Synthesis, biological properties and structural study of new halogenated azolo[4,5-b]pyridines as inhibitors of CK2 kinase

Article abstract of DOI:10.1016/j.bioorg.2020.104502

The new halogenated 1H-triazolo[4,5-b]pyridines and 1H-imidazo[4,5-b]pyridines were synthesised as analogues of known CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) and 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi). Their influence on the activity of recombinant human CK2α, CK2α’ and PIM1 kinases was determined. The most active inhibitors were di- and trihalogenated 1H-triazolo[4,5-b]pyridines (4a, 5a and 10a) with IC₅₀ values 2.56, 3.82 and 3.26 μM respectively for CK2α. Furthermore, effect on viability of cancer cell lines MCF-7 (human breast adenocarcinoma) and CCRF-CEM (T lymphoblast leukemia) of all final compounds was evaluated. Finally, three crystal structures of complexes of CK2α^1-335 with inhibitors 4a, 5a and 10a were obtained. In addition, new protocol was used to obtain high-resolution crystal structures of CK2α’^Cys336Ser in complex with four inhibitors (4a, 5a, 5b, 10a).

Full text of DOI:10.1016/j.bioorg.2020.104502

Journal Pre-proofs
Synthesis, biological properties and structural study of new halogenated azo-
lo[4,5-b]pyridines as inhibitors of CK2 kinase
K. Chojnacki, D. Lindenblatt, P. Wińska, M. Wielechowska, C. Toelzer, K.
Niefind, M. Bretner
PII:
S0045-2068(20)31800-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.104502
YBIOO 104502
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Bioorganic Chemistry
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6 November 2020
19 November 2020
Please cite this article as: K. Chojnacki, D. Lindenblatt, P. Wińska, M. Wielechowska, C. Toelzer, K. Niefind, M.
Bretner, Synthesis, biological properties and structural study of new halogenated azolo[4,5-b]pyridines as
inhibitors of CK2 kinase, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104502
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Synthesis, biological properties and structural study of new halogenated azolo[4,5-b]pyridines
as inhibitors of CK2 kinase
1
2
1
1
2,3
2
1
K. Chojnacki , D. Lindenblatt , P. Wińska , M. Wielechowska , C. Toelzer , K. Niefind , M. Bretner
1
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego St. 3, 00-664 Warsaw, Poland
2Department für Chemie, Institut für Biochemie, Universtät zu Köln, Zülpicher Straße 47, D-50674 Köln, Germany
3Present address: Bristol Synthetic Biology Centre BrisSynBio, Biomedical Sciences, School of Biochemistry, University
of Bristol, Bristol, UK
Abstract
The new halogenated 1H-triazolo[4,5-b]pyridines and 1H-imidazo[4,5-b]pyridines were
synthesised as analogues of known CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) and
4
,5,6,7-tetrabromo-1H-benzimidazole (TBBi). Their influence on the activity of recombinant human
CK2α, CK2α’ and PIM1 kinases was determined. The most active inhibitors were di- and trihalogenated
H-triazolo[4,5-b]pyridines (4a, 5a and 10a) with IC values 2.56, 3.82 and 3.26 μM respectively for
1
5
0
CK2α. Furthermore, effect on viability of cancer cell lines MCF-7 (human breast adenocarcinoma) and
CCRF-CEM (T lymphoblast leukemia) of all final compounds was evaluated. Finally, three crystal
structures of complexes of CK2α1-335 with inhibitors 4a, 5a and 10a were obtained. In addition, new
protocol was used to obtain high-resolution crystal structures of CK2α’Cys336Ser in complex with four
inhibitors (4a, 5a, 5b, 10a).
Keywords: Casein Kinase CK2, Protein Kinase PIM1, Structural Study, ATP-Competitive inhibitors
1
. Introduction
Protein kinase CK2 (formerly casein kinase 2) is an ubiquitously expressed and constitutively
active enzyme composed of two catalytic subunits (CK2α and/or CK2α’) and two regulatory subunits
CK2β). CK2 catalyzes the transfer of a phosphate group from adenosine triphosphate (ATP) or
(
guanosine triphosphate (GTP) to serine, threonine or tyrosine residues of target proteins [1].
More than 600 phosphosites are known to be generated by CK2 and almost 400 target proteins
are recognized by CK2 which illustrates high pleiotropy of this enzyme [2]. The activity or localization of
proteins important for cell life processes such as cell cycle, proliferation, apoptosis and angiogenesis is
regulated by CK2 [3-5]. Highly elevated CK2 expression was detected in a number of tumor cells, for
example breast cancer [6], leukemia [7], lung cancer [8] and prostate cancer [9]. Proviral Integration site
of Moloney Virus (PIM1) kinase is another serine/threonine kinase involved in regulation of crucial
cellular processes, for which overexpression in cancer cells like breast, prostate, leukemia was found
[
10]. Downregulation of both kinases by small molecule inhibitors induces apoptosis in cancer cells [11],
making CK2 and PIM1 promising molecular targets to explore new anti-cancer agents.
1

Two of the broadly studied CK2 inhibitors are 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) with
IC 1.3 µM, 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) with IC 0.3 µM and their derivatives. There are
5
0
50
a number of reported modifications of both inhibitors at N1, N2 (for TBBt) and N1, C2 (for TBBi) positions
or even at the benzene ring [12-17]. Many of them improved inhibitory activity of the compounds, the
most successful inhibitors are: 3-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)propan-1-ol (MB 001, IC50
0
.54 μM) [12], 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT, IC 0.14 μM) [15] and 1-
5
0
(carboxymethyl)-2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (K66, IC50 0.50 μM) [16].
However, only few modifications of TBBt lead to compounds with higher or comparable inhibitory
activity: 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol (MB 002, IC50 0.32 μM), 3-(4,5,6,7-
tetrabromo-2H-benzotriazol-2-yl)propan-1-ol (MB 003, IC 0.34 μM) [12] and 4,5,6-tribromo-7-ethyl-1H-
5
0
benzotriazole (IC 0.16 μM) [13] where one of the bromine atoms was displaced with an ethyl group.
5
0
The role of the ethyl group in a more effective binding to the CK2 active site is still unknown (Fig. 1).
OH
Br
Br
Br
Br
COOH
N
H
N
H
N
Br
Br
Br
Br
Br
Br
Br
Br
N
N
N
N
N
N
N
Br
Br
Br
Br
TBBi
MB 001
DMAT
K66
IC50 0.50 M
IC50 1.3 M
IC50 0.54 M
IC50 0.14 M
OH
Br
Br
Br
H
N
H
N
Br
Br
Br
Br
Br
Br
Br
N
N
N
N
N
N
N
N
N
N
Br
OH
Br
Br
Br
Br
TBBt
MB 002
MB 003
4,5,6-tribromo-7-ethyl-
1H-benzotriazole
IC50 0.3 M
IC50 0.32 M
IC50 0.34 M
IC50 0.16 M
Fig. 1 Structures of previously described polybrominated CK2 kinase inhibitors: TBBi, MB 001, DMAT, K66, TBBt, MB 002, MB003
and 4,5,6-tribromo-7-ethyl-1H-benzotriazole.
Previous research studied the role of each bromine atom of TBBt and TBBi in binding to the
active site of CK2. Influence of various mono-, di- and tribrominated benzotriazoles on CK2 activity
showed that bromine atoms at C5 and C6 positions contribute most effectively to CK2 binding [18],
which was proved by thermodynamic studies as well [19]. Overall, research shows that modifications at
positions C4 and C7 of TBBt are very promising and can lead to more effective inhibitors of CK2. In the
case of TBBi similar experiments were not performed, however, inhibitory and thermodynamic
properties of TBBi and 4,5,6-tribromo-7-methyl-1H-benzimidazole suggest that loss of one bromine atom
leads to weakened affinity of benzimidazole derivatives to CK2 [20].
2

Some of the CK2 kinase inhibitors can decrease PIM1 kinase activity at low concentrations as well
(Fig. 2; TDB, 6k and AMR) [21-23]. Those dual inhibitors show how similar pharmacophores and structure
of active sites of both kinases are.
Br
O
N
HOOC
COOH
OH
Br
N
O
NH
O
O
N
N
Br
Br
O
Br
OH
OH
TDB
6k
AMR
CK2 IC50 32 nM
PIM1 IC50 86 nM
CK2 IC50 28 nM
PIM1 IC50 11 nM
CK2 IC50 200 nM
PIM1 IC50 31 nM
Fig. 2. Structures of dual inhibitors of CK2 and PIM1 kinases: TDB (1-(β-D-2'-deoxyribofuranosyl)-4,7-dibromo-5,6-dichloro-1H-
benzimidazole), 6k (8-bromo-2-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}[1]benzofuro[3,2-d]pyrimidin-4(3H)-one) and AMR (2,9-
Dicarboxynaphtho[2,1-b:7,8-b']difuran).
There are reported compounds with pyridine moiety which are inhibitors of the Akt1 kinase in
low nM range, and are cytotoxic against human pancreatic cancer cell line (Mia-Pa-Ca-2) in μM range, for
example (2S)-1-{[5-(3-methyl-1H-indazol-5-yl)pyridin-3-yl]oxy}-3-phenylpropan-2-amine with K (Akt1) 11
i
nM and EC (Mia-Pa-Ca-2) 0.4 μM [24]. Even some bioactive compounds with imidazo[4,5-b]pyridine are
5
0
known which are inhibitors of PAK4 kinase (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-
imidazo[4,5-b]pyridine with IC50 8.7 μM) [25] or Aurora-A kinase, for example 6-Chloro-7-(4-(4-
chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine
Aurora-A) 38 nM [26].
with
IC50
(
The aim of this study was to synthesize and examine properties of new TBBt as well as TBBi
analogues, with pyridine nitrogen atom instead of bromine atom in the C4 position (Fig. 3). This
modification can help to elucidate the role of this bromine atom in binding to the CK2 active site and can
improve the water solubility and/or cell membrane permeability of these inhibitors.
Br
Br
H
H
Br
Br
N
N
7
7
6
5
1
6
5
1
2
2
N
3
N
3
N
4
N
4
N
Br
Br
5,6,7-tribromo-1H-triazolo[4,5-b]pyridine
5,6,7-tribromo-1H-imidazo[4,5-b]pyridine
Fig. 3. Structures of 5,6,7-tribromo-1H-]triazolo[4,5-b]pyridine and 5,6,7-tribromo-1H-imidazo[4,5-b]pyridine.
3

