A π‐halogen bond of dibenzofuranones with the gatekeeper phe113 in human protein kinase CK2 leads to potent tight binding inhibitors

Article abstract of DOI:10.3390/ph11010023

Human protein kinase CK2 is an emerging target for neoplastic diseases. Potent lead structures for human CK2 inhibitors are derived from dibenzofuranones. Two new derivatives, 7,9‐dichloro‐1,2‐dihydro‐8‐hydroxy‐4‐[(4‐methoxyphenylamino)‐methylene]dibenzo[b,d]furan‐3(2 H)‐one (4a) and (E)‐1,3‐dichloro‐6‐[(4‐methoxyphenylimino)‐methyl]dibenzo[b,d]furan‐2,7‐diol (5) were tested for inhibition of CK2 and induction of apoptosis in LNCaP cells. Both turned out to be tight binding inhibitors, with IC₅₀ values of 7 nM (4a) and 5 nM (5) and an apparent K_i value of 0.4 nM for both. Compounds 4a and 5 reduced cellular CK2 activity, indicating cell permeability. Cell viability was substantially impaired in LNCaP cells, as well as apoptosis was induced, which was not appearing in non‐neoplastic ARPE‐19 cells. Co‐crystallization of 4a and 5 revealed an unexpected π‐halogen bond of the chloro substituent at C9 with the gatekeeper amino acid Phe113, leading to an inverted binding mode in comparison to parent compound 4b, with the Cl at C6 instead, which was co‐crystallized as a control. This indicates that the position of the chloro substituent on ring A of the dibenzofuran scaffold is responsible for an inversion of the binding mode that enhances potency.

Full text of DOI:10.3390/ph11010023

pharmaceuticals
Article
A π-Halogen Bond of Dibenzofuranones with the
Gatekeeper Phe113 in Human Protein Kinase CK2
Leads to Potent Tight Binding Inhibitors
Alexander Schnitzler 1, Andreas Gratz 2, Andre Bollacke 2, Michael Weyrich 3, Uwe Kuckländer 4
,
ID
Bernhard Wünsch 2, Claudia Götz 3, Karsten Niefind 1 and Joachim Jose 2,*
ID
ID
1
Institut für Biochemie, Department für Chemie, Universität zu Köln, Zülpicher Straße 47,
D-50674 Köln, Germany; Alexander.Schnitzler@posteo.de (A.S.); karsten.niefind@uni-koeln.de (K.N.)
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus,
2
Westfälische Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany;
gratz.andreas@gmail.com (A.G.); andre.bo@web.de (A.B.); wuensch@uni-muenster.de (B.W.)
Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Kirrberger Str., Geb. 44,
D-66421 Homburg, Germany; michaelweyrich@gmx.de (M.W.); claudia.goetz@uks.eu (C.G.)
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf,
Universitätsstraße 1, D-40225 Düsseldorf, Germany; kucklaen@uni-duesseldorf.de
Correspondence: joachim.jose@uni-muenster.de; Tel.: +49-251-8332-200
3
4
*
Received: 23 November 2017; Accepted: 14 February 2018; Published: 17 February 2018
Abstract: Human protein kinase CK2 is an emerging target for neoplastic diseases. Potent lead
structures for human CK2 inhibitors are derived from dibenzofuranones. Two new derivatives,
7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)-methylene]dibenzo[b,d]furan-3(2H)-
one (4a) and (E)-1,3-dichloro-6-[(4-methoxyphenylimino)-methyl]dibenzo[b,d]furan-2,7-diol (
tested for inhibition of CK2 and induction of apoptosis in LNCaP cells. Both turned out to be tight
binding inhibitors, with IC50 values of 7 nM (4a) and 5 nM ( ) and an apparent Ki value of 0.4 nM for
both. Compounds 4a and reduced cellular CK2 activity, indicating cell permeability. Cell viability
was substantially impaired in LNCaP cells, as well as apoptosis was induced, which was not
appearing in non-neoplastic ARPE-19 cells. Co-crystallization of 4a and revealed an unexpected
-halogen bond of the chloro substituent at C9 with the gatekeeper amino acid Phe113, leading
5) were
5
5
5
π
to an inverted binding mode in comparison to parent compound 4b, with the Cl at C6 instead,
which was co-crystallized as a control. This indicates that the position of the chloro substituent
on ring A of the dibenzofuran scaffold is responsible for an inversion of the binding mode that
enhances potency.
Keywords: human protein kinase CK2; dibenzofuran; tight binding inhibitor; crystal structure;
π-halogen bond; apoptosis induction
1. Introduction
Human CK2 is an ubiquitous protein kinase, which catalyzes the phosphorylation of
serine/threonine residues within a consensus sequence which is present in a remarkable number
of human proteins [
provided and about 2300 putative phosphorylation sites have been suggested by homology to the
CK2 substrate consensus sequence [ ]. Despite the number of proteins which were experimentally
1]. Meanwhile, evidence for more than 430 CK2 phospho-sites have been
2
phosphorylated by CK2 is indeed lower, the still remaining huge number of tentative substrates
in combination with its constitutive cellular activity can explain pleiotropic effects of this enzyme
and could be a reason for its indispensable physiological role [3]. In addition there is experimental
Pharmaceuticals 2018, 11, 23; doi:10.3390/ph11010023
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evidence that the enzyme has multiple functions in signal transduction and metabolic pathways [
Studies predicting that a substantial proportion of the phosphoproteome is phosphorylated by
CK2 [ ], suggested that this kinase might be responsible for maintaining an essential level of cellular
phosphorylation [ ]. This has been supported by more recent experimental studies analyzing the CK2
4].
2
5
related phosphoproteome [6–8].
The heterotetrameric CK2 consists of two catalytically active α-isoforms (either CK2α or CK2α’)
and two
are recruited to form the quaternary complex (“holoenzyme”) with a α2β2 stoichiometry. Despite
its denomination, the “regulatory” -subunit is not directly involved in general regulating CK2
β-subunits [9]. Upon dimerization of two β-subunits via zinc-finger domains, the α-subunits
β
enzymatic activity, but appears to be rather responsible for substrate specificity and stabilization of the
holoenzyme [10]. Up to now it is unclear how CK2 activity is regulated in vivo. Some models propose
a dynamic dissociation and spatiotemporal localization as a mechanism of regulation, other reports
indicate that a controlled oligomerization of CK2 molecules may be an alternative or an additional
method to influence its activity [4,11–14].
CK2 hyperactivity has been shown to promote cell proliferation, angiogenesis and to suppress
apoptosis—three pivotal characteristics in the development of cancer. An increased CK2 level was
found in many cancer tissues [15], and in consequence it is discussed as a prognostic marker, at least
in prostate cancer [16] and acute myeloid leukemia [17]. CK2 hyperactivity can be explained by
an increased expression level of at least CK2
isozyme. It was shown that upregulated CK2 amplifies essential tumor-promoting signal transduction
pathways, such as the Wnt-, the NF- B- or the PI3K/Akt-pathway [ 18] by interfering “laterally”
with single central, but different components of these pathways [19 20]. In addition, CK2 mediates
α, but is not due to a mutated, and hence, hyperactive CK2
κ
3,
,
phosphorylation of caspase recognition sequences, masks these sites and thus protects proteins from
caspase cleavage. Consequently, CK2 interferes with caspase signaling and its activity inversely
correlates with the rate of apoptosis [21,22]. An increased cellular level of CK2 as such is not sufficient
to provoke a cancer phenotype, but it has been shown in a mouse model that cooperation of the
overexpressed enzyme with other oncogenes, such as myc or tal-1 can essentially promote cancer
development [23
,
24]. To conclude, raised CK2 activity seems to generate a cellular milieu favorable
26]. The tumor
for cancer progression, consistent with a concept of “non-oncogene addiction” [25
,
develops an undue reliance on CK2 activity to maintain the malignant phenotype [20], suggesting
CK2 to be a promising molecular target for cancer therapy [27]. In xenograft models of prostate cancer,
suppression of CK2 using antisense oligonucleotides indeed induced apoptosis of malignant cells,
which finally resulted in complete remission of the tumor [28].
The increasing evidence that human protein kinase CK2 is supposed to be a druggable pharmacological
target resulted in several drug discovery approaches. These yielded a substantial number of potent CK2
inhibitors with different chemical scaffolds [29–31]. The quinoline derivative silmitasertib (formerly known
as CX-4945) is an orally available ATP-competitive CK2 inhibitor with high affinity (Ki = 0.38 nM) [32].
Silmitasertib’s antitumor effect was suggested to be caused by the selective inhibition of CK2-dependent
signaling in diverse signal transduction pathways [33]. Clinical trials of phase 1 with silmitasertib were
recently completed with success in patients with advanced solid tumors, breast cancer, Castleman’s disease
and multiple myeloma (www.clinicaltrilas.gov identifier: NCT00891280 and NCT01199718) [32,34] and it
is at current in clinical trials of phase 2 in combination with cisplatin and gemcitabine for the treatment
of cholangiocarcinoma (www.clinicaltrials.gov identifier: NCT02128282). The still remarkable success of
silmitasertib in early drug development confirms human protein kinase CK2 to be a valid and druggable
therapeutic target. This is further confirmed by another CK2 inhibitor being in clinical trials. The peptide
CIGB-300 was identified to cover the phosphorylation site in the consensus sequence of CK2 substrates and
thereby inhibiting CK2 activity [35]. It is at current in clinical trials phase 2 for the treatment of squamous
cell carcinoma or adenocarcinoma of the cervix with local application of CIGB-300 (www.clinicaltrilas.gov
identifier: NCT01639625).

