Thiazole- and selenazole-comprising high-affinity inhibitors possess bright microsecond-scale photoluminescence in complex with protein kinase CK2

Article abstract of DOI:10.1016/j.bmc.2018.09.003

A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable room temperature phosphorescence when bound to the ATP pocket of the protei

Full text of DOI:10.1016/j.bmc.2018.09.003

Accepted Manuscript
Thiazole- and selenazole-comprising high-affinity inhibitors possess bright mi-
crosecond-scale photoluminescence in complex with protein kinase CK2
Jürgen Vahter, Kaido Viht, Asko Uri, Ganesh babu Manoharan, Erki Enkvist
PII:
S0968-0896(18)31135-0
https://doi.org/10.1016/j.bmc.2018.09.003
BMC 14528
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 June 2018
30 August 2018
3 September 2018
Please cite this article as: Vahter, J., Viht, K., Uri, A., Manoharan, G.b., Enkvist, E., Thiazole- and selenazole-
comprising high-affinity inhibitors possess bright microsecond-scale photoluminescence in complex with protein
kinase CK2, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.003
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Thiazole- and selenazole-comprising high-affinity inhibitors
possess bright microsecond-scale photoluminescence in complex
with protein kinase CK2
Jürgen Vahter, Kaido Viht, Asko Uri, Ganesh babu Manoharan,
and Erki Enkvist*
Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia
*E-mail: Erki.Enkvist@ut.ee
Abstract
A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-
yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable
room temperature phosphorescence when bound to the ATP pocket of the protein kinase CK2.
Conjugation of these fragments with a mimic of CK2 substrate peptide resulted in bisubstrate
inhibitors with increased affinity towards the kinase. Attachment of the fluorescent acceptor
dye 5-TAMRA to the conjugates led to significant enhancement of intensity of long-lifetime
(microsecond-scale) photoluminescence of both sulfur- and selenium-containing compounds.
The developed photoluminescent probes make possible selective determination of the
concentration of CK2 in cell lysates and characterization of CK2 inhibitors by means of time-
gated measurement of photoluminescence.
Introduction
CK2 kinases are ubiquitous highly conserved pleiotropic protein kinases (PKs) that have roles
in various essential and pathological biological processes.1–7 In mammals the CK2
serine/threonine kinase family is composed of 2 enzymes, CK2α and CK2α’. CK2 kinases can
function as monomeric kinases, and also within a tetrameric complex. The latter is composed
of two CK2 catalytic units (CK2α and/or CK2α’) and two regulatory units (CK2β).
In many types of cancer CK2 is overexpressed, it favors rapid proliferation and survival of
cancer cells and supports angiogenesis.8–15 In turn, pharmacological inhibition or
downregulation of CK2 by gene silencing has been shown to suppress angiogenesis and
induce apoptosis.10,16–21 Within the past decade, two inhibitors of CK2-catalysed protein
phosphorylation, CX-4945 (Silmitasertib) and CIGB-300, entered clinical trials
(www.clinicaltrials.gov identifiers: NCT02128282 and NCT01639625), both as anticancer
agents. CX-4945 is an ATP-competitive CK2 inhibitor, whereas CIGB-300 is a peptidic
inhibitor of CK2-catalysed reaction that binds to the phospho-acceptor domain of CK2
substrates, thus impairing the correct phosphorylation by the enzyme. In addition, the
overexpression of CK2 often correlates with diagnosis of cancer, suggesting that the PK is not
only a potential drug target but may also serve as a biomarker for certain types of
malignancy.8,11,12,18,22 The implication of CK2 in malaria, neurodegenerative and several other
human diseases23–25 has been described that increases the pharmacological potential of
inhibition of CK2.
