Structure-based design of novel potent protein kinase CK2 (CK2) inhibitors with phenyl-azole scaffolds

Article abstract of DOI:10.1021/jm2015167

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine protein kinase for hundreds of endogenous substrates. CK2 has been considered to be involved in many diseases, including cancers. Herein we report the discovery of a novel ATP-competitive CK2 inhibitor. Virtual screening of a compound library led to the identification of a hit 2-phenyl-1,3,4-thiadiazole compound. Subsequent structural optimization resulted in the identification of a promising 4-(thiazol-5-yl)benzoic acid derivative.

Full text of DOI:10.1021/jm2015167

Brief Article
pubs.acs.org/jmc
Structure-Based Design of Novel Potent Protein Kinase CK2 (CK2)
Inhibitors with Phenyl-azole Scaffolds
Zengye Hou,† Isao Nakanishi,*,‡ Takayoshi Kinoshita,§ Yoshinori Takei,† Misato Yasue,† Ryosuke Misu,†
Yamato Suzuki,† Shinya Nakamura,‡ Tatsuhide Kure,‡ Hiroaki Ohno,† Katsumi Murata,† Kazuo Kitaura,†
Akira Hirasawa,† Gozoh Tsujimoto,† Shinya Oishi,*,† and Nobutaka Fujii†
†Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
‡Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
§Graduate School of Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Japan
S
* Supporting Information
ABSTRACT: Protein kinase CK2 (CK2) is a ubiquitous serine/threonine protein kinase for hundreds of endogenous substrates.
CK2 has been considered to be involved in many diseases, including cancers. Herein we report the discovery of a novel ATP-
competitive CK2 inhibitor. Virtual screening of a compound library led to the identification of a hit 2-phenyl-1,3,4-thiadiazole
compound. Subsequent structural optimization resulted in the identification of a promising 4-(thiazol-5-yl)benzoic acid derivative.
INTRODUCTION
■
Protein kinase CK2 (CK2) (previously called casein kinase
II) is a ubiquitous, essential, and highly pleiotropic serine/
threonine-selective kinase for >300 protein substrates. Many of
the substrates are implicated in various important cellular func-
tions, including signal transduction and gene expression.1,2 CK2
typically forms tetrameric complexes consisting of two catalytic
α subunits (α or α′) and two regulatory β subunits in various
combinations.3 CK2α′ subtype is exclusively expressed in the
brain and testis, while ubiquitous expression of CK2α subtype
has been reported.4 Being different from most protein kinases
whose functions are activated only in response to specific stimuli,
CK2 is constitutively active. It has been well demonstrated that
CK2 is usually overexpressed in several cancer cells compared
with corresponding normal tissues.5 CK2 regulates many
antiapoptotic signaling cascades to ensure that cancer cells can
survive apoptosis.6 Another mechanism by which CK2 can
Figure 1. Structures of representative CK2 inhibitors.
promote tumor phenotypes is reported to impair the function
of cancer.17,18 ATP-noncompetitive CK2 inhibitors targeting
of tumor suppressor proteins such as promyelocytic leukemia
(PML).7 It has also been reported that a CK2 inhibitor may
the CK2β subunit or CK2α−CK2β interaction have also been
enhance the efficacy of antitumor agents such as melphalan and
imatinib.8,9 Thus, CK2 has a multifunctional role that creates a
favorable cellular environment for tumor progression, making it
a potential target for cancer treatment.
reported, providing opportunities for the development of
allosteric inhibitors.19
Alternatively, two potent CK2 inhibitors (1a,b) containing a
2,6-disubstituted pyrazine framework have been identified.20
Their derivatives were synthesized for structure−activity relation-
ship studies.21 The binding mode of pyrazine-based inhibitors 1a
and 1b to CK2α or CK2α′ was determined by X-ray crystallo-
graphy: the inhibitors were bound to the ATP binding site through
three hydrogen bonds.22,23 In the present work, we report the
structure-based design of CK2 inhibitors with a 5-phenyl-
thiazole scaffold on the basis of the structure of the CK2α−1b23
and CK2α−adenylyl-imidodiphosphate (AMPPNP)24 complexes.
