Molecular Plasticity of Crystalline CK2α′ Leads to KN2, a Bivalent Inhibitor of Protein Kinase CK2 with Extraordinary Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.1c00063

CK2α and CK2α′ are paralogous catalytic subunits of CK2, which belongs to the eukaryotic protein kinases. CK2 promotes tumorigenesis and the spread of pathogenic viruses like SARS-CoV-2 and is thus an attractive drug target. Efforts to develop selective CK2 inhibitors binding offside the ATP site had disclosed the αD pocket in CK2α; its occupation requires large conformational adaptations of the helix αD. As shown here, the αD pocket is accessible also in CK2α′, where the necessary structural plasticity can be triggered with suitable ligands even in the crystalline state. A CK2α′ structure with an ATP site and an αD pocket ligand guided the design of the bivalent CK2 inhibitor KN2. It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2 substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD pocket.

Full text of DOI:10.1021/acs.jmedchem.1c00063

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Article
Molecular Plasticity of Crystalline CK2α′ Leads to KN2, a Bivalent
Inhibitor of Protein Kinase CK2 with Extraordinary Selectivity
Dirk Lindenblatt, Violetta Applegate, Anna Nickelsen, Merlin Klußmann, Ines Neundorf, Claudia Götz,
Joachim Jose, and Karsten Niefind*
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ABSTRACT: CK2α and CK2α′ are paralogous catalytic subunits
of CK2, which belongs to the eukaryotic protein kinases. CK2
promotes tumorigenesis and the spread of pathogenic viruses like
SARS-CoV-2 and is thus an attractive drug target. Efforts to
develop selective CK2 inhibitors binding offside the ATP site had
disclosed the αD pocket in CK2α; its occupation requires large
conformational adaptations of the helix αD. As shown here, the αD
pocket is accessible also in CK2α′, where the necessary structural
plasticity can be triggered with suitable ligands even in the
crystalline state. A CK2α′ structure with an ATP site and an αD
pocket ligand guided the design of the bivalent CK2 inhibitor KN2.
It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2
substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD
pocket.
leads to significant off-target effects: CX-4945 inhibits several
other EPKs in the low nanomolar range.11 Recent efforts
resulted in more efficient and selective ATP-competitive CK2
inhibitors,12−14 but a residual selectivity problem remains as it
is inherent to so-called type-I EPK inhibitors15 that occupy the
ATP site in its active conformation.
In many EPKs, this limitation was overcome by addressing
the ATP site in an inactive kinase state in which the so-called
regulatory (R)-spine is dismantled (type-II inhibitors).15 The
R-spine and its counterpartthe catalytic (C)-spineare
stacks of hydrophobic/aromatic residues (Figure S1) that
penetrate the border of the two structural domains of an EPK
(Figure 2a).16 Both spines must be completely established
the C-spine requires the adenine moiety of ATP for thisas a
prerequisite of full activity. For CK2, type-II inhibitors are
excluded: here, the R-spine is stabilized via intramolecular
interactions of the N-terminal segment with the helix αC and
the activation segment, the canonical regulatory switches in
EPKs (Figure 2a).16 In fact, a dismantled R-spine was never
found in any CK2 structure.17
INTRODUCTION
■
Protein kinase CK2, a member of the superfamily of eukaryotic
protein kinases (EPKs), is among several viral and human
proteins considered as targets to treat COVID-191 as it
colocalizes with the nucleocapsid protein of SARS-CoV-2 in
filopodia protrusions exploited by the virus for rapid cell-to-cell
transfer.2
The benefit of CIGB325, an anti-CK2 peptide, in the
context of COVID-19 treatment was described recently.3 A
further potential anti-COVID-19 drug is the ATP-competitive
CK2 inhibitor CX-4945 (Figure 1a) that was developed for
cancer therapy4 and is subject of clinical studies in the context
of COVID-19 (NCT04668209)5 and cholangiocarcinoma
(NCT02128282).6 CK2 activity is elevated in many cancer
tissues; the enzyme promotes cell proliferation and angio-
genesis and suppresses apoptosisthree characteristics of
cancerogenesis.7 Upregulated CK2 amplifies tumor-promoting
signaling, such as the Wnt, the NF-κB, the PI3K/Akt,8 or the
Hedgehog pathway.9 In addition, CK2 mediates phospho-
rylation of caspase recognition sequences and thus protects
apoptotic proteins from caspase cleavage. Consequently, CK2
interferes with caspase signaling: its activity inversely correlates
with the rate of apoptosis,10 and tumors develop an undue
reliance on CK2 activity to maintain the malignant
phenotype.7
Special Issue: New Horizons in Drug Discovery -
Understanding and Advancing Kinase Inhibitors
Received: January 14, 2021
CX-4945 is a tight-binding CK2 inhibitor (Ki = 223 pM)11
with antiproliferative activity against a broad panel of cancer
cell lines,4 but its binding to the highly conserved ATP site
© XXXX The Authors. Published by
American Chemical Society
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Figure 1. CK2 ligands used or mentioned in this study: (a) CK2
benchmark inhibitor CX-4945;4 (b) αD pocket ligand 3,4-
dichlorophenethylamine (DPA); (c) bivalent inhibitor
CAM4066;24,25 (d) CK2α′ crystallization additive MB002;34,35 (e)
4,5,6,7-tetrabromo-1H-benzo[d]imidazol (TBI) and its derivative
TBIa, used as a synthon here; and (f) bivalent inhibitor KN2 [N1-
(3,4-dichlorophenethyl)-N4-(4-(4,5,6,7-tetrabromo-1H-benzo[d]-
imidazol-1-yl)butyl)succinamide] developed in this study.
Instead, CK2 inhibitor design moved toward allosteric
compounds18 (type-IV inhibitors),19 bisubstrate inhibi-
tors20−23 or bivalent ligands24,25 (type-V inhibitors),19,26 that
combine high affinity and selectivity.27 CK2’s quaternary
structure with two catalytic subunits (CK2α or CK2α′) bound
to a dimer of regulatory chains (CK2β)28 offers a unique
opportunity for this: CK2β antagonistic compounds addressing
the CK2β interface of CK2α (Figure 2a) disturb the CK2α/
CK2β interaction and the CK2β-dependent enzyme pro-
file.20,29−33
Figure 2. The αD pocket in CK2α/CK2α′. (a) Overview of CK2α
with helix αD in closed state (from PDB_ID 3BQC),36 in open state
(from PDB_ID 1PJK),37 and in unfolded state with αD pocket
occupied by DPA (from PDB_ID 5CLP).25 (b) CK2α′Cys336Ser with
bound MB002 and DPA [PDB_ID 7AT9 (Table S1)], both covered
with electron density (contour level 1 σ). (c) Helix αD region
mobility in crystalline CK2α′Cys336Ser during crystal soaking and
starting point of linker design for KN2. Phe121 and Tyr125 of CK2α
(panel a) are equivalent to Phe122 and Tyr126 of CK2α′ (panels
b,c). The green helix αD (from PDB_ID 6HMQ)35 marks the initial
point of crystal soaking with DPA and the magenta-colored elements
from the CK2α′Cys336Ser/DPA/MB002 structure (this work) the end
point.