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We have identified dibenzofurans as lead structures for potent inhibitors of human protein kinase
CK2 [36]. The most potent derivative, with an IC50 value of 29 nM and a Ki value of 15 nM [37],
was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo[b,d]furan-3
(2H)-one (4b, Figure 1). Compound 4b only inhibited seven other kinases (Aurora A, KDR/VEGFR2,
SGK1, FLT4/VEGFR3, PIM1, PKD2, and LCK), to similar extent as CK2, and turned out to
be cell-permeable and capable of inducing apoptosis in the prostate cancer cell LNCaP. Most
recently it was shown, that—depending on the salt concentration—4-carboxy-6,8-dibromo-flavonol,
also a nanomolar inhibitor of CK2, forms an unusual
π-halogen bond with an aromatic side chain
of the catalytic -subunit [38]. Because this -halogen bond was found only at high concentrations
α
π
of the kosmotropic salt NaCl, but not at low concentrations, and hence was dependent on
a conformational change of the protein, it appeared worth investigating whether such halogen bonds
could also be responsible for a positional or conformational adaption of the inhibitor to a constant
protein conformation. For this purpose two derivatives of 4b were synthesized: compounds 4a
(7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)methylene]dibenzo[b,d]furan-3(2H)-
one) and
5 ((E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]dibenzo[b,d]furan-2,7-diol) (Figure 1),
both containing the second chloro substituent at a different position than 4b. In addition 4b and 4a
contained a flexible connection between the dibenzofuran scaffold and the methoxyphenyl moiety,
in contrast to
5, in order to evaluate the impact of planarity of the four ring system on inhibition
of CK2. The enzymological, cellular and structural studies with 4b
,
4a and as described herein
5
revealed that the change of the halogen position as indicated above improves the inhibitory efficacy
substantially, an effect that can be rationalized by a surprising reversal of the binding mode.
Figure 1. New dibenzofuran derivatives 4a and
5 tested for CK2 inhibition. Structural differences
between the compounds are given in red. Compound 4b was published before [28,37] and was
therefore used as a reference.
2. Results
2.1. Synthesis of 4a and 5
As previously reported, 1,4-benzoquinone reacted with 2-arylaminomethylenecyclohexanone
derivatives (e.g., , Supplementary Information Scheme S1) to afford tetrahydrocarbazoles [39].
However, chlorinated 1,4-benzoquinones provided dibenzofurans under the same reaction
2
4
conditions. The higher redox potential of chlorinated 1,4-benzoquinones 1a and 1b compared to
unsubstituted 1,4-benzoquinone is responsible for the different reaction pathways, since oxidation
of the enaminoketones 2a and 2b to provide dienaminoketones 3a and 3b is the first step of the
transformation [39]. Reaction of dienaminoketones 3a and 3b with a second equivalent of 2,6- or
2,3-dichloro-1,4-benzoquinone 1a and 1b resulted in the dibenzofurans 4a and 4b, respectively [40].
The fully aromatic dibenzofuran
1a). Since the aromatic product can exist in two tautomeric forms, a 2D NMR spectrum was recorded.
The NMR spectrum in the solvent DMSO clearly indicates the presence of the phenolimine tautomer
5 was obtained by oxidation of 4a with 2,6-dichloro-1,4-benzoquinone
(
5
,
because a correlation signal between the aldimine proton at 9.16 ppm and carbon atom at 154.3 ppm
was detected. A signal beyond 162 ppm, which was expected for the carbonyl carbon atom of a keto
enamine tautomer could not be detected. Thus, the existence of the phenolimine tautomer
the solvent DMSO was shown unequivocally.
5 at least in

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2.2. Inhibition of Human Protein Kinase CK2 In Vitro
To evaluate the inhibitory effect of the two dibenzofuran derivatives 4a and
5
on human
CK2, the recombinantly expressed and purified CK2α2β2 holenzyme was used. The phosporylation
of an artificial substrate peptide (RRRDDDSDDD) by the purified holoenzyme was analyzed in
a capillary electrophoresis (CE) assay as described before [41]. This peptide is commonly used for
the determination of CK2 activity and phosphorylation was determined in the presence and in the
absence of the compounds. For this purpose a stock solution of 10 mM in DMSO was prepared for each
compound and diluted in the assay to a final concentration of 10 µM. At this concentration the residual
enzymatic activity was less than 1% in case of both compounds, when compared to the enzymatic
activity without compounds. Consequently 13 concentrations of both dibenzofurans, ranging from
0.01 nM to 10
µM were tested on their inhibition of CK2 dependent substrate phosphorylation in
the presence of 100
µM ATP. This resulted in a dose-response curve, which served for IC50 value
determination by setting the percentage of inhibition in relation to the inhibitor concentration in
a logarithmic scale (Figure 2). By this method, the IC50 value of 4a was determined to be 7 nM
and the IC50 value for
a compound “D11” obtained by a screening of the Diversity Set III of the DTP program from the
NCI/NIH, which appears to be identical with compound in this study, and determined an IC50 value
of 7 nM towards CK2 in a radiometric assay [42]. In our preceding investigation, the Ki value of 4b
(Figure 1) had been determined experimentally to be 15 nM, by applying different ATP concentrations
ranging from 6 M to 400 M, followed by IC50 value determination for each of the different ATP
concentrations [37]. When the same procedure was applied to 4a and , however, no linear dependency
5 was determined to be 5 nM. More recently, Guerra et al. reported on
5
α
µ
µ
5
of the IC50 values obtained from the different ATP concentrations was detectable (Figures 3A,B), as it
would have been expected for ATP-competitive inhibitors. Moreover, when the CK2 activity was
determined at a constant ATP concentration of 60 µM with varying substrate concentrations at very
low concentrations of 4a, the double reciprocal plot of the data indicated a non-competitive mode of
action (data not shown), which actually suggested that 4a shows similar binding affinities for both,
the free enzyme and the enzyme—substrate binary complex, in this case. As a consequence of these
controversial results, we repeated the measurements with 4b on ATP competitiveness and controlled
the 4a and
first results coming from the co-crystallization experiments (described in detail in Section 2.6) clearly
indicated an ATP-competitive binding mode of 4b and , we were confronted with the discrepancy,
that such competitiveness was not possible to show in inhibition experiments with compounds 4b
and under gradually increasing ATP-concentrations (Figures 3A,C). Therefore, it was taken under
consideration whether 4a and could be so-called “tight-binding” inhibitors. Such a “tight-binding”
5 preparations in use for impurities, both with no other outcome than before. Because the
5
5
5
effect can be observed in case the IC50 value of an inhibitor is in the same range as the concentration of
the enzyme in the assay [43]. In our assay, the concentration of CK2 was 9 nM (concentration of active
sites 18 nM, due to the α2β2 architecture) and the IC50 value for 4a turned out to be 7 nM and 5 nM
for 5. In such case the population of free enzyme molecules is significantly depleted by formation of
enzyme-inhibitor complex and the simplification that the concentration of the free enzyme is equal
to the total enzyme concentration as required for Michaelis-Menten kinetics is no longer valid [44].
As a consequence the concentration of free enzyme varies, which has a severe influence on the IC50
values as determined, and the double-reciprocal plot looks like classical non-competitive inhibitor.
But this is an artefact of measurement and does not reflect the “true” situation [44]. The IC50 value
is still dependent on the ATP-concentration, but it cannot be measured in this way. Hence, for such
“tight-binding” inhibitors a graphical determination of the Ki value using e.g., Lineweaver-Burk
plot is not possible. In order to be still able to determine the inhibition constant, the Morrisson
equation was developed which is a non-linear fit of the initial reaction velocities in dependence of
the inhibitor concentration and which considers the contemplable concentrations of enzyme and
substrate [45]. This can be used to determine the so-called apparent Ki (Ki app) for different substrate
(ATP) concentrations. These experiments have been performed for 4a and 5 with ATP concentrations

Pharmaceuticals 2018, 11, 23
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ranging for 6–400 µM, and the calculated Ki app values by the Morrission equation were plotted against
the ATP concentration (Figures 3C,D) [46].
Figure 2. Dose-response curves for the determination of IC50 values of 4a (A) and 5 (B). Human protein
kinase CK2 holoenzyme was pre-incubated for 10 min with 13 different concentrations of each compounds
ranging from 0.01 nM to 10 M before the enzymatic activity was determined. The residual activity was set
µ
into relation of the activity of the enzyme without added compounds but with the same volume of DMSO,
which was taken as 100%. The resulting inhibition in % was set into relation to the concentration of the
corresponding compound in a logarithmic scale. For the calculation of the sigmoidal curve fitting the date
GraphPad Prism (GraphPad, La Jolla, CA, USA) was used, as well as for the compound concentrations
corresponding to 50% enzyme inhibition. The IC50 values determined this way turned out to be 7 nM for 4a
and 5 nM for 5.
Figure 3. IC50 values of 4a and
5
determined at different ATP-concentrations (
A,B) and calculation
of the apparent Ki values of 4a
(C
) and ) by the Morrison equation using the initial reaction
5 (D
velocity determined for each ATP concentration used [45]. Phosphorylation of the substrate peptide
RRRDDDSDDD by human protein kinase CK2 holoenzyme was determined in the CE assay at different
ATP-concentrations (6, 30, 60, 150, 300, and 400 µM). As no Ki value was possible to determine by
this strategy, the enzymatic reactions were repeated independently three times and the initial reaction
velocities obtained thereby for each ATP concentration were put into the Morrison equation which
enables to calculate a so-called apparent Ki-value. For both compounds the interception of the best fit
line with the Y-axis yielded a Ki app 0.041 nM +/− 0.02 for 4a and 0.46 nM +/− 0.09 for 5.