The relatively small ATP binding site of CK2 compared with
those of other protein kinases has facilitated the design of selec-
tive small-molecule ATP-competitive inhibitors.10 Several
classes of ATP-competitive CK2 inhibitors have been reported
(Figure 1).11−18 Representative examples include coumarin
derivatives (including quercetin and apigenin),11,12 emodin,13
4,5,6,7-tetrabromo-1H-benzotriazole (TBB),14 (5-oxo-5,
6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid (IQA),15 and
pyrazolo[1,5-a][1,3,5]triazine derivatives.16 More recently, the
benzonaphthyridine derivative 31 (CX-4945) has been reported
to be a first-in-class, orally bioavailable ATP-competitive CK2
inhibitor and is currently in clinical trials for the treatment
Received: November 9, 2011
Published: February 17, 2012
© 2012 American Chemical Society
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Brief Article
Figure 2. Structures and binding mode of thiadiazole- and thiazole-based CK2 inhibitors. (A) Structures of the 2-amino-1,3,4-thiadiazole-based
inhibitors. (B) Crystal structure of the CK2α−thiadiazole 3b complex. (C) Simulated binding mode of thiazole 10 with CK2α.
Among 55 compounds evaluated for in vitro CK2 inhibitory
activities (3a, 3c−3s, S1−S37, SI), 18 compounds exhibited mod-
erate inhibitory activities (>90% inhibition at 32 μM against
CK2α or CK2α′; see Table 1 and SI). The inhibitory activities
of amide derivatives varied significantly depending on the acyl
group structure. The inhibitory activities of the benzoyl derivatives
relied on the substitution pattern of the benzene ring. Replacement
of the benzene ring by a thiophene (3f) or a furan ring (3g)
retained the potency, whereas replacement by a pyridine ring
(S9, SI) resulted in the loss of activities. Acyl derivatives bearing
a relatively small alkyl group (3a and 3h−k) exhibited moderate
potency. Most carbamate derivatives (3l−s) displayed moderate
potency. Among 18 compounds exhibiting moderate inhib-
itory activities, compound 3e with a 4-methoxybenzoyl group
displayed the lowest IC50 value and was employed for the
optimization process.
We next focused on the five-membered heterocyclic moiety
of 3e. The crystal structure of the CK2α−3b complex revealed
a potential electrostatic repulsion between the thiadiazole nitrogen
at the 4-position and the backbone carbonyl oxygen of Glu114
(Figures 2B and 3A). When simulations of the phenyl-azole
derivatives 29 and 30 with a thiazole or pyrazole core sub-
structure were carried out,26 higher binding affinity toward
CK2α was expected for thiazole 29 and pyrazole 30 compared
to that of thiadiazole 3a (see SI). Replacement with a thiazole
ring could remove unfavorable repulsion and append a weak
but favorable interaction between CH and the backbone
oxygen atom of Glu114 to improve binding affinity and
potency (Figures 2C and 3B). Alternatively, an NH group of
a pyrazole ring in 21 could also form an additional hydrogen
bond with the Glu114 carbonyl oxygen (Figure 3C). The
pyrazole 21 could be rearranged to the tautomer 21′, which may
form disfavored electrostatic repulsions with CK2α. 2-Amino-
oxadiazole 14 and 5-aminothiazole derivative 28 (Scheme 1)
were also designed as reference scaffolds. A potential electrostatic
repulsion between the thiazole nitrogen in 28 and the Glu114
carbonyl group was expected.
RESULTS AND DISCUSSION
■
For the development of new classes of CK2 inhibitors, a structure-
based virtual screening of an approximately three million compound
database was undertaken based on the structure of the CK2α−1b
(PDB 3AT4) and CK2α−AMPPNP (PDB 1JWH) complexes.
Among several candidate compounds selected, thiadiazole 2
exhibited moderate CK2 inhibitory activities with an IC50 of
26.8 μM toward CK2α and 32.2 μM toward CK2α′ (Figure 2A).