A further attractive allosteric site of CK2α is the so-called
αD pocket at the back of the canonical helix αD (Figure 2a).25
Typically in EPKs, the αD pocket is not accessible for small
molecules since it is part of the C-spine and filled with a side
chain from the helix αD. The latter is therefore largely
invariable in its conformation.16 The helix αD of CK2α,
however, is unusually adaptable;36 depending on its orientation
(Figure 2a), it feeds the side chain of either Phe121 or Tyr125
into the C-spine or it unfolds completely and permits access to
the αD pocket for ligands such as 3,4-dichlorophenethylamine
(DPA; Figure 1b and Figure 2a). This feature of CK2α, found
by fragment-based screening,25 was exploited to design
CAM4066, a bivalent inhibitor addressing the ATP site and
the αD pocket (Figure 1c).24,25
partner. The answer to this question finally led to KN2 (Figure
1f), a bivalent CK2 inhibitor of extremely high selectivity.
RESULTS AND DISCUSSION
■
DPA Opens the αD Pocket of CK2α′ in the Crystalline
State. A further motivation of this work had a methodical
background: to combine DPA (Figure 1b) with CK2α′, we
used a crystallographic procedure originally developed to solve
atomic resolution structures of CK2α′ with ATP-competitive
CK2 inhibitors. It depends on crystals of the oxidation-stable
mutant CK2α′Cys336Ser (Figure S1) in complex with the ATP
site ligand MB002 (Figure 1d)34 that is subsequently
The αD pocket was characterized with human CK2α only.25
In contrast, the available crystal structures of its paralog CK2α′
suggest conformational invariability in the helix αD region.35
Therefore, we considered whether CK2α′ can nevertheless
provide an accessible αD pocket if an established αD pocket
ligand such as DPA (Figure 1b) is offered as the interaction
B
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a
Scheme 1. Synthesis of KN2
a
Reagents and conditions: (a) DIPEA, HATU, DMF, 24 h, rt; (b) TIPS, DCM/TFA, 2 h, rt, 74%; (c) DIPEA, HATU, DMF, 24 h, rt, 66%.
exchanged by extensive soaking.35 The method was successful
with CX-4945 and with several other high-, medium-, and low-
affinity ATP-competitive ligands,35,38,39 but it was unclear if it
works for allosteric sites, as well. A first study to explore this
failed insofar as it proved that certain 2-aminothiazole
compounds described before to be allosteric CK2 inhibitors40
actually bind to the ATP site.38
achieve membrane penetration as reported for CAM4066.25
A
topological polar surface area of 76.02 Å2 and a lipophilicity
value (clogP) of 6.38 were calculated with SwissADME.42 The
latter is larger than recommended,43 but overall in silico
evaluation did not exclude cell permeability; rather, it
suggested to proceed with KN2 as a promising candidate for
a bivalent CK2 inhibitor in vitro and in the cell.
The use of DPA was a further attempt, and here, the
procedure, in fact, resulted in a high-resolution ternary
complex structure of CK2α′Cys336Ser, MB002, and DPA
(Table S1), in which the latter occupied the allosteric αD
pocket while the ATP site and its MB002 ligand were largely
unaffected (Figure 2b). A comparison of this structure with the
starting point of the soaking procedurerepresented by a
CK2α′Cys336Ser/MB002 structure (PDB_ID 6HMQ)35re-
veals that the occupation of the αD pocket by DPA required
a movement of the helix αD region by around 12 Å combined
with a loss of the helical geometry (Figure 2c and Movie S1).
Noteworthy, this huge displacement happened within the
crystal during soaking. Hence, CK2α′ not only is able to
emulate its paralog CK2α in opening of the αD pocket but also
provides a remarkable example of molecular plasticity of a
crystalline protein. Apparently, the ATP site and the αD
pocket of CK2α′Cys336Ser in this crystalline environment can be
independently addressed by soaking of suitable compounds.
For the future, this suggests a path to atomic resolution
structures of ternary CK2α′Cys336Ser complexes with various
couples of ATP site and αD pocket ligands.
Design, Synthesis, and Inhibitory Efficacy of the
Bivalent CK2 Inhibitor KN2. The ternary complex structure
in Figure 2b inspired us to connect the two ligands and
provided a perfect basis to model a tailored linker. The linker
design in CAM4066 had demonstrated the benefit of peptide
groups to improve solubility in an aqueous environment and to
combine the potential to form hydrogen bonds with a balanced
degree of flexibility.24 To adapt this strategy, we used two
terminal synthons with amino groups, namely, DPA itself
(Figure 1b) and TBIa41 (Figure 1e) that resembles MB002.
After TBIa was modeled into the ternary CK2α′Cys336Ser/DPA/
MB002 complex (Figure 2b) by replacing MB002 (Figure 2c),
the two amino groups were only 5.6 Å apart from each other,
suggesting succinic acid as a dicarbonic acid with a perfect
length to bridge this distance and to form two peptide bonds.
Unlike CX-4945 (Figure 1a) and CAM4066 (Figure 1c), the
resulting compound KN2 (Figure 1f) did not contain a
negative charge that could enforce a pro-drug approach to
After synthesis of KN2 according to Scheme 1, its CK2
inhibitory efficacy was tested using tetrameric CK2 holoen-
zymes based on either CK2α or CK2α′Cys336Ser (Figure 3a). At
100 μM ATP, the IC50 values were 19.3 6.4 nM with the
CK2α2β2 isoform and 15.6
isoform, corresponding to Ki values of 6.1
5.5 nM with the CK2α′2β2
2.0 nM
(CK2α2β2) and 4.0 1.4 nM (CK2α′2β2). Thus, the affinity
of KN2 to the enzyme is between those of CX-4945 (Ki =
0.223 nM with CK2α2β2)11 and CAM4066 (IC50 = 370 nM at
25 μM ATP with CK2α)25 and significantly higher than the
affinity of TBIa (Figure 1e), KN2’s synthon at the side of the
ATP cavity (Ki = 190 nM with CK2α2β2).41 Overall, these data
indicate a very strong binding of KN2 to both CK2α paralogs.