A large number of inhibitors of CK2 have been developed.26 Most of them are rigid small
molecules that contain one or more aromatic carbo- or heterocycles; these compounds target

the ATP-binding pocket of the enzyme. CK2 is an acidophilic PK that has a preference to
phosphorylate serine/threonine residues in negatively charged regions of proteins.27
Accordingly, CK2 is also inhibited by negatively charged oligomers such as heparin28,29 and
bisubstrate inhibitors comprising an anionic peptide or peptoid fragment.30–33 The activity of
CK2 and inhibitory potential of compounds towards CK2 have been mostly assessed by
monitoring the CK2-catalyzed phosphorylation of synthetic peptide substrates with
radioactively labeled ATP, the handling of which requires special precautions. Therefore,
photoluminescence-based binding and displacement assays could be valuable alternatives for
quantification of catalytically active PKs and for screening and characterization of
inhibitors.30,34
Previously, we have shown that complexes of protein kinases with inhibitors that comprised
sulfur or selenium atoms in aromatic structures were phosphorescent at room temperature,
while the long-lifetime signal emitted by the free inhibitor was negligible.34–36 This
phenomenon was caused by stabilization of the triplet excited state of the aromatic structure
of the inhibitor inside the ATP-binding pocket of PK that slowed down competing non-
radiative relaxation pathways (e.g., quenching by dissolved oxygen37). Moreover, significant
enhancement of kinase binding-responsive long lifetime photoluminescence was achieved
when the inhibitor was covalently labeled with a bright fluorescent dye. This enhancement
resulted from efficient radiationless intramolecular Förster-type resonant energy transfer
(RET) from the donor phosphor in the excited triplet state to the non-excited acceptor
fluorophore, leading it to the excited singlet state of the latter.35,38
Herein we present a new series of bisubstrate inhibitors incorporating either 4-(2-amino-1,3-
thiazol-5-yl)benzoic acid (ATB) moiety, a fragment that was previously introduced to inhibit
CK2 by others39,40, or 4-(2-amino-1,3-selenazol-5-yl)benzoic acid (ASB) moiety, the
selenium-comprising counterpart of ATB. The invented conjugates have high affinity and
selectivity in respect to CK2 and emit protein-responsive phosphorescence at room
temperature that has not been previously described for thiazole- and selenazole-containing
scaffolds. In addition, the inhibitors labeled with fluorescent dyes are tandem probes that
detect the active forms of CK2 and CK2’ in time-gated photoluminescence measurement
mode, a technique otherwise used in the case of lanthanide-based long-lifetime labels. The
developed probes have many potential applications in CK2-related studies such as screening
of inhibitors, determination of binding affinity41 and kinetics35 of the inhibitors or quantifying
the expression level of CK2 in cell lysates.
Results and discussion
Synthesis of ATB- and ASB-peptide conjugates
O
O
NHR
O
NH
HN
X
NH COOH
6
NH
HO
O
N
O
COOH
ATB or ASB
ARC-1527: X = S, R = H
ARC-1528: X = S, R = 5-TAMRA
ARC-1529: X = Se, R = H
ARC-1530: X = Se, R = 5-TAMRA
Scheme 1. Chemical structures of conjugates of peptides and ATB or ASB.

O
O
O
O
O
O
O
a
b
HO
O
1
2
3
O
NH2
NH
O
O
O
O
X
X
c, d
e
OH
N
N
O
4a: X=S
5a: X=S
4b: X=Se
5b: X=Se
Scheme 2. Synthesis of tBu-ATB-Suc and tBu-ASB-Suc (5a/5b). a) (CH3)2NCH[OC(CH3)3]2
42; b) MeOCH2-PPh3Cl, KOtBu 39; c) NBS 43; d) thiourea or selenourea 43; e) succinic
anhydride.44
Four conjugates were constructed: ARC-1527 and ARC-1529 that comprised (L-Asp)6
peptide moiety and differed only by the heavy atom (S or Se) in the aromatic fragment, and
the corresponding 5-TAMRA-labeled compounds ARC-1528 and ARC-1530 (Scheme 1 and
Supplementary S1). ATB and ASB (compounds 4a and 4b) were synthesized according to
previously published methods for the preparation of the corresponding methyl ester of ATB
(Scheme 2)43. Acylation of compound 4a or 4b with succinic anhydride resulted in
succinamides 5a or 5b, respectively. Compounds 5a and 5b were coupled to peptides on a
solid resin using Fmoc-peptide synthesis protocols. Treatment with TFA released the
conjugates from the resin and removed protecting groups. All compounds were isolated by
reverse phase HPLC. Lysine residue was included into the peptide fragment of the conjugates
for the attachment of the fluorescent label 5-TAMRA (Scheme 1).
Binding of the conjugates to CK2
Biochemical equilibrium binding studies with CK2α showed that the conjugates followed the
affinity trends inherent to previously designed bisubstrate inhibitors of CK2 (Table 1 and
Supplementary S2). First, conjugation with negatively charged peptide sequence remarkably
(about by two orders of magnitude) increased the affinity as shown by subnanomolar KD
value of compound ARC-1527 (KD = 0.58 nM) and two-digit nanomolar KD values of the
fragments ATB (KD = 48 nM). Second, attachment of 5-TAMRA label to the C-terminal
lysine residue of ARC-1527 and ARC-1529 had negligible effect on the affinity. Sulfur to
selenium substitution was well tolerated as no affinity difference was registered between
ATB- and ASB-conjugates.