During our initial structure-based design, we focused on the similar
binding mode of 1b and 2 with CK2 under molecular modeling in
which the nitro group of 2 was accommodated at the identical
site with the carboxyl group of 1b. Both polar groups are likely
to be essential for the interaction with Lys68 of CK2. As
expected, the analogue 3a, in which the nitro group of 2 was
replaced with a carboxyl group, exhibited equipotent inhibitory
activities against CK2α and CK2α′, respectively [IC50(CK2α) =
29.9 μM; IC50(CK2α′) = 5.3 μM].
To confirm the binding mode of 2-phenyl-1,3,4-thiadiazole-
type compounds with CK2α, we determined the crystal struc-
ture of CK2α complexed with 3b, which is an N-acyl group-
deficient analogue of 3a (Figure 2B). Compound 3b was bound
to the ATP binding pocket, which was surrounded by hydrophobic
amino acids. Several hydrogen bonds were observed between
CK2α and the inhibitor. The carboxyl group formed a salt
bridge with Lys68 and an additional water-mediated hydrogen
bond. The amino group was bound with the carbonyl oxygen of
Val116, while the thiadiazole nitrogen at the 3-position inter-
acted with the backbone NH group of Val116.
On the basis of the docking simulation experiment using the
CK2α−3b complex, a series of aminothiadiazoles 3 bearing various
acyl groups with higher scores for favorable interactions were
designed and synthesized (Scheme 1). Briefly, condensation
of commercially available methyl 4-formylbenzoate 4 with thio-
semicarbazide afforded the corresponding thiosemicarbazone,
which was converted to the aminothiadiazole 5 by FeCl3-mediated
oxidative cyclization followed by treatment with pyridine.25 One-
pot transformation of 5 (including treatment with various acyl
chloride followed by ester hydrolysis) afforded the desired
compounds 3 and S1−S37 (Supporting Information (SI)).
Using the standard synthetic methods for five-membered
heterocycles, the phenyl-azole compounds 10, 14, 21, and 28
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Brief Article
a
Scheme 1. Synthesis of Phenyl-azole CK2 Inhibitors
a
Reagents and conditions: (a) thiosemicarbazide, EtOH, H2O, 70 °C, then FeCl3·6H2O, 75 °C; (b) RCOCl, Et3N, THF, 65 °C (or sonication), then
LiOH·H2O, H2O, rt; (c) KOt-Bu, MeOCH2PPh3Cl, THF, 0 °C to rt; (d) conc. HCl aq, THF, rt; (e) Br2, CH2Cl2, rt; (f) thiourea, EtOH, reflux; (g)
4-methoxybenzoyl chloride, Et3N, THF, rt; (h) LiOH·H2O, H2O, THF, rt; (i) semicarbazide hydrochloride, NaOAc, MeOH, H2O, rt; (j) Br2,
AcOH, 60 °C; (k) 4-methoxybenzoyl chloride, NaH, THF, rt; (l) H2SO4, MeOH, 60 °C; (m) p-TsOH·H2O, NBS, MeCN, 85 °C; (n) KCN, EtOH,
H2O, 0 °C to rt; (o) hydrazine monohydrate, MsOH, EtOH, reflux; (p) glycine, NaHCO3, H2O, rt; (q) 2,4-dimethoxybenzylamine, (i-Pr)2NEt,
HBTU, DMF, CH2Cl2, rt; (r) Lawesson’s reagent, toluene, 110 °C; (s) TFA/CH2Cl2 (1:4), rt.
