A Selective Biligand Inhibitor of CK2 Increases Caspase-3 Activity in Cancer Cells and Inhibits Platelet Aggregation

Article abstract of DOI:10.1002/cmdc.201700457

Cancer cells express high levels of CK2, and its inhibition leads to apoptosis. CK2 has therefore emerged as a new drug target for cancer therapy. A biligand inhibitor ARC-772 was constructed by conjugating 4-(2-amino-1,3-thiazol-5-yl)benzoic acid and a carboxylate-rich peptoid. ARC-772 was found to bind CK2 with a K_d value of 0.3 nm and showed remarkable CK2 inhibitory selectivity in a panel of 140 protein kinases (Gini coefficient: 0.75 at c=100 nm). ARC-775, the acetoxymethyl ester prodrug of ARC-772, was efficiently taken up by cells. Once internalized, the inhibitor is activated by cellular esterase activity. In HeLa cancer cells ARC-775 was found to activate caspase-3 (an apoptosis marker) at sub-micromolar concentrations (EC₅₀=0.3 μm), a 20-fold lower extracellular concentration than CX-4945, the only CK2 inhibitor under clinical trials. At micromolar concentrations, ARC-775 was also found to inhibit ADP-induced aggregation of human platelets. The overall results of this study demonstrate that oligo-anionic biligand inhibitors have good potential for drug development.

Full text of DOI:10.1002/cmdc.201700457

Accepted Article
Title: Selective Biligand Inhibitor of CK2 Increases Caspase-3 Activity
in Cancerous Cells and Inhibits Platelet Aggregation
Authors: Hedi Rahnel, Kaido Viht, Darja Lavogina, Olga Mazina, Tõiv
Haljasorg, Erki Enkvist, and Asko Uri
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To be cited as: ChemMedChem 10.1002/cmdc.201700457
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10.1002/cmdc.201700457
ChemMedChem
Selective Biligand Inhibitor of CK2 Increases Caspase-3 Activity in
Cancerous Cells and Inhibits Platelet Aggregation
Hedi Rahnel[a]; Dr. Kaido Viht[a]; Dr. Darja Lavogina[a]; Dr. Olga Mazina[a]; Tõiv Haljasorg[a];
Dr. Erki Enkvist[a]; Dr. Asko Uri[a]*
[a] Institute of Chemistry, University of Tartu
Ravila 14A, 50411 Tartu, Estonia
E-mail: asko.uri@ut.ee
1
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Abstract
The pleiotropic nature, role in many cellular processes, and constitutive activity of CK2
exemplify its importance in a number of diseases including thrombosis, multiple sclerosis, but
most expressively, cancer. The aim of this paper was the development of a selective and potent
biligand inhibitor of CK2 for regulation and examination of signalling pathways that proceed
with participation of CK2.
Biligand inhibitors of CK2 were constructed by conjugating 4-(2-amino-1,3-thiazol-5-
yl)benzoic acid (ATB) and carboxylate-rich peptoids. ARC-772, a biligand inhibitor that
possesses subnanomolar affinity towards CK2 (Kd = 0.3 nM), moderate molecular weight and
relatively low structural complexity was chosen for cellular studies. Esterification of the
negatively charged carboxylate groups led to distinctively improved plasma membrane
penetration properties of the compound – more than 100 μM intracellular concentration was
obtained. ARC-772 revealed good inhibitory selectivity towards CK2 in a panel of 140 PKs as
indicated by the value of Gini coefficient of 0.752 and hit rate of 0.05.
Inhibition of CK2 with ARC-772 resulted in decreased proportion of phosphorylated Cdc37 in
a similar or slightly better extent than CX-4945. Even more, a distinctive specificity between
cancerous HeLa cells and non-cancerous CHO cells of ARC-772 to activate caspase-3 was
demonstrated. Although the binding affinities of ARC-772 and CX-4945 to CK2 are very
similar, ARC-772 possessed remarkable 20-fold lower EC50 value for HeLa cells. Finally, this
paper demonstrated for the first time the effective inhibition of ADP-induced platelet
aggregation after treatment with biligand inhibitor of CK2.
In conclusion, the results presented in this study support the use of oligoanionic biligand
inhibitors for selective targeting of intracellular CK2. Our compounds represent a novel class
of biologically active scaffold that may be developed into promising drug candidates, but also
to study the yet still unspecified pathways regulated by CK2.