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Despite the fact that for each substrate concentration the standard error of the mean (SEM) of the
three independent determinations was indeed noticeable, which could have been due to the difficult
measurement of the initial reaction velocity, a clear indication for a Ki app for both compounds was
given by extrapolation of the best fit line with the Y axis (Figure 3C,D), at an ATP concentration = 0
.
The Ki app values obtained thereby were 0.41 nM +/ 0.02 for 4a and 0.46 nM +/ 0.09 for . In order
−
−
5
to test the reliability of these values, the determination of Ki app at different ATP-concentrations was
repeated for 4a, but with double the amount of the enzyme CK2. This resulted in Ki app values
at the same level of 4 nM, but the fluctuation obtained with the different ATP concentrations was
much lower (data not shown). In consequence we considered 4a and
5 to ATP competitive inhibitors
of human protein kinase CK2 with a “tight binding” characteristic and with Ki app values in the
subnanomolar range.
2.3. Selectivity
As 4b, the lead compound for the development of 4a and
towards human protein kinase CK2, a selectivity profile study was performed by Reaction Biology
Corp. (Malvern, PA, USA) for 4a and as well. For this purpose 61 kinases from different representative
subgroups (and CK2 and CK2 ’) were tested. Testing was performed with inhibitor concentrations of
10 M and an ATP concentration of 10 M. The residual enzyme activity as obtained was set in relation
to a control without inhibitor. Under these inhibition conditions the residual activities obtained with 4a
and was less than 2% for CK2 and less than 1% for CK2 ’. As shown in Table 1, the selectivity profile
obtained was surprisingly different for the three compounds, despite their pronounced structural
similarities. 4a inhibited only 6 out of the 61 kinases to more than 70% at a concentration of 10 M. 5 of
these kinases belong to different families (Aurora A, SKG1, CAMKK2, DYRKB1, and FLT3). 5 inhibited
11 kinases out of 9 different families to more than 70% at 10 M instead (for a complete listing of
inhibition data towards the 61 kinases, see supplementary Figure S2). This could reflect the lower
flexibility between the rings C and D in , which could overcome an entropic effect with these kinases
5, appeared to be relatively specific
5
α
α
µ
µ
5
α
α
µ
µ
5
as obtained with 4b and 4a. Interestingly, only two kinases, Aurora A and SGK1 were inhibited by
all three dibenzofurans to more than 70%. No other kinase was inhibited to the same extent by 4a
and 4b, indicating that selectivity of ATP-competitive inhibitors, despite depending on a quite similar
active site, can be addressed with distinct modifications of an identical scaffold. This demands further
investigation. Nevertheless all three compounds of the dibenzofuran type as presented here can be
considered as quite selective inhibitors of human CK2.
Table 1. Selectivity profile of dibenzofuran derivatives: human kinases that were inhibited more than
70% at 10 µM concentration.
4a
5
4b
Aurora A
SKG1
Aurora A
SKG1
Aurora A
SKG1
CAMKK2
DYRKB1
FLT3 (D835Y)
FLT3
CAMKK2
DYRKB1
FLT3 (D835Y)
FLT3
FLT4/VEGFR3 FLT4/VEGFR3
KDR/VEGFR2 KDR/VEGFR2
PIM1
LCK (68%) *
PKA
PIM1
LCK
C-Met
PKD/PRKD2
* At 10 µM concentration inhibition was 68%.

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We recently reported a whole cell CK2 activity assay, which can be used for testing the selectivity
of compounds towards CK2 and CK2 ’ [47]. It is based on the surface display of the human CK2
subunits, either or ’ together with on E. coli, were they are able to form a functional tetrameric
holoenzymes, either ααββ or ββ, depending on which isoform has been expressed [48].
This technique was used to investigate whether 4a or have preference to one of the two catalytic
isoforms of CK2. As depicted in Supplementary Information, Figure S2, the IC50 values obtained
for and ’ containing holoenzymes were in the same order of magnitude for 4a and . There was
a tendency to for stronger inhibition of the CK2 ’ containing holoenzyme in comparison to CK2
α
α
α
α
β
α
’
α
’
α
5
α
α
5
α
α
containing holoenzyme by a factor of 1.5 for both compounds. However, this appeared to be not
statistically significant. Nevertheless, a similar tendency has been observed during the selectivity
testing with the kinase panel (Supplementary Information Figure S2).
2.4. Effects of the Dibenzofuran Derivatives on the Activity of Cellular CK2
Kinase inhibitors which are used as a tool in basic research or as therapeutic agent need to
penetrate the cell membrane and affect intracellular CK2. To test whether 4b, 4a and 5 are capable to
enter the cell, two different cell lines were used: LNCaP cells, an androgen-sensitive prostate carcinoma
cell line, and ARPE19, which was derived from a primary culture of retinal epithelium cells [49]. Thus,
this represented tumor cells on one hand and normal, non-transformed cells on the other.
In a first experiment, it was tested whether CK2 is indeed present in both cell lines. Cells were
grown under normal conditions, harvested and the proteins were extracted. After separation by
SDS PAGE a western blot analysis was performed with CK2 specific antibodies. The result shown
in Figure 4A demonstrated that despite loading fairly equal protein amounts for both cell lines as
indicated by the GAPDH loading control, CK2 subunits are much less expressed in the non-neoplastic
ARPE19 cells than in the prostate carcinoma cell line LNCaP. This is in agreement with previous
observations in different tumor cell lines and tumors and was most obvious for the catalytic
In addition a significant difference between both cell lines was observed for the regulatory
α
-subunit.
-subunit
β
as well. The catalytic α’-subunit developed the weakest signal in both cell lines, and was barely visible
in ARPE19 cells. Nevertheless, CK2 was detectable in both cell lines and in subsequent experiments
cells were incubated with the dibenzofuran inhibitors followed by an analysis of cellular CK2 activity.
ARPE19 and LNCaP cells were treated with 0, 25 or 50
derivatives 4b 4a and for 24 and 48 h before being harvested. Proteins were extracted and CK2 kinase
activity was measured by an in vitro phosphorylation assay using the CK2 specific peptide substrate
RRRDDDSDDD and radiolabeled [[32P]
ATP. The activity as measured was normalized to equal
µM of the different dibenzofuran
,
5
γ
protein amounts. The relative CK2 activity of at least three independent experiments is given as bar
graphs in Figure 4B. In ARPE19 cells CK2 activity was strongly repressed by all inhibitors. 4b exerted
a 60% inhibition independent of concentration and time. With 4a an inhibition of 80% was obtained,
already by a treatment with 25
could not improve the inhibitory effect. For
reduced the cellular CK2 activity to 80% and with 50
µ
M for 24 h; the application of a higher concentration for a longer time
a weaker but dose-dependant effect was observed. 25
M the activity was reduced to 40%. In LNCaP
5
µM
µ
cells the results were essentially similar to those made in ARPE19 cells. However, the repression of
activity by 4b and 4a was not as strong as in ARPE19 cells. 4b reduced the activity to 50%, 4a to
40%. The outcome using
roughly 80% when using 25
cell penetration of compounds 4a
5
was quite similar to the effects observed in ARPE19 cells: a reduction to
M, which was enhanced to 40% when using 50 M. This also indicated
4b, and . A more direct evidence for intracellular inhibition of CK2
µ
µ
,
5
could be obtained by measuring the phosphorylation of endogenous natural substrates (biomarker
analysis) [50]. This has been done by Guerra and co-workers, who investigated their inhibitor D11,
which is identical to our compound
inhibition of endogenous substrate phosphorylation [42
into consideration that the logP values of compounds 4a
5
, on its cellular effects in different cell lines by measuring
51 52]. With respect to their results and taking
4b, and as given in Table 2 are not of
,
,
,
5