Table 1. CK2 Inhibitory Activities of Aminothiadiazole
Derivatives
a
a
IC50 [μM]
IC50 [μM]
CK2α CK2α′
3k (E)-but-2-en-2-yl 3.6 2.9
3l 2-methoxyethoxy 6.8 4.3
compd
3a methyl
3c phenyl
R
CK2α CK2α′ compd
R
29.9 5.3
4.4 3.3
6.6 1.7
3.4 1.2
3d 4-fluorophenyl
3m allyloxy
8.1 2.7
5.7 3.0
3e 4-methoxy-
3n n-propoxy
phenyl
3f 2-thienyl
3g 2-furyl
6.4 5.8
5.1 1.8
5.1 2.8
3o isopropoxy
3p n-butoxy
14.1 8.2
7.7 3.1
5.1 5.7
4.5 2.4
6.1 2.1
3h hydroxymethyl
3q isobutoxy
3r n-pentyloxy
3s n-hexyloxy
3i methoxymethyl 6.0 4.3
3j cyclopropyl 3.9 1.6
Figure 3. Two-dimensional view of a plausible binding mode of
thiadiazole 3b (A), thiazole 10 (B), and pyrazole 21 (C) with CK2α.
R = 4-methoxybenzoyl.
a
Inhibition values were determined by the CK2 kinase assay.
compound 14. The 3-aminopyrazole 21 was derived from com-
mercially available 4-acetylbenzoic acid 15. Methyl ester formation
followed by α-bromination with N-bromosuccinimide (NBS) in
the presence of p-TsOH gave α-bromoketone 17.28 After cyana-
tion of 17 with KCN, MsOH-catalyzed condensation of β-keto
nitrile 18 with hydrazine monohydrate yielded aminopyrazole
19.29 The acylation and ester hydrolysis of 19 under standard
conditions yielded the desired compound 21. For the synthesis
of 5-aminothiazole 28, condensation of 4-(chlorocarbonyl)-
benzoate 22 with glycine followed by 2,4-dimethoxybenzyl-
amine coupling afforded N-dimethoxybenzyl-protected amide 24.
Cyclization of 24 in the presence of Lawesson’s reagent,30 and
the subsequent manipulation afforded the compound 28.
The in vitro inhibitory activities of the compounds toward
CK2α and CK2α′ are shown in Table 2. As expected from the
were prepared (Scheme 1). The synthesis of 2-aminothiazole
10 started from methyl 4-formylbenzoate 4. Wittig reaction of 4
with methoxymethyltriphenylphosphonium ylide followed by
acid treatment yielded phenylacetaldehyde 6. Bromination of
6 followed by condensation with the thiourea led to the
2-aminothiazole 8. Acylation of 8 with 4-methoxybenzoyl chloride
followed by ester hydrolysis provided the desired compound
10. For the synthesis of the 2-amino-1,3,4-oxadiazole analogue
14, the reaction of 4 with semicarbazide hydrochloride afforded
semicarbazone 11, which was cyclized to oxadiazole 12 by
Br2-mediated oxidative cyclization.27 Acylation of 12 with
4-methoxybenzoyl chloride under standard conditions using Et3N
resulted in low yields, which was overcome by use of NaH as
the base to yield 13. Ester hydrolysis of 13 afforded the desired
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Table 2. CK2 Inhibitory Activities of 4-Arylbenzoate
Derivatives
Table 3. Inhibitory Effects on Cell Proliferation of CK2
Inhibitors toward A549, HCT116, and MCF-7
a
IC50 [μM]
compd
A549
HCT116
MCF-7
31
1a
3e
10
21
8.2
36
5.2
24
6.5
75
>30
2.6
>30
1.6
>30
2.4
>30
>30
>30
a
IC50 values were derived from the dose−response curves generated
from triplicate data points. Cytotoxic activity against cell lines after
72 h exposure to the compound.
CONCLUSION
■
In conclusion, we undertook a structure-based virtual screening for
novel CK2 inhibitors using X-ray crystal structures of pyrazine-
based CK2 inhibitor−CK2α and AMPPNP−CK2α complexes
which led to a 2-phenyl-1,3,4-thiadiazole-type hit compound 2. The
subsequent structure-based scaffold hopping of five-membered
nitrogen heterocycles identified a highly potent 4-(2-aminothiazol-
5-yl)benzoate-type CK2 inhibitor 10. The improved potency of 10
was rationalized by potential multiple favorable interactions with
the CK2α ATP binding pocket. The thiazole-based inhibitor 10
with a relatively high selectivity toward CK2 exhibited potent
cytotoxicity, indicating that it could be a promising candidate for
cancer treatment. Further studies to improve CK2 inhibitory
activities and kinase selectivity are now in progress.