To explore if KN2 inhibits CK2 within the cell, we first
applied a toxicity assay to check if it is cell-permeable enough
to affect whole cells. For this, HeLa cells, a tumor cell line
relying on high levels of CK2, were chosen and for comparison
HEK293 cells, an artificially immortalized nontumor cell line
being less sensitive toward CK2 inhibitors.7,32 In fact, KN2
proved to be cell-permeable and cytotoxic (Figure 3b). The
EC50 value is significantly (p-value 0.0198) lower for HeLa
cells (6.19 μM) than for HEK293 cells (15.94 μM), consistent
with their higher CK2 requirement for viability. CX-4945 was
somewhat more cytotoxic for HeLa cells than KN2 (Figure
3c); the EC50 value we determined (2.10 μM) is in the same
range as reported for a panel of 13 cancer cell lines.44 We did
not detect any CX-4945 cytotoxicity with HEK293 cells up to
a concentration of 100 μM (Figure 3c). This finding is
consistent with the results of a caspase 3/7 activation assay
with CX-4945 reported earlier.4
Within cells, CK2 regulates the PI3K/Akt signaling pathway
among others via phosphorylation of Akt/PKB kinase.8 The
major CK2 phosphorylation site is Ser129,8 and the down-
regulation of the Akt phosphorylation at Ser129 indicates
reliably that an inhibitor affects CK2 intracellularly.4 In
Western blot analyses, the phosphorylation signal of Akt at
Ser129 vanished in a concentration- and time-dependent
manner for both cell lines (Figure 3c,d). In summary, KN2 can
C
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Figure 4. Selectivity screening of KN2 (3 μM) against 83 different
EPKs from all families of the human kinome. Error bars correspond to
standard deviation ( ) based on duplicate measurements (details in
Table S2). Color-coded bars plus triangles indicate EPKs inhibited by
10 μM TBI (Figure 1e) according to Pagano et al.45 Asterisks mark
EPKs inhibited by 0.5 μM CX-4945 (Figure 1a) to at least 90% as
published by Battistutta et al.11 The inset shows the Lorentzian curve
derived from the KN2 selectivity profile according to Graczyk46 for
calculation of the Gini coefficient; the latter equals the area ratio A/(A
+ B).
cocrystal structure of KN2 with the close CK1ε-relative CK1δ
(sequence identity 97.3% and no sequence differences in the
region of the ATP site and the αD pocket; Table S2). In this
structure, a KN2 ligand is only weakly visible at the ATP site,
but neither an open helix αD conformation nor an accessible
αD pocket could be observed (Figure S2). Final clarity was
provided by a dose−response curve (Figure S3), demonstrat-
ing that KN2 is only a negligible CK1ε inhibitor (IC50 value
more than 30 μM).
Figure 3. Functional characterization of KN2. (a) Inhibition of
CK2α- and CK2α′-based CK2 holoenzymes (quadruple measure-
ments). (b,c) Cytotoxic effect of KN2 (b) and for comparison CX-
4945 (c) on HeLa and HEK293 cells (experiments performed as
triplicates). (d,e) Western blot analysis of HEK293 (d) and HeLa
cells (e) to explore the effect of KN2 on the CK2-dependent
phosphorylation level of AKT/PKB at Ser129.
Notably, 3 μM KN2 did not inhibit a number of EPKs
previously reported to be significantly affected by CX-4945 at
0.5 μM11 and by TBI at 10 μM45 (colors and asterisks in
Figure 4). Rigorous selectivity comparisons would require Ki
values, which are independent of ATP and inhibitor
concentrations. Their determination is laborious, so instead,
the Gini coefficient calculated from selectivity profile data was
introduced as a quantitative indicator of kinase selectivity.46 Its
use is recommended now,47 and it was applied for several CK2
inhibitors, among them CX-494511 and CAM4066.25 A Gini
coefficient of 0.76, calculated from the kinase profile in Figure
4, suggests an extraordinary high selectivity of KN2. For
comparison, the selectivity data published for 10 μM of KN2’s
ATP-site anchor TBI45 lead to a Gini coefficient of 0.30,
whereas data from the same article45 for 1 μM 4,5,6,7-
tetrabromo-1H-benzo[d][1,2,3]triazolethe mother com-
pound of MB002 (Figure 1d)correspond to a Gini
coefficient of 0.68. The Gini coefficient published for
CAM4066 is slightly higher than that of KN2 (0.82), but it
was calculated on the basis of a smaller panel (52 EPKs) and a
lower inhibitor concentration (2 μM).25 For CX-4945, a Gini
coefficient of 0.62 was reported, determined with 102 EPKs
and an inhibitor concentration of 0.5 μM.11 Overall, the high
Gini coefficient of KN2 suggests that it mimics the binding
mode of CAM4066 by exploiting the αD pocket in favor of
selectivity.
enter cells and manipulate the cellular CK2-dependent
phosphoproteome.
KN2 Is Highly Selective for CK2α and CK2α′.
CAM4066 (Figure 1c) was designed with the aim of a high
selectivity by exploiting the αD pocket.25 To avoid that the
ATP site anchor group dominates binding, CAM4066 was
supplied only with a low-affinity anchor there.25 In the case of
KN2, we did not adapt this precautionary strategy, but we were
interested if high selectivity can be achieved on the basis of a
high-affinity ATP site fragment, as well. To explore this
quantitatively, KN2 was tested against a panel of 83 EPKs from
all families of the human kinome including those reported to
be off-targets of CK2 inhibitors (Table S2). In the screening,
the ATP concentration was in the range of the KM of the
respective kinase. Throughout, a KN2 concentration of 3 μM
was applied, which equals approximately 150−200 times the
IC50 value with respect to CK2. With such a high
concentration, even weak off-target effects should be reliably
detected. The resulting cinhibitor/IC50 ratio is close to the range
Graczyk46 recommended for the application of the Gini
coefficient as a numerical expression of selectivity.
