A novel class of selective CK2 inhibitors targeting its open hinge conformation

Article abstract of DOI:10.1016/j.ejmech.2020.112267

Protein kinase CK2 sustains cancer growth, especially in hematological malignancies. Its inhibitor SRPIN803, based on a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold, showed notable specificity. Our synthesis of the initially proposed SRPIN803 resulted in its constitutional isomer SRPIN803-revised, where the 2-cyano-2-propenamide group does not cyclise and fuse to the thiadiazole ring. Its crystallographic structure in complex with CK2α identifies the structural determinants of the reported specificity. SRPIN803-revised explores the CK2 open hinge conformation, extremely rare among kinases, also interacting with side chains from this region. Its optimization lead to the more potent compound 4, which inhibits endocellular CK2, significantly affects viability of tumour cells and shows remarkable selectivity on a panel of 320 kinases.

Full text of DOI:10.1016/j.ejmech.2020.112267

Journal Pre-proof
A novel class of selective CK2 inhibitors targeting its open hinge conformation
Andrea Dalle Vedove, Francesca Zonta, Enrico Zanforlin, Nicola Demitri, Giovanni
Ribaudo, Giulia Cazzanelli, Alberto Ongaro, Stefania Sarno, Giuseppe Zagotto,
Roberto Battistutta, Maria Ruzzene, Graziano Lolli
PII:
S0223-5234(20)30234-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112267
EJMECH 112267
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2019
Revised Date: 2 February 2020
Accepted Date: 20 March 2020
Please cite this article as: A. Dalle Vedove, F. Zonta, E. Zanforlin, N. Demitri, G. Ribaudo, G. Cazzanelli,
A. Ongaro, S. Sarno, G. Zagotto, R. Battistutta, M. Ruzzene, G. Lolli, A novel class of selective CK2
inhibitors targeting its open hinge conformation, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112267.
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A novel class of selective CK2 inhibitors targeting its open hinge conformation
Andrea Dalle Vedove1*, Francesca Zonta2*, Enrico Zanforlin3, Nicola Demitri4, Giovanni Ribaudo5,
Giulia Cazzanelli1, Alberto Ongaro5, Stefania Sarno2, Giuseppe Zagotto3, Roberto Battistutta6,
Maria Ruzzene2, Graziano Lolli1
Author Affiliations:
1Department of Cellular, Computational and Integrative Biology – CIBIO, University of Trento, Via Sommarive 9, 38123
Trento, Italy
2Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padua, Via U. Bassi 58/B, 35131
Padua, Italy
3Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via F. Marzolo 5, 35131 Padua,
Italy
4Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5 in Area Science Park, 34149 Basovizza-Trieste, Italy
5Department of Molecular and Translational Medicine, Division of Pharmacology, University of Brescia, Brescia, Italy
6Department of Chemical Sciences, University of Padua and Institute of Biomolecular Chemistry, National Research
Council (CNR), Via Marzolo 1, 35131 Padua, Italy
* The authors contributed equally to the work
Correspondence to Giuseppe Zagotto: giuseppe.zagotto@unipd.it, Roberto Battistutta: roberto.battistutta@unipd.it,
Maria Ruzzene: maria.ruzzene@unipd.it and Graziano Lolli: graziano.lolli@unitn.it.
ABSTRACT
Protein kinase CK2 sustains cancer growth, especially in hematological malignancies. Its inhibitor
SRPIN803, based on a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold, showed
notable specificity. Our synthesis of the initially proposed SRPIN803 resulted in its constitutional
isomer SRPIN803-revised, where the 2-cyano-2-propenamide group does not cyclise and fuse to
the thiadiazole ring. Its crystallographic structure in complex with CK2α identifies the structural
determinants of the reported specificity. SRPIN803-revised explores the CK2 open hinge
conformation, extremely rare among kinases, also interacting with side chains from this region. Its
optimization lead to the more potent compound 4, which inhibits endocellular CK2, significantly
affects viability of tumour cells and shows remarkable selectivity on a panel of 320 kinases.
1

