellular activity of 16 and 23, considering
that they were reasonably potent in our cell-free assays at 100
μM ATP, which was significantly above the reported KM value
of Dyrk1A (cf. Table S5, Supporting Information). Thus, we
decided to determine the general potency of the compounds to
modulate pre-mRNA splicing in intact cells by analyzing the
transcript pattern of two model genes, which are known to
undergo SR protein phosphorylation−dependent alternative
splicing, SC35 and Clk1 itself.24,25 Importantly, alteration of the
A 6-hydroxybenzothiazole isomer of 10 had been previously
described as a highly potent inhibitor of 17β-hydroxysteroid
dehydrogenase type 1 (17β-HSD1).23 Therefore, we tested our
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Letter
splicing pattern of these transcripts after treatment with the
reference compound TG003 had been reported.9,16 Catalyti-
cally active Clk1/Sty promotes skipping of exon 2 from its own
pre-mRNA via hyperphosphorylation of SR proteins, thus
producing a mRNA, which encodes the kinase-negative Clk1/
StyT.16,24 Vice versa, treatment of the model cell line, immortal
embryonic fibroblasts (STO), with Clk1 inhibitors should lead
to enhanced exon 2 inclusion. Indeed, as detected by reverse
transcription (RT)-PCR, the compounds with the highest cell-
free potency, 10, 16, and 23, strongly induced the production
of the mature Clk1 mRNA retaining exon 2 (Figure 2). It
became evident that the dual inhibitors 10, 16, 23, and TG003
were more potent in altering the splicing pattern than
compounds 14 and 19, which were less active against
Dyrk1A (Table 1). At the chosen concentration (10 μM), 23
led to a complete disappearance of the incompletely and
alternatively spliced transcripts, while such were still visible in
the case of TG003. The weakest effect among the
hydroxybenzothiophenes was noted for 8, possessing the
lowest potency for both Dyrk1A and Clk1. Hence, the efficacy
of the Clk1 splicing modulation correlated with the potency to
simultaneously inhibit Dyrk1A and Clk1. To corroborate our
findings, we also analyzed the effects of the compounds on the
splicing pattern of SC35, another substrate of Clk1 and
Dyrk1A,11,26 which regulates splicing of its own pre-mRNA
dependent on its phosphorylation status.9,16 Administration of
compounds 16 and 23 into the culture medium of STO cells
changed the splicing profile of SC35 in the same manner as
previously shown for the reference compound TG003 (Figure
3).16 The specific enhancement of a splicing product lacking
the terminal intron (represented by the 274 bp product) was
slightly weaker in the case of the less potent Dyrk1A inhibitors
TG003 and 25, suggesting again that dual inhibition of both
Dyrk1A and Clk1 results in a stronger modulation of the
alternative splicing process. The dynamic range of the visible
changes in the splicing profiles was considerably higher in the
case of the Clk1 mRNA (Figure 2), possibly supported by an
enhanced splicing of larger, intron-retaining premature Clk1
mRNA transcripts, which were not amplified under the PCR
conditions.9 Therefore, we chose the Clk1 mRNA as a PCR
target for the development of a qPCR assay in order to quantify
the strength of splicing modulation in cells. The results for the
best compounds are shown in Table 2: 23 triggered a
significant response of the Clk1 pre-mRNA splicing already at
submicromolar concentrations (C5‑fold = 1.1 μM), followed by
16. Again both compounds, being efficient dual Dyrk1A/Clk1
inhibitors, were more potent than the amide analogue 25,
which was equally active against Clk1 but not against Dyrk1A
in the cell free assay (Table 1). Similarly, significantly higher
concentrations of TG003 than of 16 or 23 were needed to elicit
a clear response, correlating with the less potent inhibition of
Dyrk1A (IC50 = 830 nM, cf. Table 1). Assuming that there were
no major differences in cell permeation between the
compounds, our data suggested an at least additive effect of
simultaneous Dyrk1A and Clk1 inhibition on the modulation of
alternative pre-mRNA splicing. Interestingly, the maximum
achievable effect on Clk1 pre-mRNA splicing was a
compound−specific variable; hence, the calculated EC50 values
appeared to be of limited significance. Next we examined the
selectivity toward a carefully selected panel of kinases
representing frequently reported off-targets and further kinases
from each subfamily of the kinome. Compounds 16 and 23
showed good selectivity for the Clks and Dyrks in this panel; 23
was only slightly less selective than 16 (Table S6, Supporting
Information) exhibiting a similar inhibitory potency among the
closely related cdc2-like kinases Clk1−4 and, in addition, a
moderate potency against the atypical kinase haspin (IC50 = 0.8
μM; Table S7, Supporting Information). Notably, our new
inhibitors were clearly more selective against some frequent off-
targets than TG003 (Table S8, Supporting Information). In
conclusion, we developed a new dual inhibitor of Clks and
Dyrks, 23, with exceptionally high ligand efficiency and notable
cellular efficacy. Because of the low molecular weight, the good
aqueous solubility and the selectivity among the panel of
frequent off-target kinases, it might become a valuable tool for
the in vitro and in vivo pharmacological validation of Dyrk1A
and Clk1 coinhibition in missplicing-related diseases, including
Alzheimer’s,10−14 viral infections,8,9 and congenital genetic
disorders such as Duchenne muscular dystrophy.27
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for all assays and syntheses; supplementary
Tables S1−S8 and Figure S1. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(M.E.) E-mail: ma.engel@mx.uni-saarland.de. Phone:
+4968130270312.