Under these conditions, KN2 inhibited CK2α and CK2α′ to
about 94% (Figure 4 and Table S3). The only apparent off-
target was CK1ε with an inhibition of 29.9%, however with a
high standard deviation of 19.84% (Figure 4 and Table S3).
To explore its putative interaction with KN2, we determined a
KN2 Binding to CK2α and CK2α′ Benefits from the
Tailor-Made Linker. To investigate the binding mode
D
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precisely, we determined crystal structures of KN2 in complex
with CK2α′Cys336Ser and CK2α1−335 (Table S1). In both cases,
crystal soaking was applied and started with DPA (Figure 1b)
plus the intention to open the αD pocket first in order to assist
the binding of KN2. Significantly, however, this strategy turned
out to be unnecessary: irrespective of the presence and the
concentration of DPA, KN2 diffused smoothly into
CK2α′Cys336Ser or CK2α1−335 crystals, induced access to the
αD pocket there (Movie S2) and was finally well-defined by
electron densities (with atomic resolution in case of the
CK2α′Cys336Ser/KN2 complex) (Figure 5a).
Whether follow-up compounds of KN2 resulting from such
efforts can improve COVID-19 or cancer therapies is a
particularly interesting question for future studies.
CONCLUSION
■
Allosteric binding sites play an increasing role in protein kinase
inhibitor design to escape the limitations of the ATP site with
respect to selectivity.15,19,26,27 The αD pocket of CK2α and its
paralog CK2α′the result of a local helix αD plasticity unique
among eukaryotic protein kinasesis such an allosteric site. Its
potential was known from the proof-of-concept inhibitor
CAM406624,25 and was emphasized here again with the
bivalent CK2 inhibitor KN2. In KN2, a high-affinity, but low-
selectivity, ATP-competitive inhibitor was coupled to a low-
affinity, but selectivity providing ligand of the αD pocket via a
tailor-made linker. The basis for the linker design was an
atomic resolution CK2α′ structure in complex with an ATP
site and an αD pocket fragment. It was provided by a
crystallographic approach exploiting the plasticity of helix αD
in crystalline CK2α′. This procedure is potentially applicable
to determine CK2α′ complex structures with various
combinations of ATP site and αD pocket ligands. This
would be particularly valuable if a recent prediction that
bivalent ligands constitute the next generation of CK2
inhibitors should come true.27
Figure 5. KN2 binding to CK2α/CK2α′. (a) KN2 and parts of its
protein environment in complex with CK2α′Cys336Ser covered by
electron density (contouring level 1.5 σ); for comparison, parts of the
CK2α1−335/KN2 complex were drawn (C atoms in black; Phe121,
Tyr125, and Ile140 of CK2α are equivalent to Phe122, Tyr126, and
Leu141 of CK2α′); crystallographic details are given in Table S1. (b)
LIGPLOT51 projection of KN2 bound to CK2α′Cys336Ser. Green
dotted lines, H-bonds; red dotted lines, halogen bonds.
EXPERIMENTAL SECTION
■
Chemicals. 3,4-Dichlorophenethylamine (DPA; Figure 1b),
succinic acid, and chemicals in general were purchased from Sigma-
Aldrich. 3-(4,5,6,7-Tetrabromo-1H-benzotriazol-1-yl)propan-1-ol
(MB002; Figure 1d)52 and 4-(4,5,6,7-tetrabromo-1H-benzo[d]-
imidazol-1-yl)butan-1-amine (TBIa; Figure 1e)41 were kind gifts
from the group of Professor Maria Bretner, Warsaw, Poland. TBIa was
used as a synthon for KN2 here; its synthesis and analytical
characterization was described in detail by Chojnacki et al.41
The binding of KN2 is essentially as anticipated by in silico
modeling. The four bromo substituents participate via halogen
bonds and hydrophobic interactions (Figure 5b), as well-
known for TBI derivatives.48 As intended, the two peptide
groups of the linker contribute to affinity by a number of
hydrogen bonds with the protein matrix (Figure 5b). One of
these H-bonds is formed with the terminal hydroxy group of
Tyr126 (Tyr125 in CK2α); this means that the helix αD
region, which is largely disordered in the published CK2α/
DPA structure25 (Figure 2a) and in the CK2α′Cys336Ser/DPA/
MB002 complex structure of this work (Figure 2b),
reassembles within the crystal when the high-affinity ligand
KN2 is bound (Movie S2).
Surprisingly, the orientation of the αD pocket anchor in the
CK2α1−335/KN2 complex differs from the CK2α′Cys336Ser/KN2
complex (Figure 5a) due to space restrictions in the αD pocket
enforced by the terminal methyl group of Ile140. In the future,
this and other subtle differences between CK2α and CK2α′ in
the αD pocket region may assist attempts to develop inhibitors
discriminating between both paralogs.35,49,50
Efforts to further improve the selectivity and affinity of KN2
plus other properties essential for a drug can include the linker
region and the ATP site, as well. For such intentions, an
excellent basis is provided not only by the complex structures
of this work but also by the option to replace the MB002
ligand of CK2α′Cys336Ser/DPA/MB002 crystals by other ATP-
competitive compounds and thus to determine further atomic
resolution structures of ternary CK2α′Cys336Ser complexes.
Analytical Chemistry. A 1H NMR spectrum of the coupling
product between succinic acid and DPA (compound 1) was measured
on a Bruker Avance I 300 spectrometer (300 MHz). The residual
protonated solvent signal of DMSO-d6 served as an internal reference.
NMR spectra of KN2 were recorded at 26.0 °C in deuterated
solvents on an Agilent DD2 600 MHz spectrometer equipped with a
cold probe (Agilent, Santa Clara, CA, USA). Chemical shifts (δ) are
reported in parts per million (ppm) against the reference substance
tetramethylsilane and calculated using the solvent residual peak of the
1
undeuterated solvent. The assignment of H and 13C NMR signals
were supported by gCOSY, gHSQC, gHMBC, and bsgHMBC spectra
(not shown). The data were analyzed using MestReNova 12.0.4
(Mestrelab, Santiago de Compostela, Spain).
An LTQ XL three-segment 2D linear ion trap mass spectrometer
(Thermo Fisher Scientific) was used to acquire mass spectra of the
coupling product between succinic acid and DPA (compound 1). The
sample was injected by a 1260 infinity autosampler (Agilent) with an
injection volume of 10 μL and ionized by an electrospray ionization
source. The measurement was conducted with a spray voltage of 4.5
keV, a N2 sheath gas pressure of 6.5 × 105 Pa, a capillary voltage of 44
V, and a capillary temperature of 275 °C. An Aeris 3.6 μm peptide
XB-C18 (150 × 4.6 mm) (Phenomenex) LC column was used for
compound separation. A linear ACN gradient, 50−100% ACN over
15 min, was applied with water containing 0.1% formic acid (A) and
ACN containing 0.08% formic acid (B). The flow rate was set to 0.6
mL/min.