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substantial difference, we conclude that the inhibitors were able to cross the cell membrane and exerted
a similar inhibitory effect on intracellular CK2 activity in both cell lines.
Figure 4. Expression of CK2 in prostate cancer cell line LNCaP and non-neoplastic retinal pigment
epithelial cell line ARPE19 and inhibition of intracellular activity by dibenzofurans. (
extracted with RIPA buffer. 50 g cell extract was separated on a 12.5% SDS polyacrylamide gel and
transferred to a PVDF membrane followed by Western blotting with anti-CK2 (1A5), anti-CK2 ’ (#30),
anti-CK2 (6D5) and anti-GAPDH antibodies. GAPDH was used as a loading control. ( ) ARPE19
or LNCaP cells were treated with DMSO as control (0 M) or with 25 or 50 4b 4a or for
24 or 48 h. Kinase activity of CK2 in the cell extracts was measured with the synthetic CK2 specific
peptide substrate, RRRDDDSDDD, in the presence of [[32P]
ATP. Results from at least three individual
experiments are shown; the activity in the control cells was set to 100%.
A) Cells were
µ
α
α
α
B
µ
µM
,
5
γ

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2.5. Induction of Apoptosis and Impact on Tumor Cell Viability
Protein kinase CK2 is essential for the survival of cells. Therefore, we tested the impact of the CK2
inhibitors on the viability of cells in both lines. The inhibitors were applied in increasing concentrations
up to 50
treatment. The effect of the compounds on the growth of the normal ARPE19 cells was rather moderate
(Figure 5A). had no impact on ARPE19 cell viability. 4b reduced the viability to 70% in its highest
concentration and 4a exerted the strongest influence by reducing the ratio of viable cells to 40% when
applying a 50 M dose. In contrast LNCaP cells were strongly impaired by the treatment with the
dibenzofurans (Figure 5B): Using 10 M of the different inhibitors, we only observed an effect with 4b
However, increasing the concentration to 20, 25 and 50 M exerted a drastic impact on the viability of
LNCaP cells. With 50 4b a viability rate of final 5% was obtained, whereas 4a and reduced the
µM to growing cell cultures and the viability was analyzed by a MTT assay after 24 h of
5
µ
µ
.
µ
µM
5
viability to 30 to 35%. Thus, the inhibitors in particular affected the viability of LNCaP carcinoma cells,
but not of normal retinal pigment epithelium cells. This observation was supported by the morphology
of cells as seen in the microscope (Supplementary Information Figure S3). Even after 48 h of growth
under CK2 inhibition ARPE19 cells appeared rather unaffected by the treatment. In contrast, LNCaP
cells were severely altered in their morphology by all inhibitors; they could not form a dense cell layer
anymore, but detached from the culture dish.
The fact that after 48 h of growth under CK2 inhibition ARPE19 cells were rather unaffected by the
treatment whereas in contrast, LNCaP cells were severely constrained by all inhibitors is supporting the
concept of the study. As can be seen in Figure 3A, CK2 subunits are strongly overexpressed in LNCaP
tumor cells in comparison to ARPE19 cells. Reducing the activity of CK2 by the inhibitors is supposed
to overcome the antiapoptotic effect of the enzyme and cells are induced to undergo apoptosis.
Whereas in “normal” ARPE19 cells, no apoptotic signals are anticipated, and hence a reduction of
CK2 activity should not lead to cell death [53]. In consequence, we analyzed in the next experiments
whether the cells were killed by apoptosis. Hallmark of apoptosis is the activation of the key effector
caspases 3 and 7 which ends in the cleavage of poly (ADP ribose) polymerase PARP1. The full-length
PARP1 is cleaved by the caspases to an 89 and a 24 kDa fragment. These events of late apoptotic
signaling were analyzed in ARPE19 and LNCaP cells grown in the presence and in the absence of
dibenzofurans. Inhibitor concentrations of 25 and 50 µM were used and were applied for 24 and 48 h.
In cell lysates of LNCaP and ARPE19 cells the activity of caspases 3 and 7 was determined subsequently
by a luminescence based assay (Figure 6A). In ARPE19 cells only basal caspase activity was observed
which never exceeded 4000 RLU (relative luminescence units), regardless of the compound, its applied
concentration or the incubation time. However, in LNCaP cells a slight increase of caspase activity was
observed already after 24 h, whereas upon treatment with 50 µM of 4b and 5 after 48 h a significant
increase of caspase activity going beyond 50,000 RLU was detectable. With 4a caspase activity in
LNCaP was also enhanced but not as significant as with the two other inhibitors (max. 17,000 RLU
in comparison to 55,000 RLU as obtained with
obviously switched on by 50 4b and in the tumor cell line LNCaP; 4a treatment activated the
caspases only to a minor degree.
5). Thus, the activity of the key effector caspases was
µM
5
In addition to the luminescence assay, Western blot analysis was performed to check
PARP1-cleavage (Figure 6B). Therefore specific antibodies for the detection of the full-length PARP1
(113 kDa) and the larger cleavage fragment of PARP1 (89 kDa) were used. In ARPE19 cells no cleavage
product of PARP1 was detectable. In LNCaP we observed the cleavage of PARP1 after treatment with
50 µM 4b as well as with 50 µM 5. Treatment with 4a, however, did not result in cleavage of PARP1.
Thus, we conclude that treatment with all dibenzofuran inhibitors affect the viability of cells,
tumor cells being much more sensitive to the treatment than normal cells. The severe drop in viability
in tumor cells is obviously due to the induction of apoptosis for 4b and
5. 4a also exerts a cytotoxic
effect on LNCaP cells; however, cells do not die by inducing the apoptotic program.

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Figure 5. Effects of dibenzofuran inhibitors on cell viability. ARPE19 (A) or LNCaP (B) cells were
incubated with the given concentrations of 4b, 4a, 5 or DMSO as control. Cell viability was determined
using the MTT assay after 24 h of incubation. Viability was set in reference with DMSO. The mean and
standard deviation of three independent experiments with two technical replicates each is shown.
Figure 6. Cont.

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Figure 6. Induction of apoptosis in prostate cancer cell line LNCaP and non-neoplastic retinal pigment
epithelial cell line ARPE19. (
A
5
) ARPE19 or LNCaP cells were treated with DMSO as control (0
for 24 or 48 h. Activity of caspases 3 and 7 was determined using the
µM)
or with 25 or 50 4b 4a or
µM
,
luminescent Caspase-Glo® 3/7 assay according to the technical bulletin of the manufacturer. Median
relative light units (RLU) + standard deviation from at least three independent experiments are shown
in the bar graph. (B) Cells were treated as described before. 75 µg cell extract was separated in a 10%
SDS polyacrylamide gel and analyzed in a Western blot analysis with a rabbit polyclonal antibody
against PARP1. + is a positive control for PARP cleavage.
2.6. Human CK2α Crystal Structures in Complex with 4a, 5 and Their Archetype 4b
In order to elucidate the binding mode of 4b
,
4a and
5
to CK2 each of the compounds was
54].
co-crystallized with hsCK2 α [
α
1–335, a fully active C-terminal deletion mutant of homo sapiens CK2
To ensure comparability of the binding modes, care was taken to minimize artifacts of the crystallization
media or introduced by crystal packing differences [54]: first we crystallized with polyethylen glycol
4000 (PEG4000) as a precipitant rather than with a kosmotropic salt that might affect potential
halogen bonds with aromatic groups [38], second we used in all three cases solutions of comparable
compositions for crystallization and for preparation of cryo diffractometry, and third we initiated
crystallization by seeding to direct the crystallization process always towards the same crystal packing.
In fact, we obtained isomorphous monoclinic crystals of the three hsCK2α
1–335/inhibitor
complexes (Table 4) grown in 32% (w/v) PEG4000, 0.2 M ammonium acetate, 0.1 M sodium citrate,
pH 5.6, i.e., in the presence of moderate salt concentrations. In all three cases well resolved X-ray
diffraction data sets were collected which allowed to determine and to refine the underlying complex
structures with high quality (Table 4). For the co-crystals with the mother compound 4b and with its
most similar derivative 4a we collected additional diffraction data with soft X-rays (wavelength 2 Å)
in order to localize the chloro substituents of the inhibitors via their anomalous signals [55,56].
As expected, all three protein matrices are structurally largely identical and the three inhibitors
bind to the canonical ATP/GTP site of CK2α which is located in a cleft between the two main
domains typical for eukaryotic protein kinases (EPKs) (Figure 7A). The three dibenzofuran moieties