All four conjugates emitted long-lifetime (microsecond-scale) luminescence upon excitation
in the UVA wavelength range when in complex with CK2 and not in the absence of CK2
(Figure 1, Table 1). As expected, the compounds with fluorescence labels revealed somewhat
shorter luminescence lifetimes and much higher initial signal intensities if compared to
unlabeled conjugates. This can be explained by efficient RET between the phosphorescent
ATB or ASB moiety and the adjacent fluorescence dye. Selenium-containing compounds
possessed significantly higher photoluminescence intensity if compared to the sulfur-
containing counterparts. Still, the efficiency of inter-chromophore triplet-to-singlet RET
(calculated from equation 2) were not much different, 24% for ARC-1528 and 29% for ARC-
1530. This could be explained by more efficient overlap of the phosphorescence emission

spectrum of thiazole fragment (Supplementary S3) with absorption spectrum of the
fluorescence dye 5-TAMRA than it is the case with selenazole fragment. For these reasons,
the RET efficiency of compounds containing thiazole is not much lower than that of
selenazole compounds, although the latter fragment possesses significantly stronger
phosphorescence in CK2 complex.
It has been previously shown that deoxygenation of samples could increase the intensity and
lifetime of emission of long-lifetime photoluminescence probes. Deoxygenation of the
solutions was conducted by the previously described method, which utilizes three
components: glucose, glucose oxidase and catalase.37 In case of ARC-1528-CK2 complex,
deoxygenation of the solution doubled the luminescence lifetime (increase from 82 µs to 165
µs). The effect observed with ARC-1530-CK2 complex was slightly smaller (increase from
36 µs to 58 µs, Supplementary S4 and S5). This indicates that even in the active site of CK2
where the phosphorescent donors ASB or ATB are hidden from the solution phase still about
half of the depopulation of the triplet excited state can be attributed to quenching by dissolved
oxygen. Previously, we have shown that even larger effect of deoxygenation of the solution to
the lifetime and intensity of photoluminescence could be observed with responsive long
lifetime photoluminescence probes of the catalytic subunit of protein kinase A.37 Although
deoxygenation somewhat increases the residual signals of the free probes, these signals still
remain much weaker than the signals of the probe-PK complexes (Supplementary S4).
Displacement curves of luminescence probe ARC-1530 from its complex with CK2 by CX-
4945 and TBBi (4,5,6,7-tetrabromobenzimidazole) are presented on Figure 2B. Achieved IC50
value for CX-4945 was 0.8 nM (0.5 – 1.2 nM) and it is close to previously reported Ki = 0.38
nM.45 Displacement IC50 for TTBi was 260 nM (170 – 390 nM) that was also similar to
previously reported values Ki = 0.5 – 1-1 μM.46 The results of the displacement studies are
comparable with previously reported results of enzyme kinetic assays. This validates the use
of ARC-1530 as a luminescent probe in displacement assays for assessing the binding of
affinities of the active site targeting compounds.
Protein kinase selectivity of ARC-1527
The inhibitory potency of the unlabeled compound ARC-1527 at 100 nM concentration was
tested in a panel of 50 PKs (Table 2). High selectivity of ARC-1527 towards CK2 was
observed, as the kinase was inhibited by the greatest extent leading to a residual activity of
1.9%. Only three other kinases were inhibited more than 50%, representing PKs of the
DYRK, GSK, and HIPK families. These PKs are common off-targets of CK2 inhibitors.32
Complexes of ARC-1530 with CK2 and CK2’ had very similar dissociation constants and
photoluminescence properties (Supplementary S5 and S6). ARC-1530 differs mainly from
ARC-1527 by the presence of fluorescence label, thus it presumably has comparable
selectivity profile that makes it a sensitive and selective reagent for determination of the
concentration of the catalytic subunits of CK2.
Determination of CK2 in cell lysates
Time-gated detection mode in combination with binding responsiveness of the long-lifetime
emission eliminates interfering background fluorescence, light scattering and signal from
ARC-1530 that may be non-specifically bound to other cellular components. That makes the
probe useful for measuring CK2 activity in complicated biological solutions. To illustrate this,
ARC-1530 was added to dilutions of lysates of NIH-3T3 cells containing either native CK2 or
over-expressed RFP-CK2 (Figure 3). As expected, higher concentration of the lysate led to
increased intensity of luminescence signal and the lysates of cells containing overexpressed

CK2 produced stronger luminescence if compared to the lysate with native CK2. Relative
expression levels could be calculated from the initial slopes of the signals and the absolute
concentrations could be calculated by using calibration with recombinant CK2 (Figure 1A).