1. Introduction
CK2 is a ubiquitous Ser/Thr kinase that catalyses phosphorylation of proteins at sites with a
motif of S/T-X-X-D/E/pS/pY, usually accompanied by additional acidic residues.[1,2] CK2 has
also been shown to exhibit protein tyrosine kinase activity in mammalian cells.[3] Although the
number of human protein kinases (PKs) exceeds 500, approximately 20% of protein
phosphorylations are performed by CK2 as such small consensus sequence is present in a large
number of proteins.[2] Unlikely to majority of the PKs, the activity of catalytic subunits of CK2
(CK2α and CK2α′) is independent on the phosphorylation status of the enzymes. Also, the
catalytic subunits of CK2 are enzymatically active in the presence or absence of the regulatory
CK2 subunit (CK2β). Thereby, the substrate specificity of CK2 is regulated by the means of its
localization, phosphorylation, and protein-protein interactions. For example, the
phosphorylation of CK2β and CK2α by Cdk1 in a cell cycle-dependent manner controls the
functional specificity of CK2 due to different protein-protein interactions.[4]
The abundance of substrate proteins of CK2 can relate the kinase to almost every signalling
pathway in a cell. For example, substrates of CK2 are regulators of protein folding (Hsp90 and
Cdc37, Figure 1.A), cell growth and proliferation (MAP2K2 and MEN1), DNA damage
response (MDC1 and RAD51), and apoptosis (CASP3 and PTEN, Figure 1.B).[5] The
pleiotropic nature, role in many cellular processes, and constitutive activity of CK2 exemplify
its importance in a number of diseases including thrombosis,[6] multiple sclerosis,[7] but most
expressively, cancer.[8]
2
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10.1002/cmdc.201700457
ChemMedChem
Figure 1. Role of CK2 in signalling pathways. A) Phosphorylation of kinase-specific co-chaperone Cdc37 is critical for binding
of PKs and for activity of chaperone. Active Cdc37 cooperates with Hsp90 (heat shock protein 90) to assist in the correct
folding and functions of many signalling PKs. CK2 itself is also a client of Cdc37, and Cdc37 is required for optimal activity
of CK2, thus together forming a positive regulatory feedback loop. In conclusion, CK2 mediated regulation of the activity of
Cdc37 has an important role in cell division, cell cycle control, but also tumour progression. The scheme is adapted from Ref.
[9]. B) The role of CK2 in the control of cell survival/apoptosis via caspase pathways. First, the phosphorylation of procaspase-
3/9 (Pro-CASP3, Pro-CASP9) protects them from cleavage by caspase-8 (CASP8); phosphorylation of procaspase-9 (Pro-
CASP9) prevents the formation of CASP9. These events, in turn, prevent the activation of CASP3. Second, the phosphorylation
of CASP3 directly inactivates it. Third, the phosphorylation of the targets of CASP3 [e.g. HS1 (actin-regulatory
adaptor protein), CX 45.6 (gap junction-forming protein, connexin 45.6), pS2 (estrogen inducible protein), and PTEN
(phosphatase and tensin homolog)] prevents the cleavage of these prosurvival proteins by CASP3. Ref [9,10]. C) CK2 plays an
important role in agonist-induced platelet activation through the regulation of PI 3K (phosphatidylinositol-4,5-bisphosphate
3-kinase) pathways. CK2-mediated inhibition of PTEN leads to activation of downstream targets of the PI 3K pathways
including PDK1 (3-phosphoinositide-dependent protein kinase-1), Akt (protein kinase B), and GSK3β (Glycogen synthase
kinase 3 beta). The scheme is adapted from Ref. [11]
.
Two aspects reflect the significance of the role of CK2 in cancer. First, CK2 regulates nearly
all cellular processes that are essential for cancer development.[5] Second, cancerous cells are
addicted to high level of CK2 and inhibition of CK2 leads to death of cancer cells.[12] The
initiation of apoptosis in the cancer cells is the therapeutic goal for cancer therapy. As a
significant proportion of substrates of CK2 is involved in cell´s death and survival, CK2 is a
very potent target for treatment.
CK2α´ is a key regulator of the activity of CASP3, a member of aspartate-specific cysteinyl
proteases (or caspases) family.[10] Caspases are mediators of apoptosis of mammalian cells.[13]
CASP3 is also activated in the beginning of apoptotic pathway by self-proteolysis and/or
cleavage by another protease. Notably, upon activation of caspases approximately 14% of the
kinome is cleaved.[14,15] Activation of the CASP3 cascade leads to initiation of apoptosis, thus
the former process can be used to characterise molecular regulators of programmed cell death.