INTRODUCTION
CK2 is a ubiquitous Ser/Thr protein kinase acting on several substrates whose phosphorylation is
involved in a wide range of cellular processes, spanning from cell differentiation to apoptosis [1]. It
is a tetramer, formed by two catalytic α and two regulatory β subunits. Historically considered a
constitutively active kinase [2], it has been proposed that CK2 is instead kept in a non-active state
by a peculiar inhibition mechanism, where the tetramers supramolecularly organize into trimers
and other more complex structures, and then revert to the active state upon necessity [3-5].
CK2 is expressed at abnormally high levels in various cancers and hematological malignancies [6-
7]. Its increased activity has been related with a wide range of responses, such as augmented
neovascularisation [8], multidrug resistance [9] and reduced apoptosis [10], all traits that
contribute to increased tumour aggressiveness and poorer overall prognosis.
Consistently with the protective role of CK2, tumour cells undergo apoptosis if its catalytic activity
is reduced [11]. Furthermore, treatment with CK2 inhibitors induces apoptosis more efficiently in
tumour cells than in healthy control cells [12]. Drug discovery efforts devoted at interfering with
CK2 activity culminated in the development of CX-4945 [13]; CX-4945 (silmitasertib) has recently
been designated as orphan drug for the treatment of cholangiocarcinoma [14] and is in clinical
trials for the treatment of various cancers [15].
Potency and selectivity of CK2 inhibitors rely on their interactions with both the hinge region, on
one side of the kinase ATP-binding pocket, and the peculiar positively charged region, on the other
side. Here we focus on a new class of CK2 inhibitors, based on a central 2-cyano-2-propenamide
scaffold variously substituted on both sides to optimize interactions with the two CK2 regions
named above. We first determined the crystal structure of CK2α in complex with SRPIN803, which
contains a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold where the 2-cyano-2-
2

propenamide is cyclised and fused to a thiadiazole ring (Fig. 1a). SRPIN803 was previously
identified in a drug development campaign against SRPK1 and kindly provided to us by the authors
of this study [16]. SRPK1 is a protein kinase involved in the regulation of various RNA-processing
pathways including RNA translation, stability and alternative splicing [17]. SRPIN803, tested
against a panel of 306 kinases, showed stronger inhibition of CK2 than of its original target SRPK1,
with IC50 values of 203 nM and 2.4 µM, respectively, and much lower activity on all other kinases
tested [16]. Due to the involvement of SRPK1 in angiogenesis [18] and of CK2 in neovascularisation
[8], the combined inhibitory effect of SRPIN803 has been successfully used in the treatment of a
mouse model of age-related macular degeneration, a progressive degenerative condition resulting
in vision loss [16].
Unexpectedly, the structure of SRPRIN803, as determined by crystallography in the complex with
CK2α, was in the open form as shown in Fig. 1b (i.e., different from the closed form, where the
thiadiazole nitrogen reacted with the nitrile carbon, as reported in [16] and in Fig. 1a). SRPIN803
was de novo synthesized in our laboratory according to the published protocol [16] and the
product obtained fully characterized, as described below, and named SRPIN803-rev (SRPIN803-
revised, confirming the open form, Fig. 1b). With the correct structure elucidated for the parent
inhibitor, we initiated its optimization by producing and characterizing various derivatives of
SRPIN803-rev with increased inhibitory potency against CK2. High-resolution crystallographic
structures of CK2α in complex with the various inhibitors presented here showed interactions with
side chains of the CK2α hinge region. This unique feature among the CK2 inhibitors developed so
far, all interacting with main chain atoms of the hinge region, constitutes the basis of the
noticeable selectivity of this series of compounds. Moreover, their binding is only compatible with
the open hinge conformation, extremely rare among protein kinases [19]. The best inhibitor
(compound 4) has IC50 = 280 nM against CK2α and only inhibits this kinase and its paralogue CK2α’
3

by more than 50% when tested at 1 µM on a panel of 320 kinases. The most promising
compounds, when tested on a cell-based assay, demonstrated to be cell permeable and to inhibit
endocellular CK2, thus promoting tumour cells death.
RESULTS AND DISCUSSION
Synthesis and characterization of SRPIN803-rev
SRPIN803-inp (SRPIN803, initially proposed), first developed and kindly provided to us by the Prof.
Hagiwara laboratory, was soaked into CK2 apo crystal and the crystallographic structure of the
complex determined at 1.5 Å resolution. Clear positive electron density was present in the kinase
ATP pocket, not compatible with the structure of SRPIN803-inp (Fig. 1a) but instead corresponding
to compound SRPIN803-rev (Fig. 1b and S1). The observed electron density did not change when
only the first diffraction images were integrated, excluding a dose-dependent X-ray radiation
damage. To confirm the X-ray evidence, we de novo synthesized the compound in agreement with
the published method for SRPIN803-inp [16] and fully characterized it. SRPIN803-inp (Fig. 1a) and
SRPIN803-rev (Fig. 1b) are constitutional isomers and cannot be distinguished by mass
spectrometry. Also, the NMR spectra for SRPIN803-rev confirm what reported for SRPIN803-inp
[16] and are compatible with both chemical structures since the amidic proton of SRPIN803-rev
cannot be distinguished from the imidic proton of SRPIN803-inp and even the quaternary carbon
of the cyano group in SRPIN803-rev from the same imidic carbon in SRPIN803-inp. However, the IR
spectrum of SRPIN803-inp (Supplementary Information, SI) shows a peak at 2222.16 cm-1
corresponding to the cyano group (the same peak is present in the IR spectra of compounds 2 –
11, Table 1 and SI) and the HPLC profile excluded a mixture of the open SRPIN803-rev and closed
SRPIN803-inp forms. Finally, and to fully exclude instability of SRPIN803-inp in the soaking
conditions, the crystallographic structure of the isolated SRPIN803-rev (SI, Fig. S3-S6 and Table S2)
4