Expression levels of transfected RFP-CK2 were usually much higher compared to
expression of native CK2 and the ratio varies between experiments.47 Presented example
(Figure 3) indicates about 10 fold higher expression of CK2α-s in transfected cells compared
to native NIH-3T3 cells. Addition of CX-4945 to the lysates reduced the long-lifetime signal
to the level of background indicating that nonspecific binding of ARC-1530 to CX-4945-
inactive components that leads to long lifetime luminescence signal is negligible. Knowing of
the number of cells and their average volumes makes also possible the estimation of
intracellular concentrations by considering the dilution factors during lysate preparation.19
Titration of 5 nM ARC-1530 with low concentrations of CK2 was performed to estimate the
detection limit (LOD) of the assay (Figure 2A). In this experiment the probe was in excess
and bound the majority of the added kinase resulting in linear dependence of TGL signal on
the concentration of CK2. On the other hand, the signal of the free probe remained
insignificant. LOD value of 25 pM was calculated from linear regression analysis of the data.
Assumingly, the value of LOD could be somewhat bigger for measurements in cell lysate
caused by formation of minor non-specific signal. Dynamic range of the measurement is
limited by the amount of the probe (5 nM in this experiment) but applying of proper
intermediate dilutions allows increase the range without limitations. It is also important to
note that the probe binds to the active site of the enzyme and therefore detects only the native
form of CK2. Thus no TGL signal will be registered when the active site is blocked or the
kinase is denatured. Therefore we could estimate a good correlation between the measured
TGL signal of CK2-ARC-1530 complex and the enzymatic activity of CK2 in the sample.
Table 1. Dissociation constants (Kd), photoluminescencea lifetimes (τ) and relative
* b
photoluminescence intensities (I0 ) of compounds in complex with CK2
*
Compound
Kd/nM
τ/µs
I0
ATB
48 ± 7
n.d.c
n.d.
1.0 ± 0.1
76 ± 8
ARC-1527
ARC-1528
ARC-1529
ARC-1530
0.58 ± 0.14
0.40 ± 0.12
0.34 ± 0.12
0.85 ± 0.15
82 ± 2
62 ± 2
55 ± 2
39 ± 6
46 ± 9
5300 ± 370
ameasured with optical module [ex 337(300-360) nm, em 590(50) nm];
bmaximal luminescence intensity of the complex, calculated from equation 1 and normalized
against the luminescence intensity of ARC-1527-CK2 complex;
cnot determined.

A
B
25000
20000
15000
10000
5000
0
20000
15000
10000
5000
0
0
50
100
50
100
150
200
c(CK2) / nM
t / s
Figure 1. Increase of the intensity of long-lifetime luminescence signal in the course of
titration of 1 nM ARC-1528 (red) or 1 nM ARC-1530 (blue) with CK2 (A) and decay of the
intensity of long-lifetime luminescence signal of 10 nM ARC-1528 (red) and 10 nM ARC-
1530 (blue) in complex with 200 nM CK2 (B).
A
B
2500
6 0 0 0
2000
T B B i
C X -4 9 45
1500
1000
500
0
4 0 0 0
2 0 0 0
0
0.00
0.05
0.10
-1 0
-8
-6
c (C K 2  ) / n M
lo g [C (in h ib ito r)/M ]
Figure 2. Titration of 5 nM ARC-1530 with CK2. Magnification of the lowest concentrations
is presented (A). Displacement of luminescence probe ARC-1530 (1 nM) from its complex
with CK2 (1 nM) by CX-4945 and TBBi (B).
A
B
2 0 0 0 0
1 5 0 0 0
1 0 0 0 0
5 0 0 0
0
150000
100000
50000
0
0
1 0
2 0
3 0
0
10
20
30
L y s a te %
L y s a te %
Figure 3. Luminescence intensity of the probe ARC-1530 (10 nM) upon binding to CK2 in
diluted cell lysates before (filled circles) and after (open circles) treatment with 10 μM CX-
4945. Lysates of NIH-3T3 cells with overexpressed RFP-tagged CK2 (A) and native NIH-
3T3 cells (B) were studied.