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lie essentially coplanar (Figures 7B,C) apart from the fact the C-atoms 1 and 2 of 4b and 4a are
sp3-hybridized which enforces a certain deviation from planarity in ring C. The dibenzofuran plane
is sandwiched by large non-polar side chains located in the
(Figure 7B). While this general arrangement is common for binding of ATP-competitive inhibitors in
EPKs, there are some CK2 residues like Val66 from the N-lobal side and Ile174 as well as Met163
β-strands of both main kinase domains
α
from the C-lobal domain (Figure 7B) that are conspicuously bulky and hydrophobic in comparison
to its equivalents in other EPKs. As a consequence there is a rather narrow hydrophobic cleft with
special features like the ability to bind flat and hydrophobic (parts of) molecules in rather diverse
ways with respect to peripheral hydrogen and halogen bonds as long as the planar hydrophobic
parts are well accommodated. Prime examples of the resulting “freedom in 2D-space” [57] are (i) the
dual co-substrate specificity of CK2, i.e., its ability to utilize either ATP or GTP as phospho donor
depending on a purine base shift within the binding plane to enable correct hydrogen bonding [58
(ii) the capability of CK2 to bind an inhibitor like emodin without the formation of any hydrogen
bond to the peptide backbone of the interdomain hinge which normally provides H-bond anchors
for ATP site ligands in protein kinases [55 58] or (iii) the flexibility to switch the halogen bonding
]
α
,
pattern by rotations within the binding plane in order to enable the coordination of diverse tetrabromo
benzimidazole derivatives [59]. We will show below that 4b and its derivatives provide a novel and
unexpected case of adaptability within CK2α’s purine base binding plane.
Thus, hydrogen bonding to the hinge region of CK2α is in principle not necessary for
ATP-competitive CK2 inhibitors. Nevertheless, in order to achieve low-nanomolar Ki values the
most potent of them like silmitasertib [32] or FLC26 [38] use polar interactions with the hinge and,
in addition, with a hidden region next to Lys68 known for its positive electrostatic potential and for
the presence of conserved water molecules [60]. Consistent with this rule, each of the tight binding
inhibitors 4b, 4a and 5
forms four hydrogen bonds with hsCK2α1–335 (Figure 7C). For this purpose
they exploit the hydrogen bonding potential of the two oxo or hydroxy substituents at positions 3
and 8 (Figure 1). Each of them is the origin for one pair of hydrogen bonds (Figure 3C) either with
the peptide groups of Val116 in the hinge region or with the side chain of Lys68 and a neighboring,
highly conserved water molecule [60]. The enzyme is pre-formed for these hydrogen bonds and
requires no structural adaptations: in the human CK2α apo-structure 3WAR which is the highest
resolved CK2α structure published so far [61] the H-bond partners on the protein side are essentially
at the same position as in the hsCK2α1–335 complexes with 4b, 4a and 5.
Remarkably, on the inhibitor side the two central H-bond positions are occupied in all three cases
by suitable oxygen substituents (H-bond anchors 1 and 2 in Figure 7D–F), irrespective of the fact
that—as illustrated in Figure 7D–F and as described below in more detail—the overall orientation of
4b is completely different from that of 4a and 5. A further note deserves the fact that Val116 is in one of
its two H-bonds the hydrogen donor, but in the other one the hydrogen acceptor: this is only possible if
a hydroxy rather than an oxo group is the H-bond partner at the inhibitor side. This condition is directly
matched for 4b and for
5 where the OH-group at position 8 (4b) or at position 3 (5) is the common
H-bond anchor for Val116. In contrast, in 4a the substituent at position 3 which is located at the
equivalent position is formally an oxo group (Figure 1), which suggests that keto-enol tautomerization
must have occurred in 4a.
While the three-ring systems of the inhibitors are well defined by electron densities this is not the
case with the distal methylphenyl or methoxyphenyl groups: only if the contouring level is lowered
to 0.5 σ, are faint and disrupted pieces of electron density visible for these parts of the molecules
(Figure 7D–F). As we do not have any experimental evidence for a general instability of the connecting
linker, the observation may be the consequence of configurational and conformational flexibilities in
this region resulting from the keto-enol tautomerization. In any case, these flexibilities are preserved
in the enzyme-bound states and obviously not disadvantageous for the affinities.

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Figure 7. Human CK2
three superimposed structures (red: 4b complex; green: 4a complex; blue:
sandwiching of the dibenzofuran framework by non-polar side chains of the N-lobal strands
3 and 5 from one side and of the C-lobal strands 7 and 8 from the other. ( ) Each of the inhibitors
forms two pairs of hydrogen bonds (drawn in detail for 4b) with the enzyme. ( ) Section of the
three inhibitors covered by the final electron densities (cutoff level 0.5 ) after superimposition of the
harboring protein matrices. To facilitate a comparison of the orientations either 4a ) or 4b ) is
additionally drawn with black C-atoms. The hydroxy/oxo groups that form hydrogen bonds and
that are positionally preserved are indicated by dashed circles. ( ) Experimental verification of the
α
crystal structures in complex with 4b
,
4a, and
5
5
. (
A
) Global overview of the
) Hydrophobic
1, 2,
complex). (
B
β
β
β
β
β
β
C
D–F
σ
(
D
(E,F
G,H
different orientations of 4b and 4a using the diffraction data sets at 2 Å wavelength (Table 4) at which
the anomalous scattering of chlorine is enhanced. In both pictures the respective ligand is covered by
its final electron density (grey mesh; cutoff 1 σ). The positions of the chlorine atoms are indicated by
red (G) anomalous difference Fourier density (cutoff level 4 σ).
In spite of the fact that each of the inhibitors contains two candidate chloro substituents neither of
them forms a halogen bond to one of the peptide carbonyl oxygens at the hinge region as it is often
found for protein kinase inhibition by halogen-containing ATP-competitive ligands [62]. Quite the
opposite, the two chlorine atoms of 4b at positions 6 and 7 (Figure 1) whose locations are clearly

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confirmed by anomalous difference density peaks (Figure 7G) point away from the hinge region
backbone; they are not halogen-bonded at all resembling in this respect the recently described binding
mode of the flavonol derivative FLC26 [38]. Intuitively one would expect that a relocation of 4b’s
Cl6-atom to position 9 (Figure 7G) as it is in 4a and in
proximity of the hinge residue Glu114 and should therefore generate a Cl-O halogen bond with the
peptide oxygen of Glu114. Surprisingly, however, neither 4a nor forms such a halogen bond to the
hinge backbone; rather, in their complexes with hsCK2
1–335 the entire dibenzofuran scaffold is rotated
5 would bring the chlorine atom into the
5
α
compared to 4b, namely by 180 degrees around the pseudo-twofold symmetry axis running in-plane
through the furan ring (ring B in Figure 1) followed by a 38 degree rotation perpendicular to this
axis (Figure 7D–F). To validate this reversal of the orientation we calculated an anomalous difference
density in the case of 4a; it unambiguously shows the two chloro substituents lying in the vicinity of
Phe113, Lys68 and Asp175 (Figure 7H), i.e., deeply inside the ATP/GTP cavity, but far away from the
hinge region. To our knowledge, no other CK2
a comparable location of the halogen atoms.
α structure with a halogenated inhibitor exists with
Thus, rather than using their novel Cl9-atom to form a Cl-O halogen bond at the hinge region,
4a and 5 prefer to establish a -halogen bond with the phenyl ring of Phe113 as indicated in Figure 7H.
In both complexes the distance of Cl9-atom to the centroid of the phenyl ring is 3.4 Å which is fairly
close compared with average values of 3.6 Å or 3.854 Å reported by Matter et al. [63] and Lu et al. [64
π
]
In addition to this vigorous halogen bond, the nearby Cl7 atom is hydrogen bonded to the Lys68 side
chain and in halogen-bond distance (3.5 Å) to one of the oxygen atoms to the side chain of Asp175.
In summary, in complex with hsCK2α1–335 all three inhibitors make very similar hydrophobic
interactions and hydrogen bonds with the enzyme. However, with respect to halogen bonds 4a and
5
differ significantly from 4b since they fit so perfectly to the structural environment provided by
the enzyme that a strong novel -halogen bond is established which reduces their affinity to the
π
subnanomolar range. This finding resembles an observation of Matter et al. [63] who reported about
the optimization of two protease inhibitors in which the switch of an aromatic chloro substituent from
the meta- to the para-position led to a drop of Ki values by about two orders of magnitude and who
presented evidence that this behavior is due to π-halogen bonds.
3. Discussion
A first outcome from this study is the confirmation that dibenzofurans are potent lead structures
for human protein kinase CK2 inhibitors [37]. This is additionally supported by recent studies
of Guerra et al. [42], who identified dibenzofuran “D11”, which appears to be identical to
5 by
screening of 1.600 compounds from the NIH/NCI Diversity Set on CK2 inhibition. However, they did
neither describe the synthesis of the compound nor clarify the structural basis of its binding to
CK2. D11 turned out to be able to induce caspase-mediated cell death in human glioblastoma and
pancreatic adenocarcinoma cells lines, which have been shown before to be resistant to conventional
chemotherapeutic agents [50]. Finally, first indications on the influence on intracellular signaling and
on the phosphoproteome by D11 treatment were obtained [50
4a and could be shown as well, in particular by the strong inhibition of intracellular CK2 activity
in the prostate cancer cell line LNCaP and in the non-neoplastic retinal pigment epithelial cell line
ARPE-19. To our surprise, showed no significantly weaker effect on intracellular CK2 activity, despite
an unfavorable high logP value of 6.02 (Table 2) in comparison to 4a (4.55) and 4b (4.95), the mother
compounds of both new structures [28 37]. Moreover, 4b reduced cell viability of prostate cancer cell
,51]. In this study, cell permeability of
5
5
,
line LNCaP with a significant stronger degree than 4a and 5, which exhibited a similar effect, despite
their quite different logP values. Here we need to emphasize, that in comparison to 4a (IC50 = 7 nM)
and
5 (5 nM), 4b (15 nM) was the weakest inhibitor of CK2. This indicates that additional effects
beyond inhibition of CK2 contribute to the observed differences in cellular activity. A first reason could
be the different cell permeability of the compounds as indicated by their different logP values and TSPA
values (Table 2), but this needs further experimental conformation, e.g., by a Caco-2 assay, as active