confirmed the structure as the one observed in complex with CK2. Extremely similar cases of i) a
synthesis aiming at producing a thiadiazolopyrimidinone and resulting in a monocyclic
thiadiazolocyanoacrylamide and ii) an incorrect assignment of the chemical structure of a
compound in a commercial library have already been reported [20-21].
Binding mode of SRPIN803-rev
The ATP binding pocket of CK2 can roughly be divided into three areas: the basic region, where
Lys68 holds in place the ATP β-phosphate; the hinge region, on the opposite side, corresponding
to the 114-120 stretch of amino acids connecting the N-terminal and C-terminal lobes of the
protein; the hydrophobic area, in between the two previous regions, comprising residues Val53,
Val66, Ile95, Phe113, Met163 and Ile174 and sandwiching the adenosine moiety of ATP.
SRPIN803-rev is fully buried in the ATP binding pocket of CK2α, spanning from the basic to the
hinge region (Fig. 1c). The molecule is anchored to the protein through its guaiacol moiety, with
both oxygen atoms tightly interacting with the positively charged ammonium tail of Lys68 side
chain. In addition, the guaiacol hydroxyl further interacts with the main chain amide of Asp175
and with a conserved water molecule (W1) [22] that is held in position by Trp176 main chain
amide nitrogen and Glu81 side chain (Fig. 1d).
5

Figure 1. Binding mode of SRPIN803-rev in the CK2 ATP-pocket. a) chemical structure of SRPIN803-inp. b) chemical
structure of SRPIN803-rev. c) SRPIN803-rev inserts deeply into the pocket contacting both the basic region (blue
surface) and the hinge region (pink surface). d) The guaiacol headgroup is involved in a number of polar interactions
with residues in the CK2 basic region. e) The central 2-cyano-2-propenamide group is sandwiched by hydrophobic
residues and contacts the hinge region through water-bridged hydrogen bonds. f) The thiadiazole ring stacks in
between side chains of Leu45 and Asn118, while the trifluoromethyl end is involved in various polar interactions.
The central 2-cyano-2-propenamide region is involved in van der Waals and hydrophobic contacts
with the aforementioned apolar residues lining the cavity (Fig. 1e). It also contacts the hinge
region through a water-mediated hydrogen bond between the propenamide carbonyl oxygen and
Glu114 main chain oxygen and Val116 main chain nitrogen. The nitrile group also contributes to
the binding affinity through its electron-withdrawal effect, with the ultimate increase in acidity of
the guaiacol hydroxy; its pKa is estimated in 8.8 with a decrease of about 1 unit with respect to
6

molecules with a methyl or an ethynyl group in place of the nitrile [23]. In the local context of the
CK2 basic region, the deprotonated form of the inhibitor should be, at least minimally, populated.
Finally, the thiadiazole ring is sandwiched between side chains of Leu45 and Asn118, while the
trifluoromethyl group gets jammed in between the hinge region and the N-terminal lobe (Fig. 1f).
Several polar interactions are established between two fluorine atoms and the protein matrix,
namely with Val116 main chain oxygen, Asn118 main chain nitrogen and the side chains of Glu55,
Asn117 and His115, although poorly oriented for the establishment of hydrogen and halogen
bonds. The observed binding mode of SRPIN803-rev in CK2 is different from the proposed docking
pose of SRPIN803-inp in complex with the same kinase (Fig. S2).
Structure-activity relationship for 2-cyano-2-propenamide compounds
IC50 for SRPIN803-rev was estimated in 1.37 µM against CK2α. In order to increase the affinity of
SRPIN803-rev, we focused on the two regions flanking the central 2-cyano-2-propenamide scaffold
(Table 1 and Scheme 1).
First, compounds 2-4 were synthesized as derivatives of SRPIN803-rev with the trifluoromethyl
group substituted with methyl, tert-butyl and bromine, respectively. Compound 2 is only slightly
more potent than SRPIN803-rev while compound 3 shows a fourfold increase in inhibitor potency
with respect to SRPIN803-rev; compound 4 is the most potent among all tested inhibitors in this
work with an IC50 = 280 nM.
7