2. Results and discussion
2.1 Synthesis
Diaminobromopyridines (1, 7) were synthesized by reduction of appropriate aminobromonitro-
pyridine with SnCl in EtOH/H O under argon atmosphere at reflux. Reaction of 5-bromo-3-nitro-2-
2
2
aminopyridine with SnCl in HCl resulted in a product of the nitro group reduction (1) in mixture with
2
aq
product of consequent chlorination in position 6 (9) (Scheme 1). According to literature, 9 can be
obtained from 5-bromo-3-nitro-2-aminopyridine in similar reaction using granulated tin instead of SnCl2
with 21% yield. [27].
3
-Amino-6-bromo-2-nitropyridine (6) was synthesized according to the procedure described
previously [28] by bromination with Br in acetic acid/sodium acetate at room temperature (56% yield)
2
(Scheme 1). Interestingly, when potassium acetate was used instead of sodium acetate, isomeric 3-
amino-4-bromo-2-nitropyridine was obtained according to the literature [29].
Di- and tribromodiaminopyridines (3a, 3b) were obtained by bromination of 1 with N-
bromosuccinimide (NBS) in HBr . 3a was synthesized by portionwise addition of NBS (1.05 eq.) at 0°C.
aq
After overnight reaction at room temperature, the product can be isolated as hydrobromic salt by
filtration. Neutralization with aqueous K CO , extraction and purification by column chromatography on
2
3
silica gel yields 3a (56%). 3b was synthesized in a similar reaction with NBS (2.05 eq.) at 80°C (yield 62%).
In general, bromination of aminopyridines for example 2-aminopyridine can be performed with NBS in
organic solvent at RT [30] (Scheme 1). In our case using acetonitrile or methanol as solvent was
unsuccessful, probably because of the presence of 2,3-diamino group. Instead of protecting the amino
groups in a usual way like with a tert-butyloxycarbonyl (Boc) group, we successfully applied bromination
in 48% HBr , which protects amino groups easily by protonation.
aq
All obtained diaminopyridines were cyclized with NaNO /H SO to the 1H-triazolo[4,5-b]pyridine
2
2
4
system (2a, 4a, 5a, 8a, 10a) with yields ranging from 36%-91%; and with HCOOH to the 1H-imidazo[4,5-
b]pyridine system (2b, 4b, 5b, 8b, 10b) with yields ranging from 82% to 98% (Scheme 1).
4

Scheme 1. Synthesis of the 1H-triazolo[4,5-b]pyridines and 1H-imidazo[4,5-b]pyridines: a) SnCl
reflux, 1h; b) NaNO , H O/H SO , 0°C-RT, overnight; c) HCOOH, reflux, overnight; d) NBS, HBr, 0°C-RT, overnight; e)
NBS, HBr, 80°C, overnight; f) Br , AcONa, AcOH, RT, 6 days; g) SnCl , HCl, 60°C, 1h.
2 2
, EtOH/H O, Ar,
2
2
2
4
2
2
2.2. Inhibition data
The influence of all final compounds on the human CK2 catalytic subunit (CK2α), isoform of the
human CK2 catalytic subunit (CK2α’) and PIM1 activity was evaluated using radiometric assay, and
results are presented in Table 1. The synthetic peptide RRRADDSDDDDD was used as the substrate of
hCK2 and peptide ARKRRRHPSGPPTA as the substrate of PIM1. Screening of potential inhibitory activity
of obtained derivatives was performed in the presence of 10 μM of the respective compound and
residual activity of hCK2α and PIM1 was normalized to the uninhibited protein activity. Furthermore, IC50
values were determined for the most active compounds at eight concentrations. The experimental data
were fitted to sigmoidal dose-response (variable slope) Y = Bottom + (Top-Bottom) / (1+10^((LogIC -
5
0
X)*HillSlope) equation in GraphPad Prism.
5

Table 1. IC50 (μM) and residual activity (%) of CK2α, CK2α’ and PIM1 kinases in the presence of
1
H-triazolo[4,5-b]pyridine and 1H-imidazo[4,5-b]pyridine derivatives (10 μM).
CK2α
CK2α’
PIM1
Compound
residual activity (%) IC / μM residual activity (%) IC / μM residual activity (%)
5
0
50
2
2
4
4
5
5
8
8
a
b
a
b
a
b
a
b
65±14
110±12
19±3
93±8
19±2
53±4
61±4
77±5
17±2
24.8±1.6
74±3
95±7
18±0.5
83±0.3
16±2
43±2
66±9
98±5
22±6
95±10
-
-
-
103±4
98±3
97±3
93±1
40±4
54±4
86±8
92±3
88±5
-
2.56±0.20
2.36±0.33
-
-
3.82±0.52
18.1±4.9
1.98±0.08
-
-
-
-
-
1
1
0a
0b
3.26±0.01
-
0.35±0.03
1.28±0.05
3.38±0.11
106±7
1.2±0.1
4.4±0.5
-
*
**
97±2
6.0±0.2
5.3±0.7
TBBt
TBBi
7±0.5
*
K 0.11[31], ** K 0.40 [31].
i
i
Biochemical in vitro assays revealed that in general analogues containing imidazole ring are not
active against CK2α or PIM1 kinases. Only derivative 5b with three bromine atoms shows moderate
inhibitory potency at 10 μM, with 53% and 54% residual activity of CK2α or PIM1, respectively. On the
other hand, analogues containing triazole ring show better inhibitory properties. Compound 5a, also
containing three bromine atoms, as well as those with two halogens in positions 5 and 6 (4a, 10a) show
good inhibitory activity against CK2α (IC 3.82; 2.56 and 3.26 μM respectively) in the same range as
5
0
TBBi. No major differences between inhibitory potency of all tested compounds against CK2α and CK2α’
can be found. Interestingly, there are some differences in selectivity of those compounds. 5a inhibits
PIM1 kinase as well (40% residual activity) while 4a and 10a are inactive against PIM1 (97% and 88%
residual activity).
The viability of two cancer cell lines: MCF-7 (human breast adenocarcinoma) and CCRF-CEM (T
lymphoblast leukemia) in presence of newly synthesised compounds was determined using MTT assay.
The cytotoxicity was determined after 72h treatment at 50 μM concentration of each compound (table
2
). Analysis of the results showed that in general these compounds have no anticancer properties, in
almost every case cell viability was higher than 80%. Only compound 5a showed weak cytotoxic
properties on CCRF-CEM cell line (74% viability). This fact may be explained by worse inhibition
selectivity of 5a. It is the only compound from the whole series which inhibits both CK2α and PIM1
kinase with a residual activity under 50%. On the other hand, TBBt, which is the most similar to 5a, has
weak cytotoxic properties as compared to TBBi which was shown previously on HeLa cells. This may be
explained by better selectivity over TBBi and/or greater acidity of triazole than imidazole ring which
makes it occur mostly in ionized form at physiological pH. However, the reasons are still not clear [31].
Additionally, LogP and solubility in water at 7.4 pH was estimated. Almost all new compounds (instead of
6

5
b) exhibit better solubility. However, LogPs of all new compounds are much lower, compared to TBBi
(table 2), what may result in worse intercellular penetration and worse cytotoxicity.
Table 2. The influence of new 1H-triazolo[4,5-b]pyridine and 1H-imidazo[4,5-b]pyridine derivatives on the viability
of MCF-7 and CCRF-CEM cell lines.
MCF-7
cell viability % ± SD
CCRF-CEM
cell viability % ± SD
50 μM
Solubilitya
Compound
cLogPa
mM
5
0 μM
2
2
4
4
5
5
8
8
a
b
a
b
a
b
a
b
93.8 ± 8.0
90.0 ± 8.0
87.3 ± 6.7
89.6 ± 6.3
92.0 ± 5.2
87.1 ± 5.0
92.3 ± 5.5
94.2 ± 5.7
85.6 ± 3.0
86.9 ± 6.6
114.1 ± 5.3
28.1 ± 0.9
113.9 ± 10.1
94.7 ± 11.3
89.0 ± 7.4
97.3 ± 7.1
74.0 ± 9.4
82.2 ± 10.6
82.0 ± 11.6
92.8 ± 7.6
84.5 ± 8.2
95.9 ± 10.4
116.5 ± 5.5
0
24.22
1.87
0.86
0.04
0.25
<0.03
8.14
0.50
0.94
0.04
<0.02
<0.02
1.18
1.14
2.15
2.11
2.92
2.88
1.38
1.34
2.00
1.99
4.38
4.33
1
1
0a
0b
TBBt
TBBi
a
Calculator plugins were used for solubility in water at 7.4 pH and cLogP. ChemAxon http://chemicalize.com.
ND - not determined
2.3 Crystal structures
We were then interested in the exact enzyme binding modes of 4a, 10a and 5a – the three best
triazolopyridine-type CK2α inhibitors (Table 1) – and of 5b – the only imidazopyridine-type compound of
this series with CK2α inhibitory potency (Table 1) – and co-crystallized them with CK2α1-335, a C-terminal-
ly truncated version of human CK2α [32]. Co-crystals and x-ray diffraction data sets were obtained for all
four complexes (Table 3). They resulted in medium-resolution complex structures for 4a, 10a and 5a (Fig.
4
a-c). In the case of 5b, residual electron density originating most probably from the bound inhibitor was
visible at the ATP-site (Fig. 4d), but it was too weak for an unambiguous interpretation by 5b [so that we
excluded the CK2α1-335/5b complex from structure refinement and data bank deposition (Table 3)]. This
observation coincides with the significantly reduced CK2α inhibition potency of 5b compared to 4a, 10a
and 5a in combination with a lower solubility in water (Table 2).
This result was only partly satisfactory and inspired us to solve high-resolution structures of the
four inhibitors in complex with CK2α’Cys336Ser, a single point mutant of a paralogous isoform of human
CK2α, according to a recently described procedure [33, 34]. CK2α and CK2α’, the two paralogs of human
CK2 catalytic subunit, have very similar primary structures in the canonical kinase domain (sequence
identity: 86%), whereas their C-terminal segments are completely unrelated. Especially, a number of
hydrophobic residues with bulky side chains that coat the binding cleft for ATP (or GTP [35]) and
ATP/GTP-competitive inhibitors and confer inhibitory selectivity compared to other eukaryotic protein
kinases [36] are absolutely conserved [33]. The only sequence differences between CK2α and CK2α’ in
7