Table 2. Residual activities of PKs in the presence of 100 nM ARC-1527
Residual activity
%
Residual activity
PK
CK2α
DYRK1a
GSK3ß
HIPK2
TAK1
SmMLCK
AMPK (hum)
SRPK1
MLK3
TBK1
PLK1
RIPK2
JAK3
TTK
JNK1
VEG-FR1
MKK1
TrkA
PIM1
PKA
PKCa
CHK2
ROCK2 (rat)
SGK1
BTK
SD (N = 2)
PK
SD (N = 2)
%
1.9
0.5 MARK3
0.6 Lck (mouse)
0.7 MST2
8 PAK4
17 RSK1
29 IRAK4
2 p38α MAPK
1 NEK6
4 Src
9 S6K1
5 PKBa
4 EF2K
18 EPH-A2
10 CK1δ
17 LKB1
13 PRK2
6 CaMK1α
14 PDK1
4 HER4
6 MSK1
1 PKD1
4 CaMKKß
0.7 SYK
4 Aurora B
8 IGF-1R
99
100.8
101
101
101
102
102
103
103
104
105
106
107
107
107
109
111
111
113
115
117
120
122.5
129
143
3
11.5
19.0
23
62
68
69
69
69
78
83
85
85
85
86
87
88
88
91
92
95
96
96.5
97
99
0.2
10
4
8
8
5
5
12
4
9
15
5
8
17
11
13
23
9
6
2
3
0.2
10
27
Summary and conclusions
For the construction of high-affinity sensors for CK2 determination based on measurement of
protein binding-responsive long-lifetime photoluminescence, 4-(2-amino-1,3-thiazol-5-
yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) were conjugated with
an oligo-(L-aspartic acid) peptide for construction of sensitive PK CK2 sensors. Conjugation
led to compounds ARC-1527 and ARC-1529, both of which revealed remarkable
phosphorescence in complex with CK2 upon excitation with near-UV radiation while free
compounds in buffer solution possessed no long-lifetime emission. Labeling of these
compounds with the fluorescent dye 5-TAMRA gave probes ARC-1528 and ARC-1530 that
possessed significantly higher long-lifetime (microsecond-scale) luminescence intensities in
complex with CK2 than their unlabeled precursors. This strong signal enhancement resulted
from efficient radiationless intramolecular Förster-type resonant energy transfer from the
thiazole- or selenazole-comprising donor phosphor in the excited triplet state to the non-
excited acceptor fluorophore 5-TAMRA, leading to the excited singlet state of the latter and
tardy light emission at the emission spectral region 5-TAMRA dye. Very efficient energy
transfer between adjacent luminophores well competes with various non-radiative decay
pathways of the triplet state resulting in significant dye-mediated enhancement of
phosphorescence signal.

ARC-1528 and ARC-1530 can be used in simple mix-and-measure type binding assays with
time-gated read-out to determine the concentration of the active form of CK2 ( and ’) and
characterize non-labeled inhibitors in displacement assays. Compared to the previously
reported benzoselenadiazole-based long-lifetime photoluminescence probe of CK2, disclosed
by us four years ago34, the ATB- and ASB-related probes have more than an order of
magnitude higher affinity towards CK2 and the probe ARC-1530 possesses an order of
magnitude stronger luminescence signal. ARC-1530 is thus the brightest highly selective
probe that can be used to measure very low concentration of CK2. In our settings, isolated
CK2 and CK2’ could be detected at concentrations starting from 25 pM. In addition, the
probes offer selectivity of detection both in the level of recognition of the target enzyme and
in the level of appearance of the long-lifetime photoluminescence signal upon binding to the
target enzyme. That makes these probes useful for determination of the concentration of the
active form of CK2 in complex biological solutions such as cell lysates, as it was also
demonstrated in the current report. Free ARC-1530 does not possess long-lifetime
photoluminescence and therefore might be used in large excess. In displacement assay it
opens the possibility to characterize very potent inhibitors whose differentiation would be
impossible with other methods because of tight-binding conditions inherent to assays in
use.41,48 Moreover, monitoring the displacement of unlabeled inhibitors by the protein-
responsive long-lifetime probes gives access to dissociation rate constants of those
inhibitors.35
It should be noted that the long-lifetime binding-responsive luminescence of ARC-1528 and
ARC-1530 becomes evident only upon illumination with radiation at near-UV range where
the excitation of ATB or ASB moiety is possible. At higher wavelength range (visible light),
the fluorophore (5-TAMRA) can be directly excited and the compounds can be used like
common fluorescent probes. The latter option extends the applicability of the probes to assays
based on the measurement of fluorescence intensity and fluorescence polarization. The
presented probes are not cell-permeable because of the large negative charge on the peptide
moiety. However, we have recently shown that esterification of the peptoid moieties of
similar compounds results in prodrugs with intracellular activity.19,31 Therefore these
luminescence probes could be converted into cell permeable compounds by using the
previously described prodrug strategy.