The position of the guaiacol group is almost identical in SRPIN803-rev and compounds 3 and 4 (Fig.
2a) in complex with CK2α, as determined by X-ray crystallography; all its interactions with the
protein matrix are conserved. In compound 3, the bulkier tert-butyl-thiadiazole group moves
significantly toward the C-terminal part of the hinge region, as compared with SRPIN803-rev, to
optimize interactions with residues from both the hinge and the CK2 N-terminal lobe. The bromo-
thiadiazole tail of compound 4 is closer to the hinge region with respect to compound 3, but also
to SRPIN803-rev (Fig. 2a); an interaction between the thiadiazole sulphur and Val116 main chain
oxygen is established. The S--O distance decreases to 3.1 Å (Car-S--O angle 127°), being 3.5 Å in the
complex with SRPIN803-rev. The thiadiazole sulfur acts as an electrophile due to the donation of
its pz electrons to the aromatic system and interacts with the carbonyl oxygen through σ-hole
bonding [24]. Strength of interaction increases with the number of the electron-withdrawing
nitrogen atoms on the ring, so that thiadiazole is much favoured over thiazole and thiophene.
Energy for the thiadiazole S--O interaction, stronger than a typical hydrogen bond, has been
computed in -27 kJ/mol at the optimal distance (3 Å) and angle (156°) [24].
On the opposite side of the molecule, the guaiacol methoxyl was then replaced by a nitro group
with the aim of increasing the acidity of the ortho hydroxyl. The predicted effect is a large
decrease of the phenolic hydroxy pKa to a value of 5.7 with the outcome of a fully deprotonated
phenolate in the basic region of the CK2 pocket [23]. The o-nitrophenolic compounds 5-7 were
synthesised, which included a thiadiazole ring derivatised with either trifluoromethyl or methyl or
bromine, respectively. Inhibitory power, with respect SRPIN803-rev, was doubled for compound 5
and the improvement was almost three times for compound 6, as compared with 2. This
confirmed the favourable contribution of the o-nitro group for these compounds. Interestingly,
this contribution is abolished in the context of the bromo-thiadiazole compounds and the o-
nitrophenolic compound 7 performed slightly worse than the guaiacol-bearing compound 4.
8

The crystallographic structure of compound 7 in complex with CK2α shows a binding mode very
similar to that observed for compound 4, with a limited sliding towards the hinge region to
accommodate the bulkier (with respect to the methoxyl) nitro group in the CK2 basic region (Fig.
2b). The o-nitrophenolic group maintains all interactions observed for the guaiacol ring, with Lys68
side chain involved in ionic interactions to both the phenolate oxygen and the charged nitro group
(Fig. 1d and 2c). The nitro group, however, gets also close to the Asp175 side chain. The
thiadiazole sulphur is at 3.1 Å (Car-S--O angle 133°) from Val116 carbonyl oxygen. The slight shift of
compound 7 with respect to 4 causes a larger protrusion of the bromine atom toward the hinge
region (Fig. 2b).
Figure 2. Comparison of binding modes for SRPIN803-rev (green) and compounds 3 (orange), 4 (purple), 7 (cyan) and
14 (yellow). a) Tert-butyl-thiadiazole (3) moves away from the hinge region with respect to SRPIN803-rev (1), bromo-
thiadiazole (4) moves closer. b) The nitrophenol moiety causes the bromothiadiazole of compound 7 to protrude more
on the top of the hinge region with respect to the bromothiadiazole of compound 4 holding a guaiacol group on the
opposite side. c) The nitrophenolic headgroup of compound 7 interacts with CK2α similarly to what observed in
compound 4. d) The o-hydroxybenzene ring in compound 14 exposes the phenolic oxygen to the solvent.
9