a
Inhibition values were determined by the CK2 kinase assay.
simulation experiment, 2-aminothiazole 10 exhibited signifi-
cantly potent inhibitory activities toward CK2α and CK2α′,
which was approximately 100-fold more active compared
with the parent thiadiazole 3e. 3-Aminopyrazole 21 also showed
more potent inhibitory activities than 3e, but it was less potent
than thiazole 10.31 Oxadiazole 14 showed less inhibitory activ-
ities, implying a significant contribution from the sulfur atom.
5-Aminothiazole 28, in which the CH and N positions on the
thiazole ring of 10 were interchanged, decreased the potency,
providing additional evidence to support the proposed binding
mode of 10 with CK2α.
The potent 2-aminothiazole 10 was screened against a panel
of 70 kinases to assess its selectivity profile. At 0.30 μM, com-
pound 10 exhibited a >50% inhibitory effect against 11 kinases,
including CK2α and CK2α′ (see SI). Highly potent inhibition
was observed only against four kinases [84% inhibition for
CK2α; >100% inhibition for CK2α′; 82% inhibition for dual-
specificity tyrosinephosphorylation-regulated kinase 1B (DYRK1B);
76% inhibition for FMS-like receptor tyrosine kinase 3 (FLT3) in
the presence of 30 nM compound 10]. Although 2-amino-
5-phenyl-thiazole and -thiadiazole scaffolds have been reported as
cyclin-dependent kinase 6 (CDK6) and AKT1 inhibitors, respec-
tively,32,33 the thiazole 10 exerted significantly less inhi-
bition at 3 μM against both kinases (32% inhibition for
CDK6; 0.8% inhibition for AKT1), indicating that this is
relatively selective for CK2.
Compounds 1a, 3e, 10, and 21 were tested for their inhib-
itory effect on the proliferation of cancer cell lines: lung cancer
cells A549, colorectal cancer cells HCT-116, and breast cancer
cells MCF-7 (Table 3). Compound 31 (CX-4945, Figure 1) has
been shown to be effective against these cell lines.17 Cells were
treated with increasing concentrations of the compounds, and
viabilities were measured by the 3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay. Thiazole 10 was found to be slightly more potent
compared with 1a and 31 toward all the cell lines tested,
whereas no inhibitory effects were observed at 30 μM of
thiadiazole 3e and pyrazole 21. The reason for the apparently
incompatible bioactivities of 3e and 21 needs to be clarified. It is
also possible that compound 10 exerts its cell growth inhibition
by a multikinase inhibition mechanism for CK2 and other
kinases such as (phosphoprotein 70 ribosomal protein S6
kinase) p70S6K.34
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, spectral characterization data, a simula-
tion procedure, and a crystallographic information file. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For S.O.: phone, +81-75-753-4561; fax, +81-75-753-4570;
E-mail, soishi@pharm.kyoto-u.ac.jp. For I.N.: fax, +81-6-6730-
1394; E-mail, isayan@phar.kindai.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Fundamental Studies in Health Sciences
of the National Institute of Biomedical Innovation (NIBIO), and
Grants-in-Aid for Scientific Research and Targeted Protein Research
Program from the MEXT, Japan. Z.H. and Y.S. are supported
by the Kobayashi International Scholarship Foundation, and by
a JSPS Research Fellowship for Young Scientists, respectively.
ABBREVIATIONS USED
■
CK2, protein kinase CK2; PML, promyelocytic leukemia;
TBB, 4,5,6,7-tetrabromo-1H-benzotriazole; IQA, (5-oxo-5,6-
dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid; AMPPNP,
adenylyl-imidodiphosphate; NBS, N-bromosuccinimide; DYRK1B,
dual-specificity tyrosinephosphorylation-regulated kinase 1B;
FLT3, FMS-like receptor tyrosine kinase 3; CDK6, cyclin-
dependent kinase 6; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
p70S6K, phosphoprotein 70 ribosomal protein S6 kinase
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