Materials and methods
Chemicals were purchased from ABCR, Acros, Alfa Aesar, Deutero GmbH, Iris Biotech
GmbH, Fluka, Macherey-Nagel, Fisher Scientific, Sigma-Aldrich and TCI Chemicals. CX-
4945 was from Synkinase. CK21-335 and CK2’ proteins were kind gifts from Prof. O.-G.
Issinger (University of Southern Denmark). THF was dried over molecular sieves. t-BuOK
was purified by dissolving the crude material in THF, centrifugation and evaporation of the
supernatant. HPLC-MS was performed with Schimadzu LC Solution (Prominence) system
connected to LCMS-2020 ESI-MS. Purification of the samples was performed with RP-HPLC
equipped with a Phenomenex Luna C18 analytical column (5 μm, 250 × 4.6 mm) at 40 °C,
and photodiode array detector SPD-M20A. Acetonitrile/water + 0.1% aqueous TFA gradient
at a flow rate of 1 mL/min was used. ESI-HRMS of compounds were measured with Thermo
Electron LTQ Orbitrap spectrometer in positive ion mode.
The concentrations of stock solutions of ligands and probes were determined
spectrophotometrically (NanoDrop 2000c, Thermo Scientific) in the same buffer used in
biochemical measurements. The following molar extinction coefficients were used: ATB-

conjugates: ε322nm = 23 000 M-1cm-1, ASB-conjugates: ε330nm = 21 000 M-1cm-1 and 5-
TAMRA-labeled conjugates: ε558nm =80 000 M-1cm-1.
The fluorescence anisotropy (FA) and/or time-gated luminescence (TGL) changes relative to
the signal of the free fluorescent probe were registered on a PHERAstar platereader (BMG
Labtech). For FA measurements, optical modules suitable for detecting PromoFluor-647 [ex
590 (50) nm, em 675 (50) nm] and 5-TAMRA [ex 540 (20) nm, em 590 (20) nm] were
applied. TGL measurements were conducted with optical module [ex 337(300…360) nm, em
590(50) nm], delay time of 50 µs and 150 µs integration time.
The phosphorescence spectra were measured with Synergy Neo platereader (BioTek) with the
following settings: 100 µs delay time and 2000 µs integration time.
Synthesis of tert-butyl 4-(2-methoxy)benzoate (compound 2)
Compound 2 was prepared according to a similar synthesis previously described.42 To the
solution of 4-carboxybenzaldehyde (521 mg, 3.45 mmol) in of benzene (6.5 ml) of N,N-
dimethylformamide di-tert-butyl acetal (2.5 eq, 1760 mg) was added dropwise over a period
of 1 h and refluxed. After two hours, the mixture was cooled down to room temperature and
water was added (10 ml). The organic layer was collected and concentrated in vacuum.
Product was purified using chromatographically (silica gel eluted with Hexane:EtOAc 6:1,
v:v). Compound 2 was obtained with the yield of 35.4%.
Synthesis of tert-butyl 4-(2-methoxyethenyl)benzoate (compound 3)
Compound 3 was prepared according to a similar procedure previously described for the
synthesis of the corresponding methyl ester.39 Under nitrogen atmosphere, a solution of t-
BuOK (1.5 eq, 341 mg) in THF (1 mL) was added dropwise to a suspension of
(methoxymethyl)triphenylphosphonium chloride (1.2 eq, 825 mg) in THF (2 mL) in ice bath.
The reaction mixture turned red. After 15 min, tert-butyl 4-formylbenzoate (1 eq, 412 mg, 2
mmol) in THF (2 mL) was added dropwise over a period of 10 min. Ice bath was removed
and the orange mixture was stirred for 2 h at r.t. Water (0.6 ml) was added, the mixture was
concentrated in vacuum (40 C) and partitioned between H2O and EtOAc. The organic layer
was washed with brine and dried over MgSO4. Chromatographic purification (silica gel eluted
with 1,2-dichloroethane) yielded the title compound as equimolecular mixture of Z- and E-
1
isomers in 75% yield. H NMR (CDCl3, 700 MHz) δ: 1.58 (s, 9H), 3.69 (s, 1.5H), 3.79 (s,
1.5H), 5.24 (d, J = 7 Hz, 0.5H), 5.81 (d, J = 13 Hz, 0.5H), 6.20 (d, J = 7 Hz, 0.5H), 7.14 (d, J
= 13 Hz, 0.5H), 7.24 (d, J = 8 Hz, 1H), 7.58 (d, J = 8 Hz, 1H), 7.87 (d, J = 8 Hz, 1H), 7.90 (d,
J = 8 Hz, 1H). 13C NMR (CDCl3, 175 MHz) δ: 28.2, 56.7, 60.9, 80.5, 80.6, 104.5, 105.0,
124.6, 127.8, 128.9, 129.0, 129.4, 129.9, 140.2, 140.9, 149.8, 150.6, 165.7, 165.9.