For the confirmation of the KN2 sum formula, a flow injection
analysis coupled to a mass spectrometer (FIA-MS) was used. The LC
system consisted of an Ultimate 3000 RS Dionex system (Dionex
Softron, Germering, Germany) with DGP-3600RS pump and WPS-
3000RS rapid separation autosampler. The high-resolution mass
E
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spectrometer was a MicrOTOF-Q II (Bruker Daltonics, Bremen,
Germany) with ESI interface (Bruker Daltonics, Bremen, Germany).
The mobile phase of the LC system consisted of acetonitrile/
ammonium formate [10 mM]/formic acid 50:50:0.1 (v/v/v) with a
flow of 0.1 mL/min. The electrospray ion source was operated in
positive ionization mode, scan range of 120−800 m/z. The capillary
was set to 4.5 kV; the nebulizer was operated at 0.4 bar, and the dry
gas was set to 4 L/min at a temperature of 180 °C. Transfer voltages:
funnels 1 and 2 were set to 200 Vpp. The hexapole RF voltage was set
to 100 Vpp. The transfer time was adapted to 70 μs. Mass calibration
was done using a 20 mM sodium formate solution.
Protein Expression and Purification. For the purpose of
crystallization, CK2α1−335, a C-terminal deletion mutant of human
CK2α,37 and CK2α′Cys336Ser, a point mutant of human CK2α′,34
without the problem of forming intermolecular disulfide bonds under
oxidative conditions,53 were used. Both constructs were prepared as
described previously.35
The enzyme kinetic measurements were performed with hetero-
tetrameric CK2 holoenzyme constructs, namely, with
(CK2α)2(CK2β1−193)2 and with (CK2α′Cys336Ser)2(CK2β1−193)2. The
latter was prepared as described,35 whereas the (CK2α)2(CK2β1−193
)
2
holoenzyme was produced as follows: full-length CK2α and
CK2β1−193 were separately expressed in Escherichia coli BL21(DE3)
using a pT7-7 plasmid system. Bacterial cells were transformed and
cultured overnight in lysogeny broth at 37 °C with 200 rpm shaking.
Cells were then grown in 1 L phosphate medium35 to an OD578 of 0.5.
Protein expression was induced by the addition of isopropyl β-D-1-
thiogalactopyranoside to a final concentration of 1 mM. After an
incubation of 4 h at 30 °C and 200 rpm shaking, the cells were
harvested, mixed in equal amounts, and suspended in lysis buffer (300
mM NaCl, 1.3 mg/mL lysozyme, 13 μg/mL DNase I, 25 mM Tris/
HCl, pH 8.5). After 30 min incubation on ice, the cells were disrupted
by sonification (6 × 30 s on ice) and the cell lysate was clarified by
centrifugation (100.000g, 4 °C, 1 h) to remove cell debris. The
supernatant was applied to a phosphocellulose column and afterward
to a HiTrap heparin HP column (GE Healthcare, Buckinghamshire,
UK) for purification according to Raaf et al.36 An analytical size
exclusion step with a Superdex 200 column (GE Healthcare,
Buckinghamshire, UK; running buffer containing 1 M NaCl, 25
mM Tris/HCl, pH 8.5) was attached to exclude the presence of free
catalytic subunit.
4-((2-(3,4-Dichlorophenyl)ethyl)amino)-4-oxobutanoic acid
(Compound 1). 4-(tert-Butoxy)-4-oxobutanoic acid (130 mg, 0.75
mmol 1.00 equiv) was dissolved in DMF (5 mL) and mixed with
DIPEA (520 μL, 2.99 mmol, 3.99 equiv). Subsequently, HATU (380
mg, 1 mmol, 1.33 equiv) was added to this solution. The mixture was
stirred at room temperature for 20 min. DPA (Figure 1b; 179 mg,
0.95 mmol, 1.27 equiv) was then added to the reaction solution. After
being stirred for 24 h, the solution was diluted with ice-cold water (25
mL). The product was extracted with DCM (3 × 15 mL). The
combined organic layers were concentrated to dryness in vacuo. The
residue was again dissolved in a mixture of DCM (5 mL) and
triisopropylsilane (0.15 mL, 0.73 mmol, 0.97 equiv). The tert-butyl
protection group was cleaved for 2 h by adding trifluoroacetic acid (5
mL), followed by neutralization with solid Na2HCO3. The solution
was mixed with half-saturated Na2HCO3 (50 mL) and washed with
cyclohexane (3 × 30 mL) and DCM (3 × 30 mL). The aqueous
layers were collected and acidified with HCl. Purification of the
precipitate was performed via preparative reversed-phase HPLC (C18
column, ACN gradient: 10−60% with 0.1% TFA, 6.0 mL/min).
Compound 1 (4-((2-(3,4-dichlorophenyl)ethyl)amino)-4-oxobuta-
noic acid) crystallized as colorless needles (162 mg, 0.56 mmol,
Enzyme Kinetics. The reduction of CK2 phosphorylation activity
by KN2 (Figure 3a) was analyzed with a nonradioactive method
based on capillary electrophoresis (CE),54 where the extent of CK2
substrate peptide (RRRDDDSDDD) phosphorylation was deter-
mined by CE with a UV detector at 195 nm. The background
electrolyte of the CE runs was 2 M acetic acid (pH 2) at a current of
30 μA.
1
74% yield). H NMR (300 MHz, DMSO-d6, Figure S4): δ (ppm) =
12.04 (br, 1H), 7.91 (t, J = 5.5 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.50
(d, J = 2.0 Hz, 1H), 7.20 (dd, J = 2.0, 8.2 Hz, 1H), 3.26 (d, J = 5.9 Hz,
1H), 2.70 (t, J = 6.9 Hz, 2H), 2.50 (quint, J = 3.6 Hz, DMSO), 2.39
(t, J = 6.2 Hz, 2H), 2.29 (t, J = 6.5 Hz, 2H). MS (ESI) m/z calcd for
C12H13Cl2NO3 [M + H]+ 290.03; found 290.10.