the ATP site region refer to His115 (Tyr116 in CK2α’) and Val116 (Ile117 in CK2α’). These residues are
part of the inter-domain hinge that participates in binding of ATP site ligands anyway via its backbone
peptide groups rather than its side chains.
In essence, CK2α’ is a good working model to study the structural basis of ATP-competitive CK2
inhibitor binding, in particular since the above-mentioned method can lead to ab initio solved structures
of atomic resolution [33, 34]. Its success involves extensive soaking of CK2α’Cys336Ser crystals with the new
ligand and depends on the replacement of the CK2 inhibitor MB002 [3-(4,5,6,7-tetrabromo-1H-
benzotriazol-1-yl)propan-1-ol] [37], which is required to facilitate CK2α’Cys336Ser crystallization, from the
ATP site. The procedure is rather new [33] and it was interesting to probe its power with inhibitors of
relatively low affinity (Table 1) such as 4a, 10a, 5a and in particular 5b. Figs. 4E-H documents that it
worked in all four cases; even 5b which was not clearly visible in complex with CK2α1-335 (Fig. 4D) could
be unequivocally identified at the ATP site of CK2α’Cys336Ser (Fig. 4H). In all four complexes, the bromo
substituent positions of the inhibitors were clearly indicated by strong anomalous difference density
peaks (red density grids in Figs. 4E-H). These peaks are normally approximately spherical; only for 5a,
one of them is an ellipsoid which coincides with the fact that this inhibitor adopts two alternative
orientations – related by a 180° rotation around the main axis of inertia of the double ring system – at
the ATP site of CK2α’Cys336Ser (Fig. 4E). For the inhibitor 4a, it was particularly valuable, that the highly
resolved CK2α’Cys336Ser structure and the anomalous difference density allowed an unambiguous
distinction between the bromo and the chloro substituent at the pyridine ring (Fig. 4G). In summary, the
crystallographic study with CK2α’Cys336Ser largely confirmed the inhibitor binding modes determined with
CK2α1-335 and could clarify some detailed issues that had remained open.
The abovementioned bulky side chains that distinguish CK2α and CK2α’ at the ATP site from
many homologous protein kinases [36] sandwich the purine base of the co-substrate ATP or GTP and
create a narrow binding cleft compatible with mainly planar ATP-competitive inhibitors [38]. A special
feature of this cleft is a significant “freedom in 2D-space” [38]: it means that as long as the planar
hydrophobic parts of the inhibitors are well accommodated, their exact orientation within the plane is
subtly dependent on hydrogen bonds, halogen bonds and ionic interactions between peripheral
substituents and the protein matrix or conserved water molecules [39].
This principle is well perceptible from the pyridine derivatives of the study presented here: the
orientation of 5a (Fig. 4A/E), for example, is clearly determined by halogen bonds from the three bromo
substituents to the hinge backbone and to the phenyl ring of the gatekeeper residue Phe113/114. As
soon as one of these bromines is missing – which is the only difference between 5a and 10a – the
orientation of the triazolo pyridine double ring system changes largely: now (in the case of 10a), only one
halogen bond is left (Fig. 4B/F), but this loss of two halogen bonds is compensated by an aromatic
stacking interaction between the pyridine six ring and the phenyl group of Phe113/114 and by a
favourable ionic interaction between the deprotonated (and thus negatively charged) triazolo moiety
and the positive side chain of Lys68/69 (Fig. 4B/F). The placement of negatively charged groups within
ATP site ligands in this region is a well-known feature [40], which was observed for the benchmark CK2
inhibitor CX-4945 [41] in all of its CK2α/CK2α’ crystal structures as well [33, 42, 43]: Fig. 4B/F illustrates
8

that the carboxylate group of CX-4945 spatially coincides with the triazolo group of 10a in the vicinity of
Lys68/69.
In general, bromine forms stronger halogen bonds than chlorine [44]. Nevertheless, since the
bromo substituent at C-atom 5 of 10a is not required for halogen bonding (Fig. 4B/F), its change to a
chlorine atom – which is the only difference between 10a and 4a – should not affect the inhibitory
efficacy, whereas it should improve the solubility in aqueous solutions. In fact, the inhibitory data for 10a
and 4a (Table 1) and their binding modes (Fig. 4B/F and Fig. 4C/G) are nearly identical (Table 1) while the
solubility of 4a is slightly enhanced (Table 2).
Fig. 4. Binding of the CK2 inhibitors 5a, 10a, 4a and 5b to the ATP sites of CK2α (A-D) and CK2α’ (E-H).
2
Fo-Fc-electron density maps covering the inhibitor (blue) and the hinge region plus a conserved lysine residue (Lys68 in CK2α
and Lys69 in CK2α’) (green) are drawn with a cutoff level of 1.0 σ (A-D) or 1.5 σ (E-H). In case of the CK2α’ structures (E-H), an
anomalous difference density map is visible with a cutoff level of 10.0 σ (red). To facilitate orientation and comparison, the
benchmark CK2 inhibitor CX-4945 is drawn from PDB file 3NGA [43] after superimposition of the structures. Of note, the residue
numbering of CK2α’ is shifted by +1 compared to CK2α.
9

Table 3. X-ray diffraction data and refinement statistics of CK2α1-335/inhibitor complexes
Inhibitor
4a
10a
5a
5b
PDB code
7A4B
7A49
7A4C
[no deposition]
X-ray diffraction data collection and quality
Wavelength [Å]
Temperature [K]
Synchrotron (beamline)
Space group
0.97625
100
ESRF (ID29)
0.97625
100
ESRF (ID29)
0.97625
100
ESRF (ID29)
0.87313
100
ESRF (ID23-2)
P4
3
P4 2
1
2
3
P4 2
1
2
3
P4 2
1
2
3 1
2 2
a, b, c [Å] 127.329, 127.329, 124.884 127.564, 127.564, 124.599 127.681, 127.681, 126.296
127.845, 127.845, 125.485
90.0, 90.0, 90.0
2
Unit cell
α, β, γ [°] 90.0, 90.0, 90.0
90.0, 90.0, 90.0
2
90.0, 90.0, 90.0
2
Protomers per asym. unit
Resolution [Å]
2
90.035 – 2.060
(2.260 – 2.060)
17.8 (210.5)
0.997 (0.483)
14.8 (1.6)
89.135 – 2.028
90.284 – 2.502
90.400 – 2.216
1
1
1
1
(highest res. shell)
(2.136 – 2.028)
(2.725 – 2.502)
(2.395 – 2.216)
1
1
1
1
R
sym [%]
7.7 (182.7)
17.3 (100.7)
24.6 (261.1)
1
1
1
1
CC1/2
1.000 (0.563)
17.9 (1.3)
0.996 (0.617)
9.2 (1.4)
0.998 (0.569)
9.7 (1.5)
1
1
1
1
Signal-to-noise ratio (I/σ
I
)
1
1
1
1
No. of unique reflections
Completeness (spherical) [%] 73.3 (15.3)
Completeness (ellipsoidal) [%] 95.3 (68.7)
46838 (2341)
60321 (2980)
28550 (1428)
42532 (2126)
1
1
1
1
90.0 (31.7)
96.5 (59.5)
78.0 (17.5)
81.5 (19.9)
95.5 (63.5)
13.3 (13.8)
1
1
1
1
1
95.3 (69.4)
1
1
1
Multiplicity
Wilson B-factor [Å ]
25.7 (18.1)
33.21
13.1 (12.7)
7.5 (4.8)
2
49.51
43.98
45.81
Structure Refinement
No. of reflections for Rwork/Rfree 45417/1392
58980/1203
18.90/21.59
5925
5634
72
219
56.21
56.19
69.09
27387/1139
20.62/23.23
5892
5645
72
175
45.89
45.97
53.68
---
---
---
---
---
---
---
---
---
---
R
work/Rfree [%]
19.11/22.26
6046
5627
Number of non-H-atoms
Protein
Ligand/ion
79
Water
Average B-factor [Å ]
Protein
Ligand/ion
340
2
39.63
39.38
51.65
40.91
water
52.41
40.11
RMS deviations
Bond lengths [Å]
Bond angles [°]
Ramachandran plot
Favoured (%)
Allowed (%)
Outliers (%)
0.001
0.40
0.002
0.50
0.002
0.47
---
---
97.13
2.87
0.00
96.99
3.01
0.00
97.74
2.26
0.00
---
---
---
1The data in brackets refer to the highest resolution shell according to autoPROC [45].
1
0