We then combined the best hinge-contacting bromo-thiadiazole tail with a o-bromophenolic
headgroup, with the aim of increasing the acidity of the phenolic oxygen (predicted pKa 7.2) with a
small and neutral substituent. Compound 8 shows a modest IC50, being significantly worse than
both compounds 4 and 7. Compounds 9-11, bearing an aniline ring instead of the guaiacol group
of SRPIN803-rev and variably substituted on the thiadiazole ring, failed to bind to the kinase when
tested by X-ray crystallography, confirming the fundamental role held by acidic groups in
complementing the basic region of the CK2 pocket.
Finally, we explored the possibility of substituting the polar thiadiazole 5-member ring with a
significantly different benzene ring. A substructure search on the ZINC15 database was performed
with the 2-cyano-3-(4-hydroxy-3-methoxyphenyl)-N-phenylprop-2-enamide scaffold. The very
diverse compounds 12-14 were selected.
Compound 12 bears a 2-chloro-5-trifluoromethyl-benzene ring, which should reproduce the
interactions observed in SRPIN803-rev via its trifluoromethyl group. The compound was not fully
soluble at the millimolar concentration needed for the soaking procedure and, although
unambiguously present in the CK2α pocket, its occupancy in the crystallographic structure was
limited (data not shown). The measured IC50 towards CK2 was 0.37 µM.
Compound 13 bears a p-nitrobenzene ring; the nitro group was selected as a possible new
specificity determinant to locate in between Arg43 side chain and the hinge downstream to the
previously explored region. No extra electron density was present in the CK2α crystal soaked with
compound 13, which then failed to bind to the kinase.
Compound 14 has a o-hydroxybenzene ring to look for hydrogen bonds with main chain atoms of
the hinge region, interactions common in various CK2 inhibitors. The crystal structure shows that
10

the o-hydroxyl group points instead towards the solvent, avoiding the higher energy conformation
with the phenolic and the amidic oxygens pointing in the same direction (Fig. 2d).
Target specificity of selected 2-cyano-2-propenamide compounds
The 2-cyano-2-propenamide compounds here reported are the first CK2 inhibitors protruding from
the active site on the top of the hinge region and in direct contact with amino acidic side chains
from this region. The peculiar binding mode should define a remarkable selectivity for these
compounds. First, SRPIN803-rev and compounds 4 and 7 were tested on the tetrameric CK2 α2β2
holoenzyme by kinase assay, obtaining very similar IC50s as those reported in Table 1 for the α
subunit; this confirms that the open hinge conformation is accessible also in the holoenzyme.
Then, the most potent compound 4 was tested on a panel of 320 kinases. Only CK2α and its
paralogue CK2α’ were inhibited by more than 50% when tested at 1 µM (SI).
CK2 has a flexible hinge/αD region which can assume two different structural arrangements: an
open conformation which is extremely rare among protein kinases [19], and a closed
conformation recapitulating the correctly assembled catalytic spine (C-spine), as seen in the vast
majority of the protein kinases [25]. Binding of the compounds here reported is only compatible
with the open conformation of the hinge/αD region (Fig. 3). Then, direct interactions with side
chains of the hinge region, a feature not previously reported for any CK2 inhibitor only contacting
main chain atoms of the same region, guarantee additional selectivity. This is evident when
comparing compound 4 with the similar tyrphostin AG99 (PDB 6QS5) [26], interacting only with
main chain atoms of the hinge region, compatible with both the open and closed hinge
conformations, and not selective (Fig. 3a).
The ability to target the CK2 open conformation has recently been exploited by the most selective
CK2 inhibitor reported to date [27-28]. However, CAM4066 (IC50 = 370 nM) has a completely
11

different binding mode and, contrary to the inhibitors reported here, targets the αD but not the
hinge region (Fig. 3b).
Figure 3. Comparison of binding modes for compound 4 (purple), tyrphostin AG99 (magenta) and CAM4066 (cyan). a)
Tyrphostin AG99 interacts with main chain atoms of the hinge region, while compound 4 is also in contact with side
chains atoms. Binding of compound 4 is only compatible with the peculiar open hinge conformation. b) Compounds 4
and CAM4066 are compatible with CK2 open conformation (white) but not with the closed conformation (yellow).
Compound 4 discriminates the two conformations at the level of the hinge region, while CAM4066 clashes with a
closed αD helix.
Cellular activity of selected 2-cyano-2-propenamide compounds
The more promising molecules were analyzed for their efficacy in cells, with special focus on the
best inhibitor, compound 4, in comparison to SRPIN803-rev. We treated Jurkat cells, a human
leukemia T-cell line that, since the beginning [29], has been frequently exploited to understand
the potential of targeting CK2 in tumour cells. We first assessed whether cells treated with the
inhibitors displayed a reduced activity of endocellular CK2. This was confirmed by the analysis of
the CK2-dependent phospho-site Akt Ser129 [30]: as shown in Fig. 4a, while the amount of the
kinase remained intact in treated cells, the Akt phosphorylation level decreased in response to
both SRPIN803-rev and compound 4. Since cell death is expected to occur in tumour cells
whenever CK2 is inhibited [12], we measured the effect of the compounds on Jurkat cell viability.
Results are shown in Fig. 4b, where also the effect of compound 7 is reported: all the inhibitors
12