Inhibitor solutions in dimethyl sulfoxide (DMSO) covering a
concentration range from 0.001 to 100 μM were prepared. Enzyme
(N1-(3,4-Dichlorophenethyl)-N4-(4-(4,5,6,7-tetrabromo-1H-
benzo[d]imidazol-1-yl)butyl)succinamide) (Compound 2,
KN2). For a second coupling step, 4-((2-(3,4-dichlorophenyl)ethyl)-
amino)-4-oxobutanoic acid (25 mg, 86 μmol, 1.00 equiv) was
dissolved in DMF (2 mL) and mixed with DIPEA (120 μL, 0.69
mmol, 8.02 equiv). The mixture was preincubated for 20 min after
HATU (42.6 mg, 112 μmol, 1.30 equiv) was added. Then, 4-(4,5,6,7-
tetrabromo-1H-benzimidazol-1-yl)butan-1-amine (TBIa, Figure 1e;
41 mg, 81 μmol, 0.94 equiv) was added, and the mixture was stirred
for 24 h at room temperature. Subsequently, 20 mL of DCM was
added, and the product was washed with water (3 × 50 mL). The final
purification of KN2 was performed by preparative reversed-phase
HPLC (Hitachi) using a VP250/16 Nucleodur 100-5 C18ec column
(Macherey-Nagel) and an ACN gradient (50−90% with 0.1% TFA;
6.0 mL/min; Figure S5). KN2 was concentrated to dryness in vacuo
using an Xcel/Vap (Horizon Technology) to isolate the product as
colorless solid (44 mg, 57 μmol, 66% yield). The purity is 97.1%
according to the integrated HPLC trace (retention time 9.17 min;
solutions containing 0.25 μg of (CK2α)2(CK2β1−193
)
or
2
(CK2α′Cys336Ser)2(CK2β1−193)2 in 100 mM NaCl, 10 mM MgCl2, 50
mM Tris/HCl, pH 7.5, were supplemented with 2 μL of inhibitor
solution to a final volume of 80 μL. The mixture was preincubated for
10 min at 37 °C with continuous shaking at 300 rpm. Kinase reaction
was started by addition of a 120 μL solution of 190 μM CK2 substrate
peptide RRRDDDSDDD and 100 μM cosubstrate ATP in 150 mM
NaCl, 5 mM MgCl2, 25 mM Tris/HCl, pH 8.5. After an incubation
time of 3 min (CK2α-based holoenzyme) or 7 min (CK2α′-based
holoenzyme) at 37 °C with continuous shaking at 300 rpm, the
reaction was stopped by addition of 5 μL of 0.5 M EDTA and by
cooling on ice. In this time, 10% of the substrate was usually
phosphorylated in the absence of KN2.
The reactions were performed as independent quadruples, and
reactions with 2 μL of DMSO without inhibitor served as a control
(100% activity). Relative inhibition was calculated as inhibition [%] =
(1 − sample/100% activity) × 100. The IC50 values were determined
from the midpoint of a sigmoidal curve [equation: Y = bottom + (top
− bottom)/(1 + 10^((LogIC50 − X) × HillSlope)), fitted by
GraphPad Prism (GraphPad, La Jolla, CA, USA)]. Ki values of KN2
were calculated according to the equation of tight-binding inhibition:
IC50 = Ki(1 + [ATP]/KM,ATP) + [enzyme]/2,55 assuming competitive
inhibition with respect to ATP. For this, a KM,ATP value of 81.6 0.2
μM as determined by Pietsch et al.20 was applied.
The inhibition of human CK1ε by KN2 (Figure S3) was quantified
by Carna Bioscience, Inc., Kobe, Japan. The catalytic domain of
human CK1ε (residues 1−348) as a GST-fusion construct was used
for the experiments performed at room temperature, pH 7.5, and with
an ATP concentration of 25 μM. Ten different KN2 concentrations
from 1 nM to 30 μM were tested; each data point was measured
twice.
1
Figure S5). H NMR (600 MHz, DMSO-d6, Figure S6): δ (ppm) =
8.49 (s, 1H), 7.86 (t, J = 5.6 Hz, 1H), 7.81 (t, J = 5.7 Hz, 1H), 7.50
(d, J = 8.2 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.15 (dd, J = 8.2, 2.0 Hz,
1H), 4.49 (t, J = 7.3 Hz, 2H), 3.24−3.20 (m, 2H), 3.08−3.03 (m,
2H), 2.65 (t, J = 7.0 Hz, 2H), 2.50 (quint, J = 3.6 Hz, DMSO), 2.24
(s, 4H), 1.82−1.74 (m, 2H), 1.41−1.34 (m, 2H). 13C NMR (151
MHz, DMSO-d6, Figure S7): δ (ppm) = 171.3 (1C), 171.2 (1C),
148.9 (1C), 143.7 (1C), 140.9 (1C), 131.3 (1C), 130.7 (2C), 130.2
(1C), 129.2 (1C), 128.6 (1C), 122.4 (1C), 120.4 (1C), 116.6 (1C),
106.6 (1C), 46.1 (1C), 39.6 (1C), 39.5 (DMSO), 37.8 (1C), 34.0
(1C), 30.73 (1C), 30.71 (1C), 29.0 (1C), 26.0 (1C). HRMS m/z
calcd for C23H22Br4Cl2N4O2 [M + H]+ 772.7926; found 772.7894
(Figure S8).
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Cell Toxicity Assay. To determine the cytotoxic effects of KN2
and for comparison CX-4945 (free acid; purchased from Sell-
eckChem, Munich, Germany) toward HeLa cells (ATCC: CCL-2)
and HEK293 cells (ATCC: CRL-1573), the cells were seeded (HeLa:
1.4 × 104; HEK293: 3.7 × 104) in a 96-well plate and grown overnight
in media (HeLa: RPMI-1640 culture medium supplemented with 4
mM L-glutamine and 10% fetal bovine serum (FBS); HEK293:
minimum essential medium Eagle supplemented with 4 mM L-
glutamine and 15% FBS) to 70−80% confluency at 37 °C, 5% CO2,
and increased air humidity. On the next day, cells were incubated with
different dilutions of KN2 or CX-4945, which were prepared in the
appropriate serum-free medium. After 24 h, cells were washed with
phosphate-buffered saline except for the positive control, which was
treated with 70% ethanol for 10 min. Cell viability was determined
using a resazurin-based cytotoxicity assay (Sigma-Aldrich), in which
the conversion of resazurin to resorufin by metabolizing cells can be
monitored at 595 nm (λex = 550 nm). Cells were incubated for 1−2 h
with a 1:10 dilution of a resazurin solution in serum-free medium (v/
v) at 37 °C, 5% CO2, and humidified atmosphere. The resulting
fluorescence of the metabolized product was monitored using a Tecan
infinite M200 plate reader. All assays were performed as triplicates.