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transport into the cells or even out of the cells again by multi-drug resistance proteins is not considered
by values as logP or TSPA. In addition, it cannot be excluded that multivalent effects, i.e., interactions
of the compounds with other cellular targets than CK2 contributed to the cellular effects. In this context,
it appears remarkable, that despite being structurally rather similar, the three compounds analyzed,
4b, 4a and 5 showed a different selectivity pattern, when tested against a panel of 61 human protein
kinases (Table 1 and Supplementary Figure S2). The only two kinases beyond CK2 that were inhibited
to more than 70% by all of the three compounds to more than 70% were Aurora 1 and SKG1. Both,
4a and 5 additionally inhibited CAMKK2, DYRKB1, FLT3 and the D835Y mutant of FLT3 to a similar
extent. In contrast, 4b as well as
PIM1 and LCK to almost 70% or more. This is surprising because from a structural point of view,
4b seems to be more similar to 4a than to (Figure 1). PKA and C-met was only inhibited by to
more than 70% at 10 M, and PKD/PRKD2 was only inhibited by 4b at the same concentration to
5 inhibited at 10 µM concentration FLT4/VEGRF3, KDR/VEGF2,
5
5
µ
a similar extent. Although these results need to be confirmed and must be considered as preliminary,
because only one concentration of each inhibitor was tested in the presence of one concentration of ATP,
independent of the different KM values for the co-substrate with each kinase, such a heterogeneous
pattern as observed was not to expect. It may be an indication, that indeed multi-target effects could
have contributed to the difference in cellular effects of the three compounds. In addition it emphasizes,
that multi-target effects, also named as polypharmacology more recently [65,66], are worth to be
considered at early stages of drug discovery and could be subjected to a rational approach. Hence,
an observed lack in selectivity must not be a flaw, but could even be beneficial if the compound
addresses multiple targets related to the same disease and the interaction can be measured and as well
be controlled by distinct structural features. First polypharmacology approaches of CK2 inhibitors
with the aim to identify the structural requirements for inhibiting additional targets such as CDC25
and ABCG2 have been published recently [67,68]. Although we are still at beginning of the rational
design of multi-target drugs, the understanding that addressing multiple targets could be beneficial
for therapeutic intervention (and as such reflecting the multifactorial genesis of disease and re-echoing
the observation of a poor correlation between in vitro drug effects and in vivo efficacy) will lead to
completely new strategies in drug discovery.
Table 2. TPSA and logP values of dibenzo[b,d]furan derivatives.
Compound
logP
TPSA (A2)
4b
4a
5
4.95
4.55
6.02
62.47
71.70
75.19
In summary, both new compounds, 4a and
human CK2 with IC50 values of 5 nM (4a) and 7 nM (
Schaefer et al. [51] investigated D11, which is structurally identical to our compound
5
turned out to be tight binding inhibitors of
5
) and an apparent Ki-value of 0.4 nM (4a, 5)
.
5
on a possible
binding mode. They supposed a Michaelis-Menten kinetics and tried to determine the Ki value of D11
by increasing concentrations of the co-substrate ATP with three different concentrations of inhibitor.
The outcome was somewhat intriguing, as this resulted in a decreased apparent maximum velocity
(Vapp max) and increased apparent KM (KMapp) values. From these results the authors concluded
a linear mixed-type inhibition. Because the enzyme concentration used in these assays was not
specified, it is difficult to evaluate, that indeed “steady state” conditions were maintained and
a Michaelis-Menten kinetics can indeed be postulated. In the light of our results—the inhibition kinetics
shown in Figure 3 and the structural results demonstrating that the inhibitor binds to the ATP-site of
CK2α and nowhere else—we conclude, that the results obtained with D11 by Schaefer et al. [51] on
the kinetics of inhibition are also reflecting “tight-binding” mode.
Compounds 4a and 5 showed a similar level of selectivity as 4b, and also induced apoptosis in
the prostate cancer cell line LNCaP, but not in ARPE-19, a non-tumor derived human retinal pigment

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epithelial cell line. Co-crystal structure determination with the three compounds and hsCK2α1–335
revealed, that changing the chloro-substituent from the 6 position of the dibenzofuran scaffold—as
in 4b—to the 9 position as in 4a and 5 resulted in the formation of a -halogen bond with the side
chain of the “gatekeeper” residue Phe113. Without precedent, the formation of this -halogen bond
[69]
π
π
led to a complete inversion of the binding mode in comparison to 4b, where the non-halogenated
phenyl-ring is a stacking partner of Phe113. To our knowledge, this is the first example for inverting
the binding mode of an inhibitor by moving the position of a chloro-substituent without reducing
its potency. It emphasizes the importance of halogen bonds—weak noncovalent interaction with
electron rich moieties and the so-called σ-hole in line with the covalent C-halogen bond [40]—within
the context of ligand/target binding, and it will redirect the view on the role of aromatic residues
for interaction with halogen containing compounds in drug discovery. The synthesis strategy as
applied here and which led to the discovery of 4b, 4a and 5, is limited concerning the pattern of chloro
substituents at ring A and the investigation of homologues compounds without a ring D. It is based on
the reaction of dichloro-p-benzoquinones with 2-[(arylamino)methylene]cyclohexanone derivatives
to yield the dibenzofurans as described here [70]. Therefore a new synthesis strategy is required in
order to systematically analyze the influence of the position of the chloro substituents at ring A for
the orientation of the dibenzofurans in the ATP pocket and inhibitor potency. It will also be needed
for elucidating the contribution of ring D to the inhibitory effect and which part the flexible or rigid
connection to the dibenzofuran moiety plays in this context. This is under current investigation with
the aim to perform QSAR studies in a more systematic approach with variants of the dibenzofuran core.
4. Materials and Methods
4.1. General Information
Spectra of the compounds were determined in a Genesys 10SUV/Vis spectrophotometer (Thermo
Scientific, Waltham, MA, USA). Purity of the comopunds was determined by HPLC (Elite LaChrom, VWR
Hitachi, Langenfeld, Germany), combining a RP18 column with a diode arry detector. The enzymatic
activity of CK2 with and without inhibitors was quantified in a CE PA800 plus (Beckman-Coulter,
Krefeld, Germany). For recording of NMR spectra, a Varian AS Mercuryplus NMR spectrometer (Agilent,
Santa Clara, CA, USA) was used and an IR Affinity-1 (Shimadzu, Kyoto, Japan) for IR spectra. Human
cell lines were cultivated in a cell culture incubator Heracell 240i (Thermo Scientific, Waltham, MA, USA)
and microscoped by a Zeiss Axiovert 25 (Zeiss, Jena, Germany). Proteins were seperated by SDS-PAGE in
a Mini-Protean Tetra System and for Western blotting a Western Blot Miniprotean II cell (both from Bio-Rad,
München, Germany) was used.
4.2. Chemistry
The synthesis of 7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)methylene]
dibenzo[b,d]furan-3(2H)-one (4a), 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)
methylene]dibenzo[b,d]furan-3(2H)-one (4b), and (E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]
dibenzo[b,d]furan-2,7-diol (
5) has been described before [37,39]. For better understanding, a brief
summary of the synthesis steps and analytics is given.
4.2.1. Synthesis of 7,9-dichloro-1,2-dihydro-8-hydroxy-4-[(4-methoxyphenylamino)methylene]dibenzo
[b,d]furan-3(2H)-one (4a).
2,6-Dichloro-1,4-benzoquinone (1.77 g, 0.01 mol) and 2-[(4-methoxyphenylamino)methylene]
cyclohexanone (4.62 g, 0.02 mol) were heated under reflux in dichloromethane (50 mL) for 60 min to
yield a green coloured solution. After cooling to room temperature 2,6-dichloro-1,4-benzoquinone
(3.54 g, 0.02 mol) dissolved in glacial acetic acid (10 mL), followed by refluxing again for 30 min.
Filtration of the ◦precipitate formed after standing overnight yielded 1.2 g (14.9%) of a pure orange
product (mp 204 C). IR (KBr) 1665 cm−1. UV (CH2Cl2) = 4.35). 1H-NMR (pyridine-d5)
λmax 406 nm (lg ε