Table 4. X-ray diffraction data and refinement statistics of CK2α’Cys336Ser/inhibitor complexes
Inhibitor
4a
10a
5a
5b
PDB code
7A1B
7A1Z
7A22
7A2H
X-ray diffraction data collection and quality
Wavelength [Å]
Temperature [K]
Synchrotron (beamline)
Space group
0.87313
100
ESRF (ID23-2)
P1
0.87313
100
ESRF (ID23-2)
P1
0.87313
100
ESRF (ID23-2)
0.87313
100
ESRF (ID23-2)
P1
P1
a, b, c [Å] 46.256, 47.593, 50.550 46.475, 47.613, 50.473
α, β, γ [°] 113.229, 89.996, 91.127 66.923, 89.637, 89.024
46.501, 47.696, 50.571
113.147, 90.460, 90.270
1
46.825, 47.604, 50.485
67.146, 89.519, 89.291
1
Unit cell
Protomers per asym. unit
1
1
4
6.434 – 1.024
Resolution [Å]
46.245 – 1.287
(1.412 – 1.287)
46.498 – 1.008
(1.106 – 1.008)
46.822 – 1.010
(1.090 – 1.010)
1
1
1
(highest res. shell)
1
(1.086 – 1.024)
1
7.1 (99.1)1
6.4 (90.9)1
R
sym [%]
7.8 (82.9)1
0.997 (0.519)
8.3 (1.4)
70878 (3544)
8.8 (138.0)
0.999 (0.500)
12.0 (1.6)
155664 (7786)
77.7 (24.2)
87.2 (17.8)
9.0 (5.3)
10.52
1
1
1
1
CC1/2
0.998 (0.555)
0.999 (0.650)
1
1
1
1
Signal-to-noise ratio (I/σ
I
)
11.7 (1.5)
16.2 (1.5)
1
1
1
1
No. of unique reflections
Completeness (spherical) [%] 70.6 (14.6)
Completeness (ellipsoidal) [%] 87.1 (45.0)
Multiplicity
Wilson B-factor [Å ]
149108 (7455)
159561 (7978)
1
1
1
1
70.8 (14.6)
75.8 (18.5)
1
1
1
1
88.7 (42.8)
85.2 (9.4)
9.1 (5.7)
1
1
1
1
3.4 (3.5)
13.30
6.4 (4.6)
2
11.40
10.81
Refinement and structure quality
No. of reflections for Rwork/Rfree 70550/1411
153915/1530
13.48/14.67
3264
147607/1495
13.79/16.62
3240
157927/1615
14.26/14.95
3304
R
work/Rfree [%]
15.73/19.29
3257
Number of non-H-atoms
Protein
2913
2861
2843
2847
Ligand/ion
38
36
76
24
Water
Average B-factor [Å ]
Protein
Ligand/ion
306
367
17.21
15.28
20.45
321
433
17.38
14.86
20.78
2
18.92
17.47
25.17
31.93
17.17
15.47
22.12
31.10
water
31.92
33.76
RMS deviations
Bond lengths [Å]
Bond angles [°]
Ramachandran plot
Favoured (%)
Allowed (%)
0.007
0.89
0.012
1.35
0.012
1.35
0.008
1.04
97.54
2.46
0.00
97.24
2.76
0.00
97.55
2.45
0.00
97.52
2.17
0.00
Outliers (%)
1
The data in brackets refer to the highest resolution shell according to autoPROC [45].
1
1

2.4 Docking studies to PIM1 kinase
It is known that PIM1 kinase is a common off target of CK2 inhibitors. In our case small
differences in the structure obstruct the inhibitory effect on PIM1 kinase. Moreover, two bromine atoms
in positions 5 and 6 in TBBt are the most essential for binding into CK2α active site. IC values for 5,6-
5
0
dibromobenzotriazole and 4,5,6,7-tetrabromobenzotriazole are in the same range (0.56 μM and 0.27
μM respectively) [18]. This fact may explain why two halogens in our compounds are enough to inhibit
CK2α. The analysis of the CK2 active site showed that it is smaller in size than in other kinases due to the
presence of bulky side chains (the most important are: Val66, Ile174 and Met163) [36]. According to this
fact we can hypothesize that compounds 4a and 10a with two halogens are too small to fill the PIM1
active site while these are big enough to interact properly with the CK2 active site. In order to investigate
this hypothesis, a molecular docking protocol was applied using the non-commercial AutoDock Vina
software [46]. The crystal structure of PIM1 kinase was taken from Protein Data Bank (PDB) with code
2
O64 [47] and used as receptor. Compounds 4a and 5a were used as ligands.
Fig. 5. Binding mode of 4a (A) and 5a (B) to PIM1 kinase (PDB: 2O64) [47] with close contacts to residues in active site.
Fig. 6. Binding mode of 4a (A) and 5a (B) to PIM1 kinase (PDB: 2O64) with surface view.
Both compounds were docked into the PIM1 active site, showing close polar contacts between
nitrogen atoms from triazole rings and Asp186 and Lys67. However, there are some differences in non-
polar interactions. Compound 4a shows close contact between Val52 and a bromine atom connected to
the C7 atom (Fig. 5A), while compound 5a containing three bromine atoms reveals additional
1
2

interactions between bromine atom connected to the C5 atom, Leu174 and Ala65 (Fig. 5B). The surface
view (Fig. 6) clearly shows the big hydrophobic pocket of the PIM1 kinase active site. Compound 5a fill
this pocket better (Fig. 6B) than compound 4a where additional empty space is visible on the top of the
pocket (Fig 6A). Those facts may explain why compound 4a as well as 10a are more specific and inhibit
CK2 but not PIM1 kinase.
3. Conclusions
In summary, we described the synthesis of new halogenated 1H-triazolo[4,5-b]pyridines and 1H-
imidazo[4,5-b]pyridines as analogues of known CK2 and PIM1 inhibitors: 4,5,6,7-tetrabromo-1H-
benzotriazole and 4,5,6,7-tetrabromo-1H-benzimidazole. Compounds with triazole ring containing two
halogen atoms (4a and 10a) inhibit CK2α activity with IC 2.56 and 3.26 μM respectively and compound
5
0
with three bromine atoms (5a) with IC50 3.82 μM. Compounds with imidazole ring showed weaker
inhibitory potency and one containing three bromine atoms (5b) inhibit CK2α activity with IC 18.1 μM.
5
0
This result demonstrates the importance of all four bromine atoms in TBBi for binding to the CK2 active
site. No major differences in activity of synthesized compounds against CK2α and CK2α’ were found.
Some differences can be noticed in inhibition of PIM1 kinase. Compounds with three bromine atoms (5a
and 5b) showed moderate inhibitory activity against PIM1 (40% and 54% residual activity respectively),
while compounds with two halogen atoms (4a and 10a) do not inhibit activity of PIM1. This observation
can be related to size of molecules and active sites architectures of the different kinases. Additionally,
we validate the extension of a recently established protocol for high-resolution structure determination
of CK2a’ in respect of low affinity ligands. The drastic improvement in resolution paves the way for
deeper and more reliable ligand binding interpretation. In the future, new protocol can be used to obtain
high-resolution crystal structures of various enzyme-inhibitor complexes.
4
. Materials and methods
.1 Chemistry
4
4
.1.1 General
Commercially available reagents were used as received without additional purification. Melting
points were determined with an MPA100 Optimelt SRS apparatus and are uncorrected. NMR spectra
were recorded using a Varian NMR System 500 MHz spectrometer. Chemical shifts (δ) are reported in
1
13
ppm related to deuterated dimethyl sulfoxide (DMSO-d , δ 2.48 for H and δ 39.51 for C) as internal
6
standard; splitting patterns have been designated as follows: s = singlet; br. s = broad singlet; d =
doublet. Thin layer chromatography (TLC) was carried out on aluminum plates with silica gel Kieselgel 60
F254 (Merck) (0.2 mm thickness film) and detection of compounds was performed with UV light at 254
nm. Silica gel with grain size 40–63 μm was used for column chromatography. Gas chromatography
analyses (GC) were performed with an instrument equipped with a flame ionization detector (FID) and
HP-50+ semipolar column (30 m, 50% phenyl-50% methylpolysiloxane); helium (2 ml/min) was used as a
carrier gas. Fourier transform mass spectrometry (FTMS) was carried out on Q Exactive Hybrid
Quadrupole-Orbitrap Mass Spectrometer, ESI (electrospray) with spray voltage 4.00 kV at IBB PAS
1
3

Warsaw. The most intensive signals are reported. HPLC analyses were performed on Shimadzu Nexera-
i (LC-2040C 3D) equipped with a photodiode array detector (PAD) using Chiralcel OD-H (4.6 mm × 250
mm, coated on 5 µm silica gel grain size, from Daicel Chemical Ind., Ltd.) column coupled with a
dedicated pre-column (4 mm × 10 mm, 5 µm, from Daicel Chemical Ind., Ltd.) using mixture of
hexane/propan-2-ole (80:20, v:v) as a mobile phase with flow rate 0.7 ml/min. HPLC analyses were
executed in an isocratic and isothermal (30°C) manner.
4
.1.2 General procedure for reduction of nitro group (1, 7)
A solution of appropriate nitroaminopyridine (1.41 mmol) and SnCl (3.5 eq.) in ethanol (3 ml)
2
and H O (0.5 ml) was refluxed for 1 h under Ar. The solution of NaOH (1.2 g) in H O (30 ml) was added
2
2
and extracted with ethyl acetate (3 x 25 ml). The combined organic layers were dried over MgSO4,
filtered, and the solvent was removed under reduced pressure to afford diaminopyridine, which was
used directly into next steps.
4
.1.3. General procedure for cyclization of diamines to 1H-triazolo[4,5-b]pyridines (2a, 4a, 5a, 8a, 10a)
H O (10 ml) and conc. H SO (3 ml) were added to the appropriate diamine (1.26 mmol; 1.0 eq.)
2
2
4
and warmed to afford a clear solution. The solution was cooled in ice/water bath and precipitate was
observed. The solution of NaNO (102 mg; 1.48 mmol; 1.17 eq.) in H O (3 ml) was added and the reaction
2
2
mixture was stirred at RT overnight. Precipitate was filtered and washed with water. Crude product was
purified by column chromatography or crystallization.
4
.1.3.1 6-Bromo-1H-triazolo[4,5-b]pyridine (2a)
Column chromatography was performed using chloroform/methanol (0-3% CH OH). Yield: 102
3
+
+
1
mg (36%). Mp 201-205°C (docomp.); MS [M+H] m/z calcd. for C H BrN 198.96139, found 198.96132. H
5
4
4
1
3
NMR (500 MHz, DMSO-d ) δ ppm: 8.75 (d, 1H, J = 2.0 Hz); 8.77 (d, 1H, J = 2.0 Hz). C NMR (125 MHz,
6
DMSO-d ) δ ppm: 117.03, 126.90, 131.75 (br.), 150.40, 190.92 (br.); HPLC: t = 9.999 min., 99.86% (λ =
6
R
2
06 nm); 99.97% (λ = 298 nm).
4
.1.3.2 5,6-Dibromo-1H-triazolo[4,5-b]pyridine (4a)
Column chromatography was performed using chloroform/methanol (0-5% CH OH). Yield: 111
3
+
+
1
mg (70%). Mp 179-181°C (decomp.); MS [M+H] m/z calcd. for C H Br N 278.8699, found 278.8586. H
5
3
2
4
1
3
NMR (500 MHz, DMSO-d ) δ ppm: 8.92 (s). C NMR (125 MHz, DMSO-d +1% H O) δ ppm: 119.29, 129.62
6
6
2
(
br.), 130.64 (br.), 141.70, 151.05 (br.); HPLC: t = 16.372 min., 99.50% (λ = 206 nm), >99.9% (λ = 305
R
nm).
.1.3.3 5,6,7-Tribromo-1H-triazolo[4,5-b]pyridine (5a)
Column chromatography was performed using chloroform/methanol (0-5% CH OH). Yield: 344
4
3
-
+
1
mg (91%). Mp 184-186°C (decomp.); MS [M-H] m/z calcd. for C Br N 354.7658, found 354.7187. H
5
3
4
1
3
NMR (500 MHz, DMSO-d ) δ ppm: none. C NMR (125 MHz, DMSO-d +1% H O) δ ppm: 122.04, 125.16
6
6
2
1
4