The p-value given in Figure 3b was calculated with an unpaired t test
using Microsoft Excel.
Western Blot Analysis of Intracellular CK2 Phosphorylation.
The human cell lines HEK293 (ATCC: CRL-1573) and HeLa
(ATCC: CCL-2) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Invitrogen GmbH, Karlsruhe, Germany)
supplemented with 10% FBS at 37 °C in an atmosphere enriched
with 5% CO2. Cells were seeded in 60 mm cell culture dishes the day
before treatment. Ten millimolar stock solutions of the CK2
inhibitors KN2 and CX-4945 (free acid; purchased from Sell-
eckChem, Munich, Germany), each dissolved in dimethyl sulfoxide
(Merck, Darmstadt, Germany), were diluted in experiments to a final
concentration of 10, 20, or 50 μM, respectively. Control experiments
were performed with an equal volume of DMSO.
After treatment for 5 or 24 h, cells were harvested and lysed with
the double volume of RIPA buffer (50 mM Tris/HCl, pH 8.0, 150
mM NaCl, 0.5% sodium desoxycholate, 1% Triton X-100, 0.1%
sodium dodecyl sulfate) supplemented with the protease inhibitor
cocktail complete (Roche Diagnostics, Mannheim, Germany) and the
phosphatase inhibitor cocktail PhosStop (Roche Diagnostics,
Mannheim, Germany). After lysis, cell debris was removed by
centrifugation. The protein content was determined according to a
modified Bradford method (BioRad, Munich, Germany). Proteins
were separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (two times for each cell line) and blotted onto
polyvinylidene fluoride membranes. For each cell line, one membrane
was incubated with a rabbit polyclonal antibody against total Akt
(#9272, Cell Signaling Technology, Frankfurt, Germany) and the
other with a rabbit monoclonal antibody recognizing phospho-S129-
Akt (ab133458, Abcam, Berlin, Germany). Incubation with the
primary antibodies was performed in a dilution 1:1000 in Tris-
buffered saline (TBS, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl) with
0.1% Tween-20 (TBS-T) and 5% bovine serum albumin for phospho-
Akt or TBS-T with 5% nonfat dry milk for total Akt at 4 °C overnight.
Then the membrane was washed twice for 10 min with TBS-T.
Incubation with the peroxidase-coupled secondary anti-rabbit anti-
body followed for 1 h in the corresponding incubation buffer. The
membrane was washed again twice for 10 min with TBS-T. Signals
were developed and visualized by the SuperSignalTM West Pico Plus
chemiluminescent substrate of Thermo Scientific (Rockford, USA).
Determination of a Protein Kinase Selectivity Profile of
KN2. The screening service of Carna Bioscience, Inc., Kobe, Japan,
was commissioned to determine a protein kinase selectivity profile of
KN2. The company offers different representative subsets of the EPK
superfamily for screening. We selected (i) the “tyrosine kinase (TK)
panel”, version 2.0, with 20 enzymes, (ii) the “serine/threonine kinase
(STK) panel”, version 3.0, with 30 enzymes, (iii) the “cell cycle
panel”, version 2.0, with 30 enzymesamong them the CK2α- and
the CK2α′-based CK2 holoenzymeinvolved in cell cycle control,
and (iv) 11 individual EPKs for which significant off-target effects
with certain CK2 inhibitors had been reported. The STK panel and
the cell cycle panel are partly redundant so that on the whole 83 EPKs
were used for the screening runs. All of them were applied as GST
fusion constructs expressed in insect cells (baculovirus expression
systems).
All EPK assays were performed in duplicate. The KN2
concentration was 3 μM in all assay mixtures. The ATP concentration
for each kinase assay is given in Table S2; it was in the range of the
KM value of the respective kinase for ATP. The substrates for
phosphorylation were EPK-specific peptides; the substrate concen-
tration was 1 μM in all cases except PLK2, where it was 0.05 μM. For
each EPK, the mean of the readout values of two control reactions
(complete reaction mixture without inhibitor) was set as 0%
inhibition, and the mean of the readout values of two background
mixtures (without enzyme) was set as 100% inhibition; then, the
percentage inhibition for each individual assay reaction was calculated.
These values are visible in columns 4 and 5 of Table S2.
For a quantitative expression of selectivity, the Gini coefficients for
3 μM KN2, 10 μM TBI, and 1 μM 4,5,6,7-tetrabromo-1H-
benzo[d][1,2,3]triazole were calculated as described by Graczyk46
using a Microsoft Excel file provided in the Supporting Information of
that publication. In the case of TBI and 4,5,6,7-tetrabromo-1H-
benzo[d][1,2,3]triazole, the selectivity profile data published by
Pagano et al.45 were taken for this purpose.
Crystal Structure Determination and Illustration. The basis
for determination of the CK2α′Cys336Ser/DPA and CK2α′Cys336Ser/KN2
complex structures of Table S1 is a procedure described recently.35
Briefly, CK2α′Cys336Ser/MB002 cocrystals were grown in 28% (w/v)
PEG 6000, 900 mM LiCl, 100 mM Tris/HCl, pH 8.5, optimized by
micro- and macroseeding. The crystals were purged from unbound
MB002, stabilized by dehydration, and finally subjected to extensive
soaking in solutions with gradually increasing concentrations of the
ligand of interest.
CK2α1−335 crystals for subsequent soaking experiments with KN2
were grown in 200 mM ammonium sulfate, 100 mM sodium
cacodylate trihydrate, pH 6.5, and 30% (w/v) PEG 8000. Unlike
CK2α′Cys336Ser, the crystallization additive MB002 was not added here.
KN2 was soaked into either CK2α1−335 or CK2α′Cys336Ser crystals by a
stepwise increase of the ligand concentration. DPA was added
simultaneously to some of the crystals with the intention to induce the
unfolding of helix αD and thus to open the αD pocket and to facilitate
the access and binding of KN2. The molar ratio of DPA to either
CK2α1−335 or CK2α′Cys336Ser in these crystallographic experiments was
varied between 0.01:1 and 1000:1.