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δ
2.80 (mc, 2H, CH2); 3.20 (mc, 2H, CH2); (DMSO-d6): 3.75 (s, 3H, OCH3); 7.00 (mc, 2H, arom. H) and
7.20 (mc, 2H, arom. H); 7.42 (s, 1H, H-6); 7.74 (d, J = 12.0 Hz, 1H, CH=N); 9.56 (s,1H, OH); 11,56 (d,
J = 12.0 Hz, 1H, NH). 13C-NMR (DMSO-d6):
17.8 (t, C-1), 37.0 (t, C-2), 197,2 (s, C-3), 104.5 (s, C-4),
δ
145.2 (s, C-4a), 147.0 (s, C-5a), 110.2 (d, C-6), 111.7 (s, C-7), 156.2 (s, C-8), 116.7 (s, C-9), 126.1 (s, C-9a),
99.4 (s, C-9b), 137.4 (d, C-10), 133.0 (s, C-1”), 118.4 (d, C-2”/C-6”), 114.8 (d, C-3”/C-5”), 154.3 (s, C-4”),
55.3 (q, OCH3). C20, H15Cl2NO4 (404.3). C, 59.42; H, 3.74; Cl, 17.54; N, 3.46. Found: C, 59.50; H, 3.72;
Cl, 17.49; N, 3.28. Purity was determined to be >95%.
4.2.2. Synthesis of 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylene]-dibenzo
[b,d]furan-3(2H)-one (4b)
2,6-Dichloro-1,4-benzoquinone (1.77 g, 0.01 mol) was mixed with 2-[(4-methylphenylamino)
methylene]cyclohexanone (4.30 g, 0.02 mol) in dichloromethane (50 mL) and heated under
reflux for 60 min to yield a green coloured solution. After cooling to room temperature,
2,6-dichloro-1,4-benzoquinone (3.54 g, 0.02 mol) dissolved in glacial acetic acid (10 mL) was added
followed by refluxing again for 30 min. After standing overnight, filtration of the precipitate yielded
1
1.55 g (20%) of pure product (mp 263 ◦C). Yellow needles (Ethanol). IR (KBr) 1650 cm−1. H-NMR
(pyridine-d5) δ 2.20 (s, 3H, CH3), 2.73 (m, 4H, CH2-CH2), 7.17 (m, 5H, arom. H), 8.00 (d, J = 12.0 Hz,
1H, CH=), 8.73 (s, 1H, OH), 11.93 (d, J = 12.0 Hz, 1H, NH). C20H15Cl2NO3 (388.25): C, 61.87; H, 3.89;
Cl, 18.26; N, 3.61. Found C, 61.72; H, 3.88; Cl, 18.39; N, 3.50. Purity was >95%.
4.2.3. Synthesis of (E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]dibenzo[b,d]furan- 2,7-diol (5)
2-[(4-Methoxyphenylamino)methylene]cyclohexanone (0.81 g, 2.0 mmol) and 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (0.5 g, 2.2 mmol) were suspended in dichloromethane (50 mL), glacial
acetic acid (30 mL) was added and the mixture was refluxed for 10 min. After cooling the red precipitate
was filtered and recrystallized from toluene to yield 0.56 g (70%). Mp 210 ◦C. IR (KBr) 1625 cm−1
.
1
C20H13Cl2NO4 (402.2). H-NMR (DMSO-d6)
δ
3.83 (3H, s, OCH3), 6.95 (1H, d, J = 8 Hz, 8-H), 7.0–7.6
(5H, m, arom. H), 7.73 (1H, s, H-C=N-), 8.14 (1H, d, J = 8 Hz, 9-H), 9.16 (1H, s, OH). C, 59.72; H, 3.26;
Cl, 17.63; N, 3.48. Found C, 59.49; H, 3.10; Cl, 18.00; N, 3.64. Purity was determined to be >95%.
4.3. CK2 Inhibition Assay and Kinetic Determinations
In order to determine the inhibition toward human protein kinase CK2 holoenzyme,
the dibenzo[b,d]furan derivatives were subjected to the CE-based CK2 assay [71], as already described
for other novel CK2 inhibitors before [70]. In summary, 78
pH 7.5, 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT) containing 0.25
supplemented with 2
L of the test-compound dissolved in DMSO and incubated at 37 ◦C for 10 min.
After the addition of 120 L of assay buffer (25 mM Tris/HCl, pH 8.5, 150 mM NaCl, 5 mM MgCl2,
1 mM DTT, 190 M substrate peptide RRRDDDSDDD and 100 M ATP) the CK2 reactions were
carried out for 15 min at 37 ◦C, before they were stopped by ice cooling and the addition of 5
L of
µL of kinase buffer (50 mM Tris/HCl,
µg CK2 holoenzyme were
µ
µ
µ
µ
µ
EDTA (10 mM, pH 8.0). The samples were then analyzed for phosphorylation of the CK2 substrate
peptide by capillary electrophoresis. For the IC50-determinations, 13 different concentrations of the
test compounds ranging from 0.01 nM to 10 µM were subjected to the test. Controls for 100% and
0% inhibition were realized by samples containing pure DMSO and samples additionally lacking the
CK2 holoenzyme. IC50 were calculated from the obtained dose response curves.
The three dibenzo[b,d]furan derivatives 4b
one of the two isoform CK2 holoenzymes (CK2α2β2 and CK2α 2
were determined following the protocol of the recently described whole-cell CK2 selectivity assay [47].
In summary, 78 L of a bacterial suspension of Escherichia coli BL21 (DE3) either surface displaying
CK2 + CK2 or CK2 ’ + CK2 in kinase buffer (50 mM Tris/HCl, pH 8.5, 100 mM NaCl, 10 mM
MgCl2 and 1 mM DTT) were supplemented with 2 L of the test compound dissolved in DMSO and
incubated at 37 ◦C and 400 rpm for 10 min. CK2 reactions were started by the addition of 120
L of
,
4a and
5
were tested on selective inhibition toward
’
β2). For this purpose, their IC50
µ
α
β
α
β
µ
µ

Pharmaceuticals 2018, 11, 23
18 of 25
assay buffer (25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 167
peptide RRRDDDSDDD and 1 mM ATP) leading to a total reaction volume of 200
µ
M substrate
µL with final
concentrations of 100
an OD578 of 2 (corresponds to
µ
M for the substrate peptide and 600
µ
M for ATP. The final cell densities had
108 cells/mL) [48]. After the samples were incubated for 120 min
≈
1.7
×
at◦37 ◦C and 400 rpm, the CK2 presenting bacteria were removed by centrifugation (3850
×
g, 5 min,
4 C). The obtained supernatants were transferred to the wells of a 96 well-microplate and finally
supplemented with EDTA to a concentration of 12.5 mM to definitely stop the enzymatic reactions.
The samples were then subjected to a Beckman Coulter pa800 plus (Krefeld, Germany) CE system
and analyzed for phosphorylation of the substrate peptide. Samples containing pure DMSO served as
controls for 0% inhibition and samples additionally lacking ATP served as controls for 100% inhibition.
Nine different concentrations of the compounds ranging from 1 nM to 100
to 10 M in case of 4a and were applied to the test. IC50 values were calculated from the resulting
dose-response curves.
µM in case of 4b and 0.1 nM
µ
5
4.4. Cell Culture and Treatment of Cells with Dibenzofuran Derivatives
ARPE19 cells (ATCC Number: CRL-2302), retinal pigment epithelia (RPE) cells, were maintained
◦
at 37 C and 5% CO2 in Dulbecco’s modified Eagles medium (D◦MEM) supplemented with 2 mM
L-glutamine, 1 mM gentamicin and 10% fetal calf serum (FCS) at 37 C in an atmosphere enriched with
5% CO2. The hormone-sensitive prostate cancer cell line LNCaP (ATCC: CRL-1740, passage number
30–40) was cultured in RPMI1640 (Sigma-Aldrich Chemie GmbH, Munich, Germany) supplemented
◦
with 10% fetal bovine serum and 2 mM L-glutamine at 37 C in an atmosphere enriched with 5% CO2.
4b, 4a and 5 were dissolved in DMSO as a 10 mM stock solution and cells were treated in the
concentrations and times as indicated. Pure DMSO was applied as control. For the evaluation of cell
morphology ARPE19 and LNCaP cells were analysed with bright field microscopy using a Axiovert
microscope from Zeiss (Jena, Germany).
4.5. MTT Viability Assay
Cell proliferation and viability was determined using a colorimetric MTT-based assay (MTT:
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich, Deisenhofen, Germany)
as described in [72]. The assay is based on the activity of mitochondrial dehydrogenases.
4.6. Extraction of Proteins
For harvesting cells—including floating cells—were scraped off the plate with a rubber policeman
◦
and sedimented by centrifugation (7 min, 4 C, 400
×
g). Cells were washed with cold phosphate
buffered saline (PBS) and the cell pellet was 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
dodecylsulfate) supplemented with the protease inhibitor cocktail completeTM (Roche Diagnostics
GmbH, Mannheim, Germany). After lysis cell debris was removed by centrifugation. The protein
content was determined according to a modified Bradford method (BioRad, Munich, Germany).
4.7. Determination of Cellular CK2 Activity by an In Vitro Phosphorylation Assay
To determine the activity of CK2 after application of the inhibitor, cells were treated with or without
4b, 4a or 5, lysed and the extracts used in a kinase filter assay as described by Schwind et al. [73].
4.8. Western Blot Analysis
Proteins were separated by SDS polyacrylamide gel electrophoresis and blotted onto a PVDF
membrane. After blocking (1 h with PBS-Tween20 with 5% dry milk) the membrane was incubated
with appropriate dilutions of primary antibodies in PBS-Tween20 with 1% dry milk for another
hour at room temperature or at 4 ◦C overnight. Then the membrane was washed twice for 10 min