(
br.), 133.72 (br.), 142.00, 148.87 (br.); HPLC: t = 28.560 min., 99.87% (λ = 210 nm), 99.46% (λ = 305
R
nm).
.1.3.4 5-Bromo-1H-triazolo[4,5-b]pyridine (8a)
Column chromatography was performed using chloroform/methanol (0-5% CH OH). Yield: 197
4
3
+
+
1
mg (78%). Mp 181-184°C (decomp.); MS [M+H] m/z calcd. for C H BrN 200.9594, found 200.9645. H
5
4
4
1
3
NMR (500 MHz, DMSO-d ) δ ppm: 7.66 (d, 1H J = 8.5, Hz), 8.39 (d, 1H, J = 8.5 Hz). C NMR (125 MHz,
6
DMSO-d +1% H O) δ ppm: 125.76, 127.63 (br.), 130.03 (br.), 140.63, 152.52 (br.); HPLC: t = 11.509 min.,
6
2
R
9
9.58% (λ = 206 nm), 99.97% (λ = 291 nm).
4
.1.3.5 6-Bromo-5-chloro-1H-triazolo[4,5-b]pyridine (10a)
Product was purified by triple recrystallization from water/methanol 3/1 v/v. Yield: 69 mg (30%
+
+
1
from 9). Mp 162-163°C (decomp.); MS [M+H] m/z calcd. for C H BrClN 234.9204, found 234.9321. H
5
3
4
1
3
NMR (500 MHz, DMSO-d ) δ ppm: 9.00 (s). C NMR (125 MHz, DMSO-d ) δ ppm: 115.83, 130.57, 147.86;
6
6
HPLC: t = 13.984 min., 99.81% (λ = 206 nm), >99.9% (λ = 304 nm).
R
4
.1.4 General procedure for cyclization of diamines to 1H-imidazo[4,5-b]pyridines (2b, 4b, 5b, 8b, 10b)
The solution of appropriate diamine (1.22 mmol; 1.0 eq.) in HCOOH (2.5 ml) was refluxed
overnight. The formic acid was removed under reduced pressure and crude product was purified by
column chromatography or crystallization.
4
.1.4.1 6-Bromo-1H-imidazo[4,5-b]pyridine (2b)
Column chromatography was performed using chloroform/methanol (0-5% CH OH). Yield: 214
3
+
+
1
mg (89%). Mp 230-233°C; MS [M+H] m/z calcd. for C H BrN 197.9662, found 197.9498. H NMR (500
6
5
3
MHz, DMSO-d ) δ ppm: 8.27 (br. s, 1H), 8.06 and 8.410 (1H, tautomers or H-D exchange), 8.47 (s, 1H),
6
1
3
1
1
2.91 and 13.27 (1H, tautomers). C NMR (125 MHz, DMSO-d +10% 5M HCl ) δ ppm: 116.54, 125.54,
6
aq
26.64, 143.97, 144.04, 148.31; HPLC: t = 8.692 min., >99.9% (λ = 206 nm), >99.9% (λ = 294 nm).
R
4
.1.4.2 5,6-Dibromo-1H-imidazo[4,5-b]pyridine (4b)
Product was purified by recrystallization from water/isopropanol 5/4 v/v. Yield: 110 mg (82%).
+
+
1
Mp 267-268°C; MS [M+H] m/z calcd. for C H Br N 277.8747, found 277.8819. H NMR (500 MHz,
6
4
2
3
1
3
DMSO-d +1% H O) δ ppm: 8.44 (s, 1H), 8.52 (s, 1H), 13.22 (br. s, 1H). C NMR (125 MHz, DMSO-d +10%
6
2
6
5
2
M HCl ) δ ppm: 118.19, 126.01, 129.25, 138.99, 145.11, 145.65; HPLC: t = 11.150 min., 99.53% (λ =
08 nm), 99.81% (λ = 300 nm).
aq
R
4
.1.4.3 5,6,7-Tribromo-1H-imidazo[4,5-b]pyridine (5b)
Column chromatography was performed using chloroform/methanol (0-10% CH OH). Yield: 180
3
+
+
1
mg (98%). Mp >295°C; MS [M+H] m/z calcd. for C H Br N 355.7852, found 355.8089. H NMR (500
6
3
3
3
1
3
MHz, DMSO-d ) δ ppm: 8.59 (s). C NMR (125 MHz, DMSO-d +1% H O) δ ppm: 118.30, 136.27, 146.91;
6
6
2
HPLC: t = 10.939 min., >99.9% (λ = 213 nm), 99.76% (λ = 302 nm).
R
1
5

4
.1.4.4 5-Bromo-1H-imidazo[4,5-b]pyridine (8b)
Column chromatography was performed using chloroform/methanol (0-10% CH OH). Yield: 211
3
+
+
1
mg (87%). Mp 249-251°C; MS [M+H] m/z calcd. for C H BrN 197.9662, found 197.9486. H NMR (500
6
5
3
MHz, DMSO-d ) δ ppm: 7.39 (d, 1H, J = 8.3 Hz), 7.96 (br. s, 1H), 8.47 (br. s, 1H), 12.98 and 13.27 (1H,
6
1
3
tautomers). C NMR (125 MHz, DMSO-d +10% 5M HCl ) δ ppm: 123.98, 126.15, 127.31, 138.92, 143.28,
6
aq
1
45.18; HPLC: t = 10.552 min., >99.9% (λ = 207 nm), >99.9% (λ = 289 nm).
R
4
.1.4.5 6-Bromo-5-chloro-1H-imidazo[4,5-b]pyridine (10b)
Product was purified by triple recrystallization from water/methanol 3/4 v/v. Yield: 116 mg (39%
+
+
1
from 9).mp 262-264°C; [M+H] m/z calcd. for C H BrClN 233.9252, found 233.9173. H NMR (500 MHz,
6
4
3
1
3
DMSO-d ) δ ppm: 8.46 (s, 1H), 8.53 (s, 1H), 13.21 (br. s, 1H). C NMR (125 MHz, DMSO-d +10% 5M
6
6
HCl ) δ ppm: 114.87, 126.01, 129.85, 145.23, 145.44, 145.88; HPLC: t = 9.985 min., >99.9% (λ = 208
aq
R
nm), >99.9% (λ = 299 nm).
4
.1.5 2,3-Diamino-5,6-dibromopyridine (3a)
,3-Diamino-5-bromopyridine 1 (265 mg; 1.41 mmol) was dissolved in warm conc. HBr (15 ml)
2
and cooled in ice/water bath. NBS (263 mg; 1,48; 1.05 eq.) was added in portions during 1 h. The
reaction mixture was stirred at RT overnight. The precipitate was filtered, washed with diethyl ether,
taken up with 50% K CO (30 ml) and extracted with ethyl acetate (3x25 ml). The combined organic
2
3
layers were dried over MgSO , filtered, and the solvent was removed under reduced pressure. Crude
4
product was purified by column chromatography (hexane:ethyl acetate, 7:3) to afford titled compound
as a pink solid; yield: 153 mg (56%). Mp 136-139°C.
4
.1.6 2,3-Diamino-4,5,6-tribromopyridine (3b)
2
,3-Diamino-5-bromopyridine (1) (140 mg; 0.74 mmol; 1.0 eq.) was dissolved in warm HBr (7 ml).
NBS (272 mg; 1.53 mmol; 2.05 eq.) was added and heated in 80°C overnight. After cooling to room
temperature precipitate was filtered and washed with cold diethyl ether. Solid was suspended in 15%
K CO (20 ml) ad extracted with ethyl acetate (3 x 15 ml). Combined organic layers were dried over
2
3
MgSO , filtered, and the solvent was removed under reduced pressure, to afford titled compound. Yield:
4
1
60mg (62%). Mp 196-198°C.
4
.1.7 3-Amino-6-bromo-2-nitropyridine (6)
To the solution of 3-amino-2-nitropyridine (506 mg; 3.64 mmol) and sodium acetate (317 mg;
3
.86 mmol; 1.06 eq.) in acetic acid (4 ml) was added solution of Br (0.2 ml; 3.90 mmol; 1.07 eq.) in
2
acetic acid (2 ml). The reaction mixture was stirred at RT for 6 days. Resulted precipitate was filtered,
washed with cold diethyl ether and dried on air affording product as a yellow solid; yield: 611 mg (77%).
4
.1.8 2,3-Diamino-5-bromo-6-chloropyridine (9)
Conc. HCl (2 ml) was added to 2-amino-5-bromo-3-nitropyridine (1.00 g; 4.45 mmol; 1.0 eq.)
with cooling. To a resulting suspension a solution of SnCl (3.31 g; 17.02 mmol; 3.8 eq.) in conc. HCl (8.8
2
1
6