turned out to significantly reduce cell viability in a dose-dependent manner. The DC50 values
(concentrations inducing 50% of cell death compared to vehicle-treated cells in 24h) indicate very
similar efficacies for SRPIN803-rev and compound 4, while 7 is less effective. The lower cellular
activity of compound 7 can be ascribed to a reduced membrane permeability caused by the
negative charge of the o-nitrophenolate ring.
Figure 4. Effects of compounds in cells. A) Jurkat cells were treated with the indicated compounds for 16h. 20 µg of
proteins from total lysate were analysed by WB with anti phospho-Ser129 Akt antibody (upper), or an antibody
recognizing both CK2 catalytic subunit α and α’ (middle). 5 µg proteins were analysed for β-actin (lower WB), as
loading control. Representative WB of three independent experiments are shown. b) Jurkat cells were treated for 24h
with increasing concentrations of the indicated compounds. Cell viability was assessed by the MTT method, and is
shown as % of vehicle-treated (Control) cells. On the right, the calculated DC50 values are reported (mean values +
SEM of at least four experiments).
Similar results were observed in another tumour cell line; in accordance with the principle of
tumour cell addiction to CK2 [12], viability of non-tumour cells was hardly affected (SI, Fig. S7).
CONCLUSIONS
The structural bases for the binding mode of SRPIN803-inp to CK2 and its reported specificity [16]
were puzzling. Its co-crystallization with CK2α identified a chemical structure different from the
one reported (SRPIN803-rev). De novo synthesis of SRPIN803-inp also resulted in SRPIN803-rev.
Based on our characterization of SRPIN803-rev and SRPIN803-inp, and on previous reports
describing very similar discrepancies, in a very similar molecular context, between the expected
13

and the obtained synthetic products [20-21], we conclude that SRPIN803-inp has indeed the
chemical structure of SRPIN803-rev. Moreover, very similar biologically active compounds based
on the 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold have been described, which
are commercially available or obtained through a very similar synthetic scheme (examples in [31-
32]). Chemical formulas for these compounds, as well as for the commercially available SRPIN803,
could have been similarly misinterpreted.
The crystallographic structure of SRPIN803-rev in complex with CK2α revealed that the observed
specificity relies on the exploration of the CK2 open hinge conformation, particularly sporadic in
other kinases, combined with interactions with side chains from the same region. Most of the
synthesized derivatives of SRPIN803-rev showed increased inhibition of CK2α. The most potent
compound 4 retained significant selectivity when tested on a panel of 320 kinases, only inhibiting
CK2α and CK2α’ by more than 50% at 1 µM concentration. Compound 4 is cell permeable, inhibits
endocellular CK2 and significantly reduces tumour cells viability.
Compounds 4, in comparison with the clinical-grade CX-4945 inhibitor and despite the lower
potency in kinase assay (IC50 280 nM and 1 nM, respectively), shows a similar efficacy in the
cellular context: the 12.8 µM DC50 that we measured in a 24h-assay in Jurkat cells for compound 4
is not far from the 2.5 µM value reported for CX-4945 in a 4-days assay [33]. Moreover, CX-4945
inhibits at least other 12 kinases in the low nanomolar range [27, 33-35].
The cyanoacrylamide group, present in all compounds here described, is a known covalent binding
group. It has been used also in kinase inhibitors for covalent binding to cysteine residues in the
ATP-binding pocket [36-40]. In the CK2 pocket, there are no cysteines and the binding mode is
non-covalent. Given the selectivity profile of compound 4 on a panel of 340 kinases (SI), significant
off-target covalent inhibition can be excluded, at least for that compound and set of kinases,
which include Cys-containing ATP-binding pockets (PLK1, NEK2, JAK3, EGFR, BTK1, RPS6KA3,
14