CK2 also plays a role in the regulation of thrombus formation. Nakanishi et al.[16] reported that
stimulation of platelets with PAR1-activating peptide and thrombin resulted in an increase in
the activity of CK2. Later, Ryu et al.[11] confirmed the role of CK2 in platelet aggregation by
regulating the level of phosphorylated phosphatase and tensin homolog (PTEN), and, in turn,
PI 3K-dependent signalling (Figure 1.C). Ampofo et al.[6] showed that inhibition of CK2 leads
to down-regulation of P-selectin and GPIIb/IIIa. These findings indicate that CK2 regulates
3
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10.1002/cmdc.201700457
ChemMedChem
multiple interaction mechanisms that mediate the activity of platelets. Accordingly, inhibition
of CK2 may contribute to the future treatment of diseases which accompany thrombosis.
The development of inhibitors of CK2 has been mainly focussed to nucleotide-competitive
inhibitors. CX-4945, the only ATP-competitive CK2 inhibitor that has reached clinical trials,
is the most successful example. Several inhibitors possess satisfactory inhibitory potency and
cell plasma membrane penetration properties. However, the disadvantage of nucleotide-
competitive inhibitors is the similarity of binding sites of the target kinase and other PKs,
leading to poor specificity. The development of substrate protein competitive inhibitors of CK2
could surpass the problem, but the acidophilic nature of CK2 dictates the need for negatively
charged peptide sequences, making these compounds poorly plasma membrane permeable.
Combining the substrate protein- and nucleotide-competitive inhibitors into a biligand inhibitor
could increase the inhibitory potency as well as cell plasma membrane penetration properties
of the compound.[17–20] Both good cellular accumulation and appropriate intracellular
compartmentalization are critical factors for a successful PK-inhibitor-based drug. As an
example, the intracellular concentration of imatinib (Gleevec) is substantially higher than in the
incubation solution or blood plasma, which could explain the effectiveness of the drug in
causing apoptosis of leukemic cells.[21,22]
The aim of this paper was the development of a selective and potent biligand inhibitor of CK2
for regulation and examination of signalling pathways that proceed with participation of CK2.
Biligand inhibitors (designated as ARCs) of CK2 were constructed by conjugating an ATP-
competitive inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and carboxylate-rich
peptoids (that mimicks the consensus sequence of CK2). These two fragments were conjugated
via an optimized hydrophobic linker yielding ARC-772, a conjugate that possesses
subnanomolar affinity towards CK2α, moderate molecular weight, and relatively low structural
complexity. Five different strategies were analysed to enhance its cellular uptake: conjugation
of folate, RPARPAR, or (L-Arg)9 moiety via a disulfide bridge; masking the negatively charged
carboxyl groups with esters; encapsulating ARC-772 in micelles of the transfection reagent
TurboFect. Esterification of the negatively charged carboxylate groups (compound ARC-775)
led to substantially improved plasma membrane penetration properties of the compound that
enabled the achievement of approximately 100 μM intracellular concentration of the inhibitor.
The inhibitory potency of the internalised ARC-inhibitor was demonstrated by its effect on the
phosphorylation level of a CK2 substrate protein Cdc37 by Western blot analysis, efficient
activation of caspase-3 (to induce apoptosis) in cancerous HeLa cells, and potent suppression
of ADP-induced platelet aggregation. The results of this study point to the applicability of the
biligand approach for the development of drug candidates. Such compounds can also be used
as research tools for studying the role of CK2 in signalling pathways.
2. Results and discussion
2.1. Construction of high-affinity inhibitors for CK2
Prior to this work we showed that biligand inhibitors, oligoaspartate conjugates of ATB bound
to CK2 with subnanomolar affinity and were of superior selectivity if compared to ATB.[23,24]
ATB moiety possesses several outstanding properties for the construction of biligand inhibitors.
First, it is less hydrophobic compared to some other potent ATP-competitive compounds, e.g.
4,5,6,7-tetrabromobenzimidazole. Second, it possesses remarkable affinity towards CK2 and
its cellular activity has been demonstrated.[24] Third, the moiety possesses good
photoluminescence (fluorescence) properties