ml) was added portion wise at 0°C , followed by heating at 70°C for 2 h. The mixture was basified with a
5% NaOH solution with cooling. Resulted precipitate was filtered, washed with warm water and dried
3
aq
to afford 9 in mixture with 2,3-diamino-5-bromopyridine (428 mg) in ratio 3.1:1 according to GC.
4
.2 Molecular docking
Molecular Docking was carried out using 1.1.2. AutoDock Vina program
http://autodock.scripps.edu/) [46]. All ligands were drawn with MarvinSketch
http://www.chemaxon.com/marvin/) and saved as .mol2 files. The hydrogens and Gesteiger partial
(
(
charges were added by 1.5.6. AutoDock tools (http://mgltools.scripps.edu/) [48] and the ligand files
were saved in .pdbqt format. The crystal structure of PIM1 kinase was taken from Protein Data Bank with
PDB code 2O64 [47]. All non-protein molecules (inhibitor, water) were removed, the polar hydrogens
were added and Gesteiger charges were calculated using AutoDock tools to get a file in .pdbqt format.
AutoGrid was used to find an appropriate grid box size. The box centre was set at 75.000, 35.000 and
0
.000 (x, y, z coordinates respectively) and final size space dimension x = 25 Å, y = 25 Å, z= 25 Å. Dockings
were performed with an exhaustiveness level of 80. The receptor-ligand interactions were visualized
using AutoDock tools.
4
.3 Crystal structure determination
4
.3.1 Proteins for crystallographic studies
The CK2α constructs used for crystallization (CK2α1-335 and CK2α’Cys336Ser) were prepared as
described by Lindenblatt et al. [33]. The storage solution for both proteins was 25 mM Tris/HCl, 500 mM
NaCl, pH 8,5. The final protein concentrations were 6 mg/mL for CK2α’Cys336Ser and 7 mg/mL for CK2α1-335
according to UV-absorption at 280 nm.
4
.3.2 Crystallization
To grow CK2α1-335 crystals, 90 µL of the CK2α1-335 stock solution were mixed with 10 µL of a 10
mM inhibitor solution in DMSO. The mixture was incubated for 30 min at room temperature; afterwards
precipitated material was removed by centrifugation. CK2α1-335/inhibitor co-crystals (Table 3) were
obtained using the sitting drop variant of vapour diffusion at 20°C. The optimized drop composition was
1
µL of the preincubated CK2α1-335/inhibitor mixture and 1 µL reservoir solution. Typically, the optimized
reservoir solution was 25 % (w/v) PEG3350, 0.2 M ammonium sulfate, 0.1 M BIS-TRIS buffer, pH 5.5.
In contrast, the crystalline CK2α’Cys336Ser/inhibitor complexes leading to the structures of Table 4
were generated by replacement of the ATP-site ligand MB002 [3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-
yl)propan-1-ol] [37] originally used as crystallization chaperone [33]. The CK2α’Cys336Ser/MB002 co-crystals
grew in 28 % (w/v) PEG 6000, 900 mM LiCl, 100 mM Tris/HCl, pH 8.5; they were optimized by micro- and
macro-seeding, purged from unbound MB002, stabilized by dehydration und subjected to extensive
soaking in solutions with gradually increasing concentrations of the inhibitor of interest (either 4a, 10a,
5
a or 5b).
4
.3.3 X-ray diffractometry
1
7

For X-ray diffraction data collection, all CK2α’Cys336Ser or CK2α1-335 crystals were flash-frozen in
liquid nitrogen. Cryo conditions for the CK2α1-335/inhibitor crystals were obtained by adding (R)-butane-
1
,3-diol into the drops. In the case of the CK2α’Cys336Ser crystals, the respective mother liquor plus 30 %
(v/v) ethylene glycol served as a cryo-protection solution.
Preliminary X-ray diffraction data were collected at the beamline X06DA of the Swiss Light
Source (SLS), Paul Scherrer Institut, in Villigen, Switzerland. The final diffraction data sets were obtained
at the beamlines ID29 (CK2α1-335 co-crystals with 4a, 10a and 5a; Table 3) or ID23-2 (CK2α1-335/5b crystals
in Table 3 and CK2α’Cys336Ser crystals in Table 4) of the European Synchrotron Radiation Facility (ESRF) in
Grenoble, France, using the wavelengths indicated in Table 3 and Table 4. The data collection
temperature was 100 K in all cases.
The diffraction data were processed with the autoPROC toolbox (version 1.0.5) [45] using default
settings. The pipeline integrates XDS [49] for indexing and integration, POINTLESS [50] and AIMLESS [51]
from the CCP4 suite [52] for space group determination as well as scaling and STARANISO [53] for
anisotropy analysis.
4
.3.4 Structure determination, refinement and deposition
The structures were solved either ab initio with Arcimboldo [54] within the CCP4 suite [52]
(
(
CK2α’Cys336Ser complex structures) or with molecular replacement using PHASER [55] within PHENIX [56]
CK2α1-335 complex structures). They were optimized with several rounds of PHENIX refinement[56]
alternating with manual modelling using COOT [57]. The inhibitor molecules were parameterized with
the eLBOW workbench [58] of PHENIX [56].
The atomic coordinates after refinement and the experimental structures factors were deposited with
the Protein Data Bank [59]; the PDB codes assigned by the data base are given in Table 3 and Table 4.
4
4
.4 Biological assays
.4.1 Cloning, expression and purification of human CK2α and PIM1
CK2α and PIM1 for biological assays were obtained according to Borowiecki [60] and Chojnacki
[
(
61]. The protein concentration in final solution was 12.68 mg/mL for CK2α and 3.0 mg/ml for PIM1
determined by Bradford method and bovine serum albumin as a standard) [62].
4
.4.2 Cloning, expression and purification of human wildtype CK2α’
The coding region of human wildtype CK2α’ was amplified by polymerase chain reaction using
the following primers: 5’- GGAATTCCATATGCCCGGCCCGGCCGCG (upstream primer) and 5’-
CCCAAGCTTGTTATCG-TGCTGCCGTGAGACCAC (downstream primer) and I.M.A.G.E. clone as a template.
The product was cloned into the vector pET28a using the restriction sites NdeI and HindIII and the
bacterial strain DH5α. The sequence of the obtained clone was confirmed. Expression of the resulting N-
terminal histidine-tagged hCK2α’ was done in the bacterial strain BL21(DE3)pLysS growing in superbroth
1
8

medium after induction with 1 mM IPTG for 20 h at 20 °C. The cell pellet was resuspended in extraction
buffer [composed of: 20 mM NaH PO (pH 8.0), 500 mM NaCl, 10 mM imidazole, O-complete inhibitor
2
4
cocktail (Roche), lysozyme (1 mg/mL)] and sonicated. The supernatant of the pellet from 400 mL of
bacterial culture was loaded onto HisTrap HP 5ml column (GE Healthcare) mounted on a AKTA Purifier10
FPLC system (GE Healthcare). hCK2α’ was eluted with imidazole gradient in extraction buffer (10-500
mM). Fractions containing His-tagged hCK2α’ were dialyzed against 25 mM Tris-HCl (pH 8.5), 300 mM
NaCl, 1 mM DTT, 0-25% glycerol and stored at -20 °C. The protein concentration in final solution was 5.86
mg/mL (determined by Bradford method and bovine serum albumin as a standard) [62].
4
.4.3 Phosphorylation assays (isotopic) and IC determination
5
0
The new synthesized azolo[4,5-b]pyridine derivatives were tested for their inhibitory activity
against human CK2α, CK2α’ and PIM1 kinases using isotopic assay [63]. CK2α activity was tested in final
volume of 50 μL containing CK2α (21.2 nM) or CK2α’ (23.2 nM), Tris-HCl (pH 7.5; 23 mM), MgCl (20
2
3
2
mM), DTT (0.4 mM), synthetic peptide substrate (RRRADDSDDDDD 40 μM; Biaffin GmbH), [γ- P]-ATP (10
μM, 200–300 cpm/pmol) and appropriate concentrations of inhibitor in 2 μL DMSO or 2 μL DMSO as
control. After 15 min incubation at 30°C, 5 μL of the reaction mixture was spotted onto P81 paper, which
was subsequently washed with 0.6% o-phosphoric acid three times and allowed to dry before counting in
a scintillation counter (Canberra-Packard).
PIM1 activity was tested in final volume of 50 μL containing PIM1 (25.30 nM), Tris-HCl (pH 7.5, 23
3
2
mM), MgCl (20 mM), DTT (0.4 mM), [γ- P]-ATP (20 μM, 200–300 cpm/pmol), and synthetic peptide
2
substrate (ARKRRRHPSGPPTA, 30 μM) and appropriate concentrations of inhibitor in 2 μL DMSO or 2 μL
DMSO as control. The reaction was initiated with enzyme, incubated for 20 min at 30°C, and next 5 μL of
the reaction mixture was spotted onto P81 paper. The filter papers were washed 3 × with 0.6% o-
phosphoric acid and once with 95% ethanol before counting in a scintillation counter (Canberra-
Packard).
The IC values for studied compounds were determined at 4% DMSO with 8 concentrations of
5
0
each tested inhibitor at the range of 0.0256–1000 μM and calculated by fitting the data to sigmoidal
dose-response (variable slope) Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X) * HillSlope) equation in
GraphPad Prism.
4
.4.4 Cell culture and treatment
MCF-7 (human breast cancer cell line) were cultured in DMEM with high glucose medium (Lonza)
with 10 μg/mL of human recombinant insulin. CCRF-CEM (human peripheral blood T lymphoblast cells),
were cultured in RPMI 1640 medium (Lonza). All cell lines were supplemented with 10% fetal bovine
serum (EuroClone), 2mM L-glutamine and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) and
2
grown in 75 cm cell culture flasks (Sarstedt), in a humidified atmosphere of CO /air (5/95%) at 37 °C.
2
4
.4.5 Cell viability assays (MTT-based test)
1
9