among others). Moreover, the presence of the cyanoacrylamide group is not sufficient for
covalent binding: the inhibitor must be designed to dock into the protein with a binding mode that
brings the reactive double bond close to the Cys thiol and with the proper orientation for the
covalent bond formation. Finally, the resulting covalent bond is reversible [36-40] with both the
reacted and unreacted species visible in the crystal structure in one case (PDB 5LWN) [40]. The
cyano group transforms an irreversible conjugate addiction (with the simple acrylamide moiety) to
a reversible reaction (with the α-cyano acrylamide) [41].
The guaiacol group has been reported as a radical scavenger and a metal chelating group flagging
it as a possible source of unwanted interactions. However, these unspecific activities are common
to many phenolic compounds, some of them also used in specific clinic settings, viz. carbidopa and
levodopa.
MATERIALS AND METHODS
Chemistry
The synthetic procedures for the preparation, isolation and characterization of SRPIN803-rev and
compounds 2-11 are reported in the SI. Compounds 12-14 were purchased from VITAS-M
Laboratory. All compounds were negative as PAINS or aggregators when tested on dedicated web
servers [42-43].
Protein production
The expression of human CK2 α and β subunits was induced in E. coli BL21-DE3 with 0,5 mM IPTG
0
for 4 hours at 30 C. CK2α pellet mixed with an equal amount of pellet expressing β subunit was
resuspended in buffer A (50 mM Tris-HCl pH 8.5 and 7 mM 2-Mercaptoethanol). After sonication
and centrifugation, the supernatant was adjusted at the salt concentration of 0,4 M NaCl and
15

loaded on Heparin-Sepharose (5 ml, GE Healthcare). The column was eluted with a linear gradient
from 0.4 to 1 M NaCl in buffer A. Holoenzyme α2β2 was purified to homogeneity by size-exclusion
chromatography on Superdex 200 (26/60, GE Healthcare) in buffer A + 0,5 M NaCl. The fractions
containing the purified holoenzyme were pooled and dialyzed for 4 hours against 25 mM Tris-HCl
0
pH 7,5 and 50% glycerol and stored at -80 C. The same protocol was applied to purify CK2α used
in activity assays.
CK2α for crystallization purposes was produced as previously reported [44]. Briefly, after
expression in E. coli BL21-DE3, human CK2α (aa. 1-336) was purified by sequential affinity, anion
exchange and size-exclusion chromatography (HiTrap Heparin, MonoQ and Superdex 75). Protein
was concentrated to 10 mg/ml and frozen in liquid nitrogen.
Crystallization and structure solution
Crystals of apo CK2 and binding of inhibitors in its ATP pocket were obtained as previously
described [45]. Briefly, CK2 crystals were first obtained by vapor diffusion sitting drop and then
soaked in the cryoprotective solution containing 5 mM inhibitor in 1% DMSO. Diffraction data
were collected at the Elettra Synchrotron Light Source (Trieste, Italy), XRD1 beamline and at the
ESRF (Grenoble, France), beamlines ID30-A1 and ID30-A3. Data were processed with XDS [46] and
Aimless [47]; structures were solved by molecular replacement with Phaser [48] using PDB 4KWP
[49] as a search model. Initial models were refined alternating cycles of automatic refinement with
Phenix [50] and manual model building with COOT [51].
Data collection and refinement statistics are reported in Table S1.
In vitro CK2 activity assay
16

Recombinant α CK2 (20–50 ng) was incubated with 0.1 mM synthetic peptide substrate
RRRADDSDDDDD (CK2-tide) in a phosphorylation buffer containing 50 mM Tris–HCl pH 7.5, 10 mM
MgCl2, 20 μM [γ-33P]ATP (1000–2000 cpm/pmol), in a final volume of 30 μl, with increasing
concentrations of each inhibitor. Controls were performed in the absence of any inhibitor, but
with the addition of the solvent DMSO (3%, v/v), which was ineffective on CK2 activity. Reactions
were performed at 30 °C for 10 min, and stopped by sample absorption on phospho-cellulose
papers. Papers were washed three times with 75 mM phosphoric acid and counted in a
scintillation counter (Perkin Elmer). The same protocol was applied for the activity of recombinant
α2β2 CK2, except for the addition of 0.1 M NaCl in the phosphorylation assay. Results were
analysed by GraphPad Prism 7.0a software, for the calculation of the IC50 (concentrations inducing
50% inhibition).
Kinase panel
The activity of compound 4 was tested at 1 µM in duplicate on a 320 protein kinases panel at
ProQinase GmbH (Freiburg – Germany, https://www.proqinase.com). A radiometric protein kinase
33
®
assay ( PanQinase Activity Assay) was used for measuring the kinase activities [52].
Cell culture, treatments and lysis
Jurkat and CEM cells (human T lymphoblastoid cell line) were cultured in an atmosphere
containing 5% CO2, maintained in RPMI 1640 medium (Sigma), supplemented with 10% (v/v) fetal
calf serum (FCS), 2mM L-glutamine, 100U/mL penicillin, and 100mg/mL streptomycin. Cell
treatments with inhibitors were performed in the culture medium, but with 1% (v/v) FCS. Control
cells were treated with equal amounts of the inhibitor solvent (DMSO), which never exceeded 1%
17