■
Author Contributions
C.S, P.M., M.M., and M.E. designed and performed research.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
We are grateful for the financial support by the DFG to M.E.
(EN 381/3-1).
Notes
Table 2. Modulation of Clk/Sty Pre-mRNA Splicing by 16,
23, and 25 in Comparison to TG003
TG003
23
16
25
The authors declare no competing financial interest.
a
C5‑fold (μM)
3.1
6.6
17
1.1
1.9
1.8
9.5
2.4
2.1
8.1
b
ACKNOWLEDGMENTS
The authors thank Mrs. Nadja Weber and Tamara Paul for
their assistance in performing splicing assays.
EC50 (μM)
max. induction
8.9
■
c
21.4
a
Concentration required to induce a 5-fold increase of the mature
Clk1 transcript levels as determined by qPCR; SD ≤ 10%.
ABBREVIATIONS
b
■
Concentration required for half-maximal generation of the mature
c
CMGC, cyclin-dependent kinases, mitogen-activated protein
kinases, glycogen-synthase kinases, and CDC-like kinases; SR
proteins, serine- and arginine-rich family of splicing proteins;
qPCR, quantitative real-time polymerase chain reaction
Clk1 mRNA splicing product; SD ≤ 10%. Maximum increase of
mature Clk1 transcripts achievable with the inhibitors (fold increase
over DMSO-treated samples); SD< 20%. Shown are means of three
independent experiments performed in triplicates.
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Letter
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.
REFERENCES
■
(1) Kelemen, O.; Convertini, P.; Zhang, Z.; Wen, Y.; Shen, M.;
Falaleeva, M.; Stamm, S. Function of alternative splicing. Gene 2013,
514, 1−30.
́
(19) Pilch, B.; Allemand, E.; Facompre, M.; Facompre, M.; Bailly, C.;
Riou, J.; Soret, J.; Tazi, J. Specific inhibition of serine- and arginine-rich
splicing factors phosphorylation, spliceosome assembly, and splicing
by the antitumor drug NB-506. Cancer Res. 2001, 61, 6876−6884.
(20) Ogawa, Y.; Nonaka, Y.; Goto, T.; Ohnishi, E.; Hiramatsu, T.;
Kii, I.; Yoshida, M.; Ikura, T.; Onogi, H.; Shibuya, H.; Hosoya, T.; Ito,
N.; Hagiwara, M. Development of a novel selective inhibitor of the
Down syndrome-related kinase Dyrk1A. Nat. Commn. 2010, 1, 86.
(21) Taira, N.; Mimoto, R.; Kurata, M.; Yamaguchi, T.; Kitagawa, M.;
Miki, Y.; Yoshida, K. DYRK2 priming phosphorylation of c-Jun and c-
Myc modulates cell cycle progression in human cancer cells. J. Clin.
Invest. 2012, 122, 859−872.
́
(2) Tollervey, J. R.; Wang, Z.; Hortobagyi, T.; Witten, J. T.; Zarnack,
K.; Kayikci, M.; Clark, T. A.; Schweitzer, A. C.; Rot, G.; Curk, T.;
Zupan, B.; Rogelj, B.; Shaw, C. E.; Ule, J. Analysis of alternative
splicing associated with aging and neurodegeneration in the human
brain. Genome Res. 2011, 21, 1572−1582.
(3) Dowling, D.; Nasr-Esfahani, S.; Tan, C.; O’Brien, K.; Howard, J.;
Jans, D.; Purcell, D.; Stoltzfus, C. M.; Sonza, S. HIV-1 infection
induces changes in expression of cellular splicing factors that regulate
alternative viral splicing and virus production in macrophages.
Retrovirology 2008, 5, 18.
(4) Fukuhara, T.; Hosoya, T.; Shimizu, S.; Sumi, K.; Oshiro, T.;
Yoshinaka, Y.; Suzuki, M.; Yamamoto, N.; Herzenberg, L. A.;
Herzenberg, L. A.; Hagiwara, M. Utilization of host SR protein
kinases and RNA-splicing machinery during viral replication. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 11329−11333.
(22) Gao, Y.; Davies, S. P.; Augustin, M.; Woodward, A.; Patel, U. A.;
Kovelman, R.; Harvey, K. J. A broad activity screen in support of a
chemogenomic map for kinase signalling research and drug discovery.
Biochem. J. 2013, 451, 313−328.
(23) Spadaro, A.; Frotscher, M.; Hartmann, R. W. Optimization of
hydroxybenzothiazoles as novel potent and selective inhibitors of 17β-
HSD1. J. Med. Chem. 2012, 55, 2469−2473.
(5) Zhou, Z.; Fu, X.-D. Regulation of splicing by SR proteins and SR
protein-specific kinases. Chromosoma 2013, 122, 191−207.