Before the treatment, adherent cells (MCF-7) were trypsinized in 0.25% trypsin-EDTA solution
4
(
2
Sigma-Aldrich) and seeded into 96-well microplates at 0.6×10 cells/well. CCRF-CEM were seeded at
4
×10 cells/well. Leukemia cells after seeding and adherent cells 24 h after plating (at 70% of confluency)
were treated with tested compounds at the appropriate concentration or DMSO at 0.5% final
concentration. After 72 h incubation with compounds or DMSO, suspension cells were centrifuged and
supernatants of CCRF-CEM and MCF-7 cells were discarded; subsequently MTT stock solution (Sigma-
Aldrich) was added to each well to a final concentration of 0.5 mg/mL. After 2 h of incubation at 37 °C,
water-insoluble dark blue formazan crystals were dissolved in DMSO (200 μL) (37°C/10 min incubation).
Optical densities were measured at 570 nm using BioTek microplate reader. All measurements were
carried out in eight repetitions and the results are expressed in percentage of cell viability relative to
control (cells without inhibitor in 0.5% DMSO).
Acknowledgments
This work was supported by Warsaw University of Technology and by the Deutsche
Forschungsgemeinschaft (grant NI 643/4-2). We would like to thank Paweł Borowiecki from Warsaw
University of Technology, Faculty of Chemistry for HPLC analyses.
We thank the staff of the beamline X06DA at the SLS (Villigen, Switzerland) and of the beamlines ID23-2
and ID29 at the ESRF (Grenoble, France) for support with X-ray data collection. We are grateful to
Professor Ulrich Baumann (University of Cologne) for access to protein crystallography infrastructure and
continuous support.
References
[
[
[
[
[
[
1] D.W. Litchfield, Protein kinase CK2: structure, regulation and role in cellular decisions of life and
death, Biochem J 369(Pt 1) (2003) 1-15.
2] C. Franchin, C. Borgo, S. Zaramella, L. Cesaro, G. Arrigoni, M. Salvi, L.A. Pinna, Exploring the CK2
Paradox: Restless, Dangerous, Dispensable, Pharmaceuticals (Basel) 10(1) (2017).
3] N.A. St-Denis, D.R. Derksen, D.W. Litchfield, Evidence for regulation of mitotic progression through
temporal phosphorylation and dephosphorylation of CK2alpha, Mol Cell Biol 29(8) (2009) 2068-81.
4] M. Faust, S. Kartarius, S.L. Schwindling, M. Montenarh, Cyclin H is a new binding partner for protein
kinase CK2, Biochem Biophys Res Commun 296(1) (2002) 13-9.
5] G. Wang, G. Unger, K.A. Ahmad, J.W. Slaton, K. Ahmed, Downregulation of CK2 induces apoptosis in
cancer cells--a potential approach to cancer therapy, Mol Cell Biochem 274(1-2) (2005) 77-84.
6] B.T. Kren, G.M. Unger, M.J. Abedin, R.I. Vogel, C.M. Henzler, K. Ahmed, J.H. Trembley, Preclinical
evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference
targets for triple negative breast cancer therapy, Breast Cancer Res 17 (2015) 19.
[
[
7] G. Di Maira, F. Brustolon, J. Bertacchini, K. Tosoni, S. Marmiroli, L.A. Pinna, M. Ruzzene,
Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a
CEM cell line characterized by high CK2 level, Oncogene 26(48) (2007) 6915-26.
8] M.S. Hung, Z. Xu, Y. Chen, E. Smith, J.H. Mao, D. Hsieh, Y.C. Lin, C.T. Yang, D.M. Jablons, L. You,
Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft
model, Int J Oncol 43(5) (2013) 1517-22.
2
0

[
[
9] J.Y. Yoo, B.J. Lim, H.K. Choi, S.W. Hong, H.S. Jang, C. Kim, K.H. Chun, K.C. Choi, H.G. Yoon, CK2-NCoR
signaling cascade promotes prostate tumorigenesis, Oncotarget 4(7) (2013) 972-83.
10] F. Brasó-Maristany, S. Filosto, S. Catchpole, R. Marlow, J. Quist, E. Francesch-Domenech, D.A. Plumb,
L. Zakka, P. Gazinska, G. Liccardi, P. Meier, A. Gris-Oliver, M.C. Cheang, A. Perdrix-Rosell, M. Shafat,
E. Noël, N. Patel, K. McEachern, M. Scaltriti, P. Castel, F. Noor, R. Buus, S. Mathew, J. Watkins, V.
Serra, P. Marra, A. Grigoriadis, A.N. Tutt, PIM1 kinase regulates cell death, tumor growth and
chemotherapy response in triple-negative breast cancer, Nat Med 22(11) (2016) 1303-1313.
11] M. Koronkiewicz, Z. Chilmonczyk, Z. Kazimerczuk, A. Orzeszko, Deoxynucleosides with
benzimidazoles as aglycone moiety are potent anticancer agents, Eur J Pharmacol 820 (2018) 146-
[
[
1
55.
12] M. Bretner, A. Najda-Bernatowicz, M. Łebska, G. Muszyńska, A. Kilanowicz, A. Sapota, New inhibitors
of protein kinase CK2, analogues of benzimidazole and benzotriazole, Mol Cell Biochem 316(1-2)
(2008) 87-9.
[
[
13] M. Makowska, E. Łukowska-Chojnacka, P. Wińska, A. Kuś, A. Bilińska-Chomik, M. Bretner, Design and
synthesis of CK2 inhibitors, Mol Cell Biochem 356(1-2) (2011) 91-6.
14] E. Łukowska-Chojnacka, P. Wińska, M. Wielechowska, M. Bretner, Synthesis of polybrominated
benzimidazole and benzotriazole derivatives containing a tetrazole ring and their cytotoxic activity,
Monatsh Chem 147(10) (2016) 1789-1796.
[
[
[
15] M.A. Pagano, M. Andrzejewska, M. Ruzzene, S. Sarno, L. Cesaro, J. Bain, M. Elliott, F. Meggio, Z.
Kazimierczuk, L.A. Pinna, Optimization of protein kinase CK2 inhibitors derived from 4,5,6,7-
tetrabromobenzimidazole, J Med Chem 47(25) (2004) 6239-47.
16] M.A. Pagano, J. Bain, Z. Kazimierczuk, S. Sarno, M. Ruzzene, G. Di Maira, M. Elliott, A. Orzeszko, G.
Cozza, F. Meggio, L.A. Pinna, The selectivity of inhibitors of protein kinase CK2: an update, Biochem.
J. 415 (2008) 353-365.
17] M. Janeczko, A. Orzeszko, Z. Kazimierczuk, R. Szyszka, A. Baier, CK2α and CK2α' subunits differ in
their sensitivity to 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazole derivatives, Eur J
Med Chem 47(1) (2012) 345-50.
[
[
18] R. Wąsik, P. Wińska, J. Poznański, D. Shugar, Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles
as inhibitors of human protein kinase CK2α, PLoS One 7(11) (2012) e48898.
19] R. Wąsik, P. Wińska, J. Poznański, D. Shugar, Synthesis and physico-chemical properties in aqueous
medium of all possible isomeric bromo analogues of benzo-1H-triazole, potential inhibitors of
protein kinases, J Phys Chem B 116(24) (2012) 7259-68.
[
[
[
20] M. Winiewska, M. Makowska, P. Maj, M. Wielechowska, M. Bretner, J. Poznański, D. Shugar,
Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase
CK2, Biochem Biophys Res Commun 456(1) (2015) 282-7.
21] G. Cozza, C. Girardi, A. Ranchio, G. Lolli, S. Sarno, A. Orzeszko, Z. Kazimierczuk, R. Battistutta, M.
Ruzzene, L.A. Pinna, Cell-permeable dual inhibitors of protein kinases CK2 and PIM-1: structural
features and pharmacological potential, Cell Mol Life Sci 71(16) (2014) 3173-85.
22] A.L. Tsuhako, D.S. Brown, E.S. Koltun, N. Aay, A. Arcalas, V. Chan, H. Du, S. Engst, M. Franzini, A.
Galan, P. Huang, S. Johnston, B. Kane, M.H. Kim, A.D. Laird, R. Lin, L. Mock, I. Ngan, M. Pack, G.
Stott, T.J. Stout, P. Yu, C. Zaharia, W. Zhang, P. Zhou, J.M. Nuss, P.C. Kearney, W. Xu, The design,
synthesis, and biological evaluation of PIM kinase inhibitors, Bioorg Med Chem Lett 22(11) (2012)
3
732-8.
[
23] M. López-Ramos, R. Prudent, V. Moucadel, C.F. Sautel, C. Barette, L. Lafanechère, L. Mouawad, D.
Grierson, F. Schmidt, J.C. Florent, P. Filippakopoulos, A.N. Bullock, S. Knapp, J.B. Reiser, C. Cochet,
New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights, FASEB J 24(9)
(2010) 3171-85.
2
1