(v/v). For lysate preparation, cells were lysed as described in [53]. Protein concentration was
determined by the Bradford method.
Cell viability assay
Cell viability was detected by means of MTT (3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltriazolium
bromide) reagent: cells (105 cells/100 µl) were incubated for 24h in a 96-well plate under the
indicated conditions. 1 h before the end of the incubation, 10 µl of MTT solution (5 mg/ml in
phosphate buffered saline) were added to each well. Incubations were stopped by addition of 20
µl of lysis solution at pH 4.7, as described elsewhere [53]. Plates were read for OD at λ 590 nm, in
a Titertek Multiskan Plus plate reader (Flow Laboratories). DC50 values (concentrations inducing
50% cell death in 24h) were calculated by analysing the results with GraphPad Prism 7.0a
software.
Endocellular CK2 activity assay
Endocellular CK2 activity was evaluated by assessing the phosphorylation state of the CK2
substrate Akt phosphor-Ser129 (Abcam) as in [54]. For this purpose, equal amounts of proteins
from treated cells were loaded on 11% SDS-PAGE, blotted on Immobilon-P membranes (Millipore),
processed in Western blot (WB), and detected by chemiluminescence. Endocellular CK2 amount
was assessed by WB with an antibody recognizing the two catalytic isoforms α and α’ (Biorad
Laboratories). β-actin antibody, used for normalization, was from Sigma. Quantitation of the signal
was obtained by chemiluminescence detection on a Kodak Image Station 440MMPRO and analysis
with the Kodak 1DImage software.
ASSOCIATED CONTENT
18

The Supporting Information contains further experimental details, tables and figures, as
consecutively outlined. Protein Crystallography: collection and refinement statistics, electron
density maps, comparison with SRPIN803 docking pose. Small molecule crystallography: methods,
results and collection and refinement statistics. Kinase assay: IC50 curves. Kinase panel profiling.
1
Synthetic methods, H-NMR, 13C-NMR, IR and HPLC traces for SRPIN803-inp, SRPIN803-rev and
compounds 2-11.
DECLARATION OF INTEREST
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Massimo Bellanda and Prof. Danilo Pedron (University of Padua, Italy) for their help
with NMR and IR spectra acquisition and analysis. We are grateful to the staff of the XRD1
beamline at Elettra (Trieste, Italy) and of the ID30-A1 and ID30-A3 beamlines at ESRF (Grenoble,
France), for on-site assistance. G.L. is supported by the Associazione Italiana per la Ricerca sul
Cancro (AIRC “MFAG 2017”, code 19882). We are grateful to Prof. Masatoshi Hagiwara (Kyoto
University, Japan) for providing us with SRPIN803.
Accession Codes
Structures were deposited to the PDB with accession numbers 6RB1 (SRPIN803-rev), 6RCM (cmp
3), 6RFE (cmp 4), 6RFF (cmp 7) and 6RCB (cmp 14). Authors will release the atomic coordinates
and experimental data upon article publication.
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Table 1: Chemical structures of tested compounds
Compound
X
Y
IC50 (µM) (SEM)
26

SRPIN803-rev
1.37 (0.20)
0.88 (0.44)
0.36 (0.10)
0.28 (0.09)
0.71 (0.28)
0.32 (0.08)
0.37 (0.08)
2
3
4
5
6
7
8
9
1.3
ND
ND
ND
10
11
12
0.37
13
14
ND
0.33
IC50 values were determined for CK2α inhibition; compound 4 and 7 were also tested on the tetrameric
α2β2 holoenzyme obtaining very similar IC50 values.
ND = not determined; compounds failed to bind the kinase in the crystal were not tested in kinase assay
27

•
•
•
•
The correct chemical structure of a previously reported CK2 inhibitor (SRPIN803) has been
elucidated.
Its crystallographic structure in the complex with CK2 identified the molecular basis of the
reported selectivity.
A derivative (compound 4) has been synthesized and characterized with increased inhibitor
activity, while retaining selectivity on a panel of 340 kinases.
Compound 4, although less potent on kinase assay than the clinical grade CK2 inhibitor CX-
4945, showed similar potency in a cell-based assay.