Pharmaceuticals 2018, 11, 23
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with PBS-Tween20 with 1% dry milk. Incubation with the peroxidase-coupled secondary antibody
(anti-rabbit 1:30,000, anti-mouse 1:10,000) followed for 1 h. The membrane was washed again twice for
10 min with PBS-Tween20. Signals were developed and visualized by the Lumilight system of Roche
Diagnostics GmbH. For the detection of the apoptose marker protein PARP we used a polyclonal PARP
antibody (Cell Signaling Technology, Frankfurt, Germany), a GAPDH specific antibody (Santa Cruz
Biotechnologies, Dallas, TX, USA) was used for loading control. For the detection of the different CK2
subunits we used the mouse monoclonal antibody 1A5 for the catalytic
α
-subunit [74], the mouse
monoclonal antibody 6D5 for the regulatory
β-subunit [75] and the rabbit anti-peptide serum #30 for
the catalytic α’-subunit [76].
4.9. Assay of Caspase Activity
The activity of the apoptotic key effector caspases 3 and 7 in cell extracts was determined using the
Caspase-Glo 3/7 assay according to the protocol of the manufacturer (Promega, Mannheim, Germany).
4.10. Crystallization and Structure Determination
Each of the three inhibitors 4b, 4a and 5 were separately crystallized with hsCK2
the crystallization these inhibitors were solubilized in 100% DMSO in a concentration of 10 mM. 4a
and 5 were mixed with hsCK2
1–335 (8–10 mg/mL in 500 mM sodium chloride, 25 mM Tris/HCl pH
α
1–335. Prior to
α
8.5) in a ratio of 1:10. 4b was mixed in a ratio of 1:5. After a short time of incubation, these mixtures
were merged with reservoir solution (32% (w/v) PEG4000, 0.2 M ammonium acetate, 0.1 M citrate
pH 5.6) in a ratio of 2.5:1. 3.5
µL of these mixtures were then equilibrated against the reservoir solution
(4b and 4a: 1 mL; 5: 100 L). The crystal growth was induced by seeding with 150 nL seeding solution
µ
after an equilibration time of two days. Grown crystals were harvested after one week.
The crystals obtained in this way were cryo-protected by incubation in a vitrification solution
[32% (w/v) PEG4000, 0.2 M ammonium acetate, 5% (v/v) glycerol, 0.1 M citrate pH 5.6, 2 mM of the co
crystallized inhibitor] for 1 min followed by vitrification in liquid nitrogen.
The subsequent X-ray diffractometry experiments were performed at beamline ID23-2 of the
European Synchrotron Radiation Facility (ESRF) in Grenoble, France, and at beamline X06DA (PXIII)
of the Swiss Light Source (SLS) in Villigen, Switzerland. We collected high resolution data from each
of the hsCK2α
1–335/inhibitor crystals (Table 4); in the case of the hsCK2α1–335 complexes with 4b and
4a we afterwards used the same crystal, respectively, to collect a “soft” X-ray diffraction data set
(wavelength 2 Å) in order to unambiguously validate the positions of the inhibitors’ Cl-substituents
via their contribution to anomalous diffraction. PDB codes: 4a: 5N9N; 4b: 5N9L; 5: 5N9K.
All X-ray diffraction data were integrated with XDS [77] followed by POINTLESS [78] for
symmetry determination, AIMLESS [78] for scaling and CTRUNCATE as well as some other programs
of the CCP4 software suite [79] for the final steps of data reduction to structure factor amplitudes.
The structures were solved by molecular replacement with PHASER [80] using the PDB file
2PVR [81] as a template. For refinement we used PHENIX [82], manual modelling was made with
Coot [83]. The topology of the three inhibitors were constructed with the help of PRODRG [84].
The anomalous difference density maps for the hsCK2
with the corresponding map calculation routine of PHENIX [82].
α
1–335 complexes with 4b and 4a were generated
Table 3. X-ray diffraction data collection and refinement statistics.
Complex
hsCK2α1–335/4b
hsCK2α1–335/4a
hsCK2α1–335/5
X-ray diffraction data collection
1.0
2.0
1.0
2.0
1.0
Wavelength [Å]
Synchrotron (beamline)
Space group
SLS (PX-III)
P21
Lattice constants
57.44, 45.71, 63.19 57.43, 45.38, 63.33 57.48, 45.51, 63.49 57.67, 45.50, 63.32 57.88, 45.67, 63.75
90, 110.94, 90 90, 110.81, 90 90, 110.97, 90 90, 110.81, 90 90, 111.14, 90
a, b, c [Å]
α, β, γ [ ]
◦

Pharmaceuticals 2018, 11, 23
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Table 4. X-ray diffraction data collection and refinement statistics.
Complex
hsCK2α1–335/4b
hsCK2α1–335/4a
hsCK2α1–335/5
X-ray diffraction data collection
Protomers perasymmetric unit
Resolution [Å] (highest res. shell)
1
1
1
1
1
34.79–1.79
(1.85–1.79)1
5.4 (51.3)
0.998 (0.818)
12.86 (1.74)
27,735 (2290)
95.24 (79.14)
3.5 (3.1)
36.01–2.90
(3.00–2.90)
3.3 (6.1)
0.999 (0.998)
43.5 (26.8)
6609 (610)
95.3 (88.2)
6.5 (6.8)
34.16–1.84
(1.91–1.84)
5.5 (70.6)
0.999 (0.705)
14.02 (1.55)
25,781 (2428)
96.10 (90.42)
3.5 (3.2)
36.10–2.99
(3.10–2.99)
9.2 (28.6)
0.996 (0.958)
16.00 (5.20)
6058 (525)
94.4 (84.2)
6.5 (6.1)
36.22–1.64
(1.70–1.64)
3.5 (61.5)
0.999 (0.738)
18.71 (1.70)
37,646 (3505)
98.85 (93.39)
3.3 (3.0)
Rsym [%]
CC1/2
Signal-to-noise ratio (I/σI)
No. of unique reflections
Completeness [%]
Multiplicity
Wilson B-factor [Å2]
25.37
29.55
28.71
48.50
23.85
Structure refinement with high-resolution data sets
No. of reflections for Rwork/Rfree
Rwork/Rfree [%]
Mean coordinate error [Å]
No. of non-H-atoms
Protein
26,336/1389
16.35/19.92
0.180
14,456/1317
16.70/20.05
36,809/1826
16.38/18.43
0.180
0.190
3055
2803
77
3094
2818
58
3147
2841
57
Ligands/ions
Water
218
33.67
32.91
47.47
175
249
33.60
32.74
48.47
Mean B-factors [Å2]
Protein
39.42
38.67
59.56
42.46
Ligands/ions
Water
39.83
40.07
RMS deviations
0.002
0.55
0.003
0.53
0.004
0.65
Bond lengths [Å]
Bond angles ( )
◦
Ramachandran plot
favored [%]
97.25
2.45
0.31
97.86
2.14
0.00
97.55
2.45
0.00
allowed [%]
outliers [%]
1
The numbers in brackets refer to the highest resolution shell.
Supplementary Materials: The following are available online at www.mdpi.com/1424-8247/11/1/21/s1, Scheme S1:
Synthesis of dibenzofurans 4 and , Figure S1: Selectivity profiles of 4a and , Figure S2: IC50 values of 4a 4b and
’ containing human CK2 holoenzyme, Figure S3: Morphological changes of ARPE19 and LNCaP
5
5
,
5
with CK2α and CK2α
cells induced by 4a, 4b, and 5; PDB Codes.
Acknowledgments: We thank the staff of the beamline ID23-2 at the European Synchrotron Radiation Facility
(ESRF) in Grenoble, France, and of the beamline X06DA at the Swiss Light Source (SLS), Paul Scherrer Institute,
in Villigen, Switzerland, for assistance with X-ray diffraction data collection. We are grateful to U. Baumann
(University of Cologne, Germany) for access to protein crystallography equipment, to R. Birus for HPLC support
and to C. Nienberg (WWU Münster, Germany) for assistance with graphical programs. The work was funded by
the Deutsche Forschungsgemeinschaft (grants NI 643/4-1 and NI 643/4-2).
Author Contributions: A.S. performed the co-crystallization experiments and solved the crystal structures, A.G.
performed CK2 testing with the inhibitors, A.B. performed the experiments to determine
α
and
α’ selectivity,
M.W. did cell culture experiments with LNCaP and ARPE19, U.K. synthesized 4a and
5, B.W. analyzed data
and critically read the manuscript, K.N. supervised parts of the study, analyzed the data and wrote parts of the
manuscript, C.G. supervised parts of the study, made cell culture experiments, analyzed data and wrote parts of
the manuscript, J.J. conceived the study, analyzed data and wrote the manuscript.
Conflicts of Interest: The authors declare no competing financial interest.
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