[
24] S.A. Thomas, T. Li, K.W. Woods, X. Song, G. Packard, J.P. Fischer, R.B. Diebold, X. Liu, Y. Shi, V.
Klinghofer, E.F. Johnson, J.J. Bouska, A. Olson, R. Guan, S.R. Magnone, K. Marsh, Y. Luo, S.H.
Rosenberg, V.L. Giranda, Q. Li, Identification of a novel 3,5-disubstituted pyridine as a potent,
selective, and orally active inhibitor of Akt1 kinase, Bioorg Med Chem Lett 16(14) (2006) 3740-4.
25] J.K. Park, S. Kim, Y.J. Han, S.H. Kim, N.S. Kang, H. Lee, S. Park, The discovery and the structural basis
of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor, Bioorg Med Chem Lett 26(11)
[
[
(2016) 2580-3.
26] V. Bavetsias, S. Crumpler, C. Sun, S. Avery, B. Atrash, A. Faisal, A.S. Moore, M. Kosmopoulou, N.
Brown, P.W. Sheldrake, K. Bush, A. Henley, G. Box, M. Valenti, A. de Haven Brandon, F.I. Raynaud, P.
Workman, S.A. Eccles, R. Bayliss, S. Linardopoulos, J. Blagg, Optimization of imidazo[4,5-b]pyridine-
based kinase inhibitors: identification of a dual FLT3/Aurora kinase inhibitor as an orally bioavailable
preclinical development candidate for the treatment of acute myeloid leukemia, J Med Chem
5
5(20) (2012) 8721-34.
27] A.H. Berrie, G.T. Newbold, F.S. Spring, Some reactions of substituted 2-bromopyridines J. Chem. Soc.
1952) 2042-2046.
28] S. Björk, V. Delisser, P. Johnström, N.A. Nilsson, K. Ruda, P.M. Schou, B.-M. Swahn, New benzofurans
suitable as precursors to compounds that are useful for Imaging amyloid deposits 2010.
29] M. Antoine, M. Czech, M. Gerlach, E. Günther, T. Schuster, P. Marchand, Preparation of Novel 2,3,8-
Trisubstituted Pyrido[3,4-b]pyrazines and Pyrido[2,3-b]pyrazines, Synthesis 5 (2011) 794-806.
30] T.C. Leboho, S.F. van Vuuren, J.P. Michael, C.B. de Koning, The acid-catalysed synthesis of 7-
azaindoles from 3-alkynyl-2-aminopyridines and their antimicrobial activity, Org Biomol Chem 12(2)
[
[
[
[
(
(
2014) 307-15.
31] P. Zien, J.S. Duncan, J. Skierski, M. Bretner, D.W. Litchfield, D. Shugar, Tetrabromobenzotriazole
TBBt) and tetrabromobenzimidazole (TBBz) as selective inhibitors of protein kinase CK2: evaluation
of their effects on cells and different molecular forms of human CK2, Biochim Biophys Acta 1754(1-
) (2005) 271-80.
[
(
2
[
[
32] I. Ermakova, B. Boldyreff, O. Issinger, K. Niefind, Crystal structure of a C-terminal deletion mutant of
human protein kinase CK2 catalytic subunit, J Mol Biol 330(5) (2003) 925-934.
33] D. Lindenblatt, A. Nickelsen, V.M. Applegate, J. Hochscherf, B. Witulski, Z. Bouaziz, C. Marminon, M.
Bretner, M. Le Borgne, J. Jose, K. Niefind, Diacritic binding of an indenoindole inhibitor by CK2α
paralogs explored by a reliable path to atomic resolution CK2α′ structures, ACS Omega 4(3) (2019)
5
471-5478.
[
34] D. Lindenblatt, A. Nickelsen, V.M. Applegate, J. Jose, K. Niefind, Structural and Mechanistic Basis of
the Inhibitory Potency of Selected 2-Aminothiazole Compounds on Protein Kinase CK2, J Med Chem
6
3(14) (2020) 7766-7772.
[
[
35] K. Niefind, M. Pütter, B. Guerra, O.G. Issinger, D. Schomburg, GTP plus water mimic ATP in the active
site of protein kinase CK2, Nat Struct Biol 6(12) (1999) 1100-3.
36] R. Battistutta, E. De Moliner, S. Sarno, G. Zanotti, L.A. Pinna, Structural features underlying selective
inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole, Protein Sci 10(11)
(2001) 2200-6.
[
[
[
37] N. Bischoff, B. Olsen, J. Raaf, M. Bretner, O.G. Issinger, K. Niefind, Structure of the human protein
kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit
CK2β, J Mol Biol 407(1) (2011) 1-12.
38] C.W. Yde, I. Ermakova, O.G. Issinger, K. Niefind, Inclining the purine base binding plane in protein
kinase CK2 by exchanging the flanking side-chains generates a preference for ATP as a cosubstrate, J
Mol Biol 347(2) (2005) 399-414.
39] J. Hochscherf, D. Lindenblatt, B. Witulski, R. Birus, D. Aichele, C. Marminon, Z. Bouaziz, M. Le Borgne,
J. Jose, K. Niefind, Unexpected Binding Mode of a Potent Indeno[1,2-b]indole-Type Inhibitor of
2
2

Protein Kinase CK2 Revealed by Complex Structures with the Catalytic Subunit CK2α and Its Paralog
CK2α', Pharmaceuticals (Basel) 10(4) (2017).
[
[
40] R. Battistutta, M. Mazzorana, L. Cendron, A. Bortolato, S. Sarno, Z. Kazimierczuk, G. Zanotti, S. Moro,
L.A. Pinna, The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and
conserved water molecules, Chembiochem 8(15) (2007) 1804-9.
41] A. Siddiqui-Jain, D. Drygin, N. Streiner, P. Chua, F. Pierre, S.E. O'Brien, J. Bliesath, M. Omori, N. Huser,
C. Ho, C. Proffitt, M.K. Schwaebe, D.M. Ryckman, W.G. Rice, K. Anderes, CX-4945, an orally
bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling
and exhibits antitumor efficacy, Cancer Res 70(24) (2010) 10288-98.
[
42] R. Battistutta, G. Cozza, F. Pierre, E. Papinutto, G. Lolli, S. Sarno, S.E. O'Brien, A. Siddiqui-Jain, M.
Haddach, K. Anderes, D.M. Ryckman, F. Meggio, L.A. Pinna, Unprecedented selectivity and
structural determinants of a new class of protein kinase CK2 inhibitors in clinical trials for the
treatment of cancer, Biochemistry 50(39) (2011) 8478-88.
[
[
[
43] A.D. Ferguson, P.R. Sheth, A.D. Basso, S. Paliwal, K. Gray, T.O. Fischmann, H.V. Le, Structural basis of
CX-4945 binding to human protein kinase CK2, FEBS Lett 585(1) (2011) 104-10.
44] P. Politzer, P. Lane, M.C. Concha, Y. Ma, J.S. Murray, An overview of halogen bonding, J Mol Model
1
3(2) (2007) 305-11.
45] C. Vonrhein, C. Flensburg, P. Keller, A. Sharff, O. Smart, W. Paciorek, T. Womack, G. Bricogne, Data
processing and analysis with the autoPROC toolbox, Acta Crystallogr D Biol Crystallogr 67(Pt 4)
(2011) 293-302.
[
[
46] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading, J Comput Chem 31(2) (2010) 455-61.
47] S. Holder, M. Zemskova, C. Zhang, M. Tabrizizad, R. Bremer, J.W. Neidigh, M.B. Lilly, Characterization
of a potent and selective small-molecule inhibitor of the PIM1 kinase, Mol Cancer Ther 6(1) (2007)
1
63-72.
[
48] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, AutoDock4
and AutoDockTools4: Automated docking with selective receptor flexibility, J Comput Chem 30(16)
(2009) 2785-91.
[
[
49] W. Kabsch, XDS, Acta Crystallogr D Biol Crystallogr 66(Pt 2) (2010) 125-32.
50] P. Evans, Scaling and assessment of data quality, Acta Crystallogr D Biol Crystallogr 62(Pt 1) (2006)
7
2-82.
[
[
51] P.R. Evans, G.N. Murshudov, How good are my data and what is the resolution?, Acta Crystallogr D
Biol Crystallogr 69(Pt 7) (2013) 1204-14.
52] M.D. Winn, C.C. Ballard, K.D. Cowtan, E.J. Dodson, P. Emsley, P.R. Evans, R.M. Keegan, E.B. Krissinel,
A.G. Leslie, A. McCoy, S.J. McNicholas, G.N. Murshudov, N.S. Pannu, E.A. Potterton, H.R. Powell, R.J.
Read, A. Vagin, K.S. Wilson, Overview of the CCP4 suite and current developments, Acta Crystallogr
D Biol Crystallogr 67(Pt 4) (2011) 235-42.
[
[
53] I.J. Tickle, C. Flensburg, P. Keller, W. Paciorek, A. Sharff, C. Vonrhein, G. Bricogne, STARANISO, Global
Phasing Ltd., Cambridge, United Kingdom, 2018.
54] D.D. Rodríguez, C. Grosse, S. Himmel, C. González, I.M. de Ilarduya, S. Becker, G.M. Sheldrick, I.
Usón, Crystallographic ab initio protein structure solution below atomic resolution, Nat Methods
6
(9) (2009) 651-3.
[
[
55] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, Phaser
crystallographic software, J Appl Crystallogr 40(Pt 4) (2007) 658-674.
56] P.D. Adams, P.V. Afonine, G. Bunkóczi, V.B. Chen, I.W. Davis, N. Echols, J.J. Headd, L.W. Hung, G.J.
Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson,
J.S. Richardson, T.C. Terwilliger, P.H. Zwart, PHENIX: a comprehensive Python-based system for
macromolecular structure solution, Acta Crystallogr D Biol Crystallogr 66(Pt 2) (2010) 213-21.
2
3

[
[
57] P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of Coot, Acta Crystallogr D
Biol Crystallogr 66(Pt 4) (2010) 486-501.
58] N.W. Moriarty, R.W. Grosse-Kunstleve, P.D. Adams, electronic Ligand Builder and Optimization
Workbench (eLBOW): a tool for ligand coordinate and restraint generation, Acta Crystallogr D Biol
Crystallogr 65(Pt 10) (2009) 1074-80.
[
[
59] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne,
The Protein Data Bank, Nucleic Acids Res 28(1) (2000) 235-42.
60] P. Borowiecki, A.M. Wawro, P. Wińska, M. Wielechowska, M. Bretner, Synthesis of novel chiral TBBt
derivatives with hydroxyl moiety. Studies on inhibition of human protein kinase CK2α and
cytotoxicity properties, Eur J Med Chem 84 (2014) 364-74.
[
61] K. Chojnacki, P. Wińska, M. Wielechowska, E. Łukowska-Chojnacka, C. Tölzer, K. Niefind, M. Bretner,
Biological properties and structural study of new aminoalkyl derivatives of benzimidazole and
benzotriazole, dual inhibitors of CK2 and PIM1 kinases, Bioorg Chem 80 (2018) 266-275.
62] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding, Anal Biochem 72 (1976) 248-54.
63] B. Olsen, T. Rasmussen, K. Niefind, O. Issinger, Biochemical characterization of CK2 alpha and alpha '
paralogues and their derived holoenzymes: evidence for the existence of a heterotrimeric CK2 alpha
[
[
'
4
-holoenzyme forming trimeric complexes, Molecular and Cellular Biochemistry 316(1-2) (2008) 37-
7.
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Highlights





New CK2α and CK2α’ inhibitors based on halogenated imidazo[4,5-b]pyridine and
triazolo[4,5-b]pyridine were synthesized.
Residual activity of CK2α, CK2α’ and PIM1 kinases in the presence of final compounds
was determined.
Seven crystal structures of CK2α and CK2α’ complexes with some final compounds were
obtained.
Influence of final compounds on the viability of MCF-7 and CCRF-CEM cell lines was
determined.
Docking studies showing possible reason for differences in the inhibitory activity of final
compounds against CK2α and PIM1 kinases was performed.
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


Declaration of interests
 The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.



☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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