Synthesis of tert-butyl 4-(2-amino-1,3-thiazol-5-yl)benzoate (tBu-ATB, 4a) and
tert-butyl 4-(2-amino-1,3-selenazol-5-yl)benzoate (tBu-ASB, 4b)
Compounds 4a and 4b were prepared according to similar procedures previously described.43
N-bromosuccinimide (1.1 eq) was added in ice bath to a suspension of compound 3 (1 eq) in a
mixture of H2O:dioxane (1:1, 1.5-2.4 mL). Ice bath was removed and after 8 min thiourea or
selenourea (1.1 eq) were added. The reaction mixture was heated over a hotplate (100 C) for
2 h. The mixture was transferred into 15-ml centrifuge tube and aqueous ammonia (25 %, 0.1-
0.3 mL) was added. The pale-yellow precipitate was separated by centrifugation, washed with
water, dried and purified chromatographically (silica gel eluted with CHCl3:MeOH 9:1, v:v).
1
Compound 4a was obtained in 77% yield. H NMR (DMSO-d6, 700 MHz) δ: 1.54 (s, 9H),
13
7.35 (br, 2H), 7.50 (m, 2H), 7.58 (s, 1H), 7.82 (m, 2H). C NMR (DMSO-d6, 175 MHz) δ:

27.8, 80.5, 123.8, 124.1, 128.2, 129.7, 136.9, 137.7, 164.6, 169.2. Compound 4b was obtained
1
in 73% yield. HMRS calculated for C14H16N2O2Se 324.0377, found 324.0368, H NMR
(DMSO6d, 700 MHz) δ 1.54 (9H, s), 7.45 (2H, d, J = 8.5 Hz), 7.47 (1H, s), 7.59 (2H, br s),
13
7.79 (2H, d, , J = 8.5 Hz). C NMR (DMSO6d, 175 MHz) δ 27.8, 80.5, 125.0, 128.1, 128.8,
129.7, 138.6, 139.3, 164.7, 171.2.
Synthesis of 4-({5-[4-(tert-butoxycarbonyl)phenyl]-1,3-thiazol-2-yl}amino)-4-
oxobutanoic acid (tBu-ATB-Suc, 5a) and 4-({5-[4-(tert-butoxycarbonyl)phenyl]-
1,3-selenazol-2-yl}amino)-4-oxobutanoic acid (tBu-ASB-Suc, 5b)
Compound 5a and 5b was prepared according to similar synthesis previously described.44
Compound 4a or 4b (1 eq) was dissolved in DMF (2 ml) and succinic anhydride (1.5 eq) was
added. Reaction mixture was stirred at 65 ºC overnight. The mixture was concentrated in
vacuum and partitioned between H2O and EtOAc. Product was purified using
chromatographically (silica gel eluted with CH3Cl:MeOH 9:1, v:v). Compound 5a was
1
obtained in 33.5% yield. H NMR (DMSO6d, 700 MHz) δ 1.55 (9H, s), 2.56 (2H, t, J = 6.7
Hz), 2.69 (2H, t, J = 6.7 Hz), 7.72 (2H, d, J = 8.5 Hz), 7.90 (2H, d, J = 8.5 Hz), 8.01 (1H, s).
13C NMR (DMSO6d, 175 MHz) δ 27.8, 28.6, 30.0, 80.8, 125.4, 129.70, 129.74, 129.9, 135.8,
136.0, 155.4, 158.1, 164.6, 173.7. Compound 5b was obtained in 32.2% yield. HMRS
1
calculated for C18H20N2O5Se 424.0537, found 424.0522. H NMR (DMSO6d, 700 MHz) δ
1.54 (9H, s), 2.59 (2H, t, J = 6.7 Hz), 2.71 (2H, t, J = 6.7 Hz), 7.67 (2H, d, J = 8.5 Hz), 7.87
(2H, d, J = 8.5 Hz), 7.97 (1H, s). 13C NMR (DMSO6d, 175 MHz) δ 27.9, 28.4, 30.1, 80.8,
126.2, 129.7, 129.9, 135.7, 136.4, 138.4, 160.3, 164.7, 171.1, 173.6.