(6) Stamm, S.; Ben-Ari, S.; Rafalska, I.; Tang, Y.; Zhang, Z.; Toiber,
D.; Thanaraj, T. A.; Soreq, H. Function of alternative splicing. Gene
2005, 344, 1−20.
(24) Duncan, P. I.; Stojdl, D. F.; Marius, R. M.; Bell, J. C. In vivo
regulation of alternative pre-mRNA splicing by the Clk1 protein
kinase. Mol. Cell. Biol. 1997, 17, 5996−6001.
́
(7) Misteli, T.; Caceres, J. F.; Clement, J. Q.; Krainer, A. R.;
́
(25) Sureau, A.; Gattoni, R.; Dooghe, Y.; Stevenin, J.; Soret, J. SC35
Wilkinson, M. F.; Spector, D. L. Serine phosphorylation of SR proteins
is required for their recruitment to sites of transcription in vivo. J. Cell
Biol. 1998, 143, 297−307.
autoregulates its expression by promoting splicing events that
destabilize its mRNAs. EMBO J. 2001, 20, 1785−1796.
(26) Jiang, K.; Patel, N. A.; Watson, J. E.; Apostolatos, H.; Kleiman,
E.; Hanson, O.; Hagiwara, M.; Cooper, D. R. Akt2 regulation of Cdc2-
like kinases (Clk/Sty), serine/arginine-rich (SR) protein phosphor-
ylation, and insulin-induced alternative splicing of PKCβII messenger
ribonucleic acid. Endocrinology 2009, 150, 2087−2097.
(8) Wong, R.; Balachandran, A.; Mao, A.; Dobson, W.; Gray-Owen,
S.; Cochrane, A. Differential effect of CLK SR Kinases on HIV-1 gene
expression: potential novel targets for therapy. Retrovirology 2011, 8,
47.
(9) Ninomiya, K.; Kataoka, N.; Hagiwara, M. Stress-responsive
maturation of Clk1/4 pre-mRNAs promotes phosphorylation of SR
splicing factor. J. Cell Biol. 2011, 195, 27−40.
(10) Shi, J.; Zhang, T.; Zhou, C.; Chohan, M. O.; Gu, X.; Wegiel, J.;
Zhou, J.; Hwang, Y.-W.; Iqbal, K.; Grundke-Iqbal, I.; Gong, C.-X.; Liu,
F. Increased dosage of Dyrk1A alters alternative splicing factor (ASF)-
regulated alternative splicing of tau in Down syndrome. J. Biol. Chem.
2008, 283, 28660−28669.
(27) Nishida, A.; Kataoka, N.; Takeshima, Y.; Yagi, M.; Awano, H.;
Ota, M.; Itoh, K.; Hagiwara, M.; Matsuo, M. Chemical treatment
enhances skipping of a mutated exon in the dystrophin gene. Nat.
Commun. 2011, 2, 308.
(11) Qian, W.; Liang, H.; Shi, J.; Jin, N.; Grundke-Iqbal, I.; Iqbal, K.;
Gong, C.-X.; Liu, F. Regulation of the alternative splicing of tau exon
10 by SC35 and Dyrk1A. Nucleic Acids Res. 2011, 39, 6161−6171.
(12) Yin, X.; Jin, N.; Gu, J.; Shi, J.; Zhou, J.; Gong, C.-X.; Iqbal, K.;
Grundke-Iqbal, I.; Liu, F. Dual-specificity tyrosine phosphorylation-
regulated kinase 1A (Dyrk1A) modulates serine/arginine-rich protein
55 (SRp55)-promoted Tau exon 10 inclusion. J. Biol. Chem. 2012, 287,
30497−30506.
(13) Hartmann, A.; Rujescu, D.; Giannakouros, T.; Nikolakaki, E.;
Goedert, M.; Mandelkow, E. M.; Gao, Q. S.; Andreadis, A.; Stamm, S.
Regulation of alternative splicing of human tau exon 10 by
phosphorylation of splicing factors. Mol. Cell Neurosci 2001, 18, 80−
90.
(14) Jain, P.; Karthikeyan, C.; Moorthy, N. S. H. N.; 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, 539−550.
(15) Zaharieva, E.; Chipman, J. K.; Soller, M. Alternative splicing
interference by xenobiotics. Toxicology 2012, 296, 1−12.
(16) Muraki, M.; Ohkawara, B.; Hosoya, T.; Onogi, H.; Koizumi, J.;
Koizumi, T.; Sumi, K.; Yomoda, J.; 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.
(17) Yomoda, J.; Muraki, M.; Kataoka, N.; Hosoya, T.; Suzuki, M.;
Hagiwara, M.; Kimura, H. Combination of Clk family kinase and
SRp75 modulates alternative splicing of Adenovirus E1A. Genes Cells
2008, 13, 233−244.
(18) Fedorov, O.; Huber, K.; Eisenreich, A.; Filippakopoulos, P.;
King, O.; Bullock, A. N.; Szklarczyk, D.; Jensen, L. J.; Fabbro, D.;
967
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