A C-terminally truncated CK1δ construct was prepared as
described.56 The recombinant protein was diluted with storage buffer
to a concentration of 8 mg/mL. A 10 mM KN2 solution in DMSO
was mixed in a 1:9 ratio with protein solution to give a final inhibitor
concentration of 1 mM. After a 30 min incubation at ambient
temperature, the solution was centrifuged to remove any precipitation.
The clear supernatant was mixed in different ratios with reservoir
solution G8 of the PEG/Ion HT crystal screen (Hampton Research),
containing 2% (v/v) Tacsimate, pH 5.0, 100 mM sodium citrate
tribasic dihydrate, pH 5.6, and 16% (w/v) PEG 3350. The
crystallization was conducted using the sitting drop variant of vapor
diffusion at a constant temperature of 20 °C.
All CK2α′Cys336Ser, CK2α1−335, and CK1δ crystals were cryo-
protected in the respective mother liquor plus 30% (v/v) ethylene
glycol and finally flash-frozen in liquid nitrogen. X-ray diffraction data
were collected at the synchrotron beamlines given in Tables S1 and
S2. The data were collected at 100 K in all cases; the X-ray
wavelengths are indicated in Tables S1 and S2.
The diffraction data were processed with the AUTOPROC toolbox
(version 1.0.5)57 using default settings. The pipeline applied XDS58
for indexing and integration, POINTLESS59 and AIMLESS60 from
the CCP4 suite61 for symmetry determination and scaling, and finally
STARANISO62 for anisotropy analysis. The statistics in Tables S1 and
S2 refer to the ellipsoidal data sets that were used for the further
procedure.
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The two CK2α′Cys336Ser structures were solved ab initio with
ARCIMBOLDO63 followed by automated model building with the
autobuild module of PHENIX.64 The other structures were
determined with molecular replacement using PHASER65 within
PHENIX64 for search calculations. Chain A of a CK2α1−335 structure
recently deposited at the PDB66 (PDB_ID 6TEI)38 and a CK1δ
structure (PDB_ID 5MQV)56 served as search models. All structures
were optimized with several rounds of PHENIX refinement,64
alternating with manual modeling using COOT.67 The structural
parametrization of the KN2 molecule was performed with the
eLBOW module68 of PHENIX,64 whereas the parameters for MB002
and DPA were taken from the PDB66 via COOT.67
The structures pictured in Figure 2, Figure 5a, and Figure S2 were
prepared with PyMol.69 The two-dimensional projection of KN2
binding to CK2α′Cys336Ser in Figure 5b was generated with LigPlot+.51
Movies S1 and S2 were produced with UCSF Chimera (version
1.15)70 and the open source cross platform video editor Shotcut
(version 19.04.30).
Merlin Klußmann − Department fur Chemie, Institut fur
̈ ̈
Biochemie, Universität zu Köln, D-50674 Köln, Germany;
orcid.org/0000-0001-6041-0501
Ines Neundorf − Department fur Chemie, Institut fur
̈ ̈
Biochemie, Universität zu Köln, D-50674 Köln, Germany;
orcid.org/0000-0001-6450-3991
Claudia Götz − Medizinische Biochemie und
Molekularbiologie, Universität des Saarlandes, D-66421
Homburg/Saar, Germany; orcid.org/0000-0001-7115-
0485
Joachim Jose − Institut fu
Chemie, Westfälische Wilhelms-Universität Mu
48149 Munster, Germany; orcid.org/0000-0002-0666-
2676
̈
r Pharmazeutische und Medizinische
̈
nster, D-
̈
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00063
ASSOCIATED CONTENT
Notes
■
sı
The authors declare no competing financial interest.
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00063.
ACKNOWLEDGMENTS
■
We thank Professor Ulrich Baumann (University of Cologne)
for a CK1δ expression strain and for access to protein
crystallography infrastructure (c2f.uni-koeln.de), Professor
Maria Bretner (Warsaw Technical University) for MB002
Figures S1−S8 with CK2α/CK2α′ sequence alignment,
CK1δ/KN2 crystal structure, dose−response curve for
the CK1ε inhibition by KN2, 1H NMR spectra of
compound 1 and KN2, HPLC chromatogram of KN2,
13C NMR spectrum of KN2, and HRMS spectrum of
KN2; Tables S1 and S2 with X-ray diffraction data and
refinement statistics; Table S3 with selectivity profiling
data (PDF)
and TBIa, Dr. Jörg Fabian (University of Mu
measurement and Dr. Jens Köhler (University of Mu
̈
nster) for HRMS
nster) for
̈
NMR spectroscopy with KN2. We are grateful to the staff of
the European Synchrotron Radiation Facility (ESRF), of the
Helmholtz-Zentrum Berlin and of the European Molecular
Biology Laboratory (EMBL) at DESY for assistance with
diffraction data collection. The work was funded by the
Deutsche Forschungsgemeinschaft (DFG; Grant NI 643/4-2).
Movies S1 and S2 illustrating the local mobility of the
helix αD region of crystalline CK2α′ upon binding of
DPA and KN2, respectively (MP4, MP4)
SMILES molecular formula strings and Ki values (CSV)
Atomic coordinates and experimental structure factors of
the three CK2α/CK2α′ structures in Table S1 (PDB,
CIF, PDB, CIF, PDB, CIF)
ABBREVIATIONS
■
CK1δ, casein kinase 1, isoform δ; CK1ε, casein kinase 1,
isoform ε; CK2, casein kinase 2; CK2α, catalytic subunit of
protein kinase CK2; CK2α′, paralogous isoform of CK2α;
CK2β, regulatory subunit of protein kinase CK2; PDB, Protein
Data Bank
Accession Codes
The PDB codes of the three crystal structures (Table S1) are
7AT9 (CK2α′Cys336Ser/DPA/MB002), 7ATV (CK2α′Cys336Ser
KN2), and 7AT5 (CK2α1−335/KN2).
/
REFERENCES
■
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Karsten Niefind − Department fu
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̈ ̈
Biochemie, Universität zu Köln, D-50674 Köln, Germany;
orcid.org/0000-0002-0183-6315; Phone: +49-221-470-
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221-470-3244
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Biochemie, Universität zu Köln, D-50674 Köln, Germany;
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Anna Nickelsen − Institut fu
Medizinische Chemie, Westfälische Wilhelms-Universität
Munster, D-48149 Munster, Germany; orcid.org/0000-
0001-9887-6403
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r Pharmazeutische und
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