Synthesis of ATB-peptide conjugates
The peptides were synthesized starting from Fmoc-Lys(Boc)-Wang resin according to Fmoc
solid-phase peptide synthesis methods. The following coupling conditions were applied:
carboxylic acid (3 eq), HBTU (2.9 eq), HOBt (3 eq) and NMM (6 eq) in DMF. The
conjugates were cleaved from the resin by treatment with TFA:TIS:H2O (95:2.5:2.5, v:v:v, 3
h), isolated by HPLC and the molecular masses were verified by HRMS (Supplementary S1).
Binding/displacement assay and photoluminescence measurements
The binding affinities of ligands were determined in the binding/displacement assay, as
described earlier.41,49 The assays were carried out in 4-component buffer (50 mM HEPES, 150
mM NaCl, 0.005% Tween® 20, 5 mM DTT, pH = 7.5) in the final volume of 20 μL on black
384-well polystyrene microplates with nonbinding surface (Corning #4514). Experiments
were run in 2 parallels. The concentration of CK2α1-335 and CK2’ were determined by
titration of a fixed concentration (20 nM) of PromoFluor-647-labeled fluorescent probe ARC-
150430 or 5-TAMRA-labeled ARC-1530 with the solution of the enzyme (2-fold dilutions).
Dissociation constants of the complexes of fluorescent probes with the catalytic subunits of
CK2 were established in similar conditions at 1 nM concentration of ARC-1528 or ARC-
1530. Displacement experiments with unlabeled compounds were performed by addition of
the competing inhibitor (3-fold dilutions) to either ARC-1504 (2 nM) in complex with CK2α1-
335
(3 nM) or ARC-1513-5O (1 nM) in complex with CK2α1-335 (2 nM). Displacement
experiments with ARC-1530 were carried out similarly by using time gated luminescence
readout. CX-4945 was used as a reference compound in all experiments. The microplates
were incubated for 15 or 60 min at 30 °C. The obtained data were analyzed as previously
described to yield the concentration of the catalytically active CK2α and dissociation constant
values for the complexes between the investigated compounds and CK2α.41,49

Deoxygenation of samples was conducted as described earlier.37 The plate containing samples
(10 nM ARC-1528 or 1 nM ARC-1530 with 40 nM CK2) with glucose oxidase (final acitivity
2 U/ml) and catalase (final concentration 0.05 mg/ml) in 15 μL volume were pre-incubated at
30 °C for 15 minutes. After incubation 5 μL of D-glucose solution (final concentration 25
nM) was added and samples were measured using same equipment as described before.
Graphpad Prism software (v 6.05, GraphPad Software, USA) was used for data analysis.
The luminescence decay curves were fitted to the equation:
I = (I0 – Ibg)  e – t/ + Ibg
(1)
where I is the intensity of luminescence signal measured at time t, I0 is the intensity of the
luminescence signal at t = 0, Ibg is the intensity of the signal of the background and  is the
luminescence lifetime.
The efficiency of triplet-singlet RET was calculated using equation:
E = 1 – τ(DA)/τ(D)
(2)
where E is triplet-singlet RET efficiency; τ(D) the luminescence lifetime of donor molecule
without the acceptor; τ(DA) the luminescence lifetime of the molecule, when donor and
acceptor are both present.
Expression of TagRFP-CK2α in NIH-3T3 cells and lysate preparation were performed as
described earlier.47 Dilution series of initial lysates were made into buffer containing ARC-
1530 and the time-gated luminescence intensities were measured. Dilution series that
contained displacer CX-4945 (10 µM) were measured for check the non-specific signal. The
results were plotted as TGL intensity vs percentage of lysate content in measurement wells.47
Selectivity profile
The selectivity panel was run on commercial basis by International Centre for Kinase
Profiling (University of Dundee) using radiometric filter-binding assay. 50 PKs applied for
measurements were of human origin unless stated otherwise. The ATP concentration applied
for inhibition measurements was at or below the Km,ATP value of the particular PK. The final
total concentration of ARC-1527 used in the selectivity panel was 100 nM. Residual activities
are expressed as the percentage retained activity compared to the control without inhibitor.
Acknowledgements
This work was funded by a grant from the Estonian Research Council (IUT20-17).
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Protein kinase CK2
O
O
HN
Linker
O
Se
Oligo-aspartic peptide
O-
N
O
Fluorescence dye
570 nm
328 nm
558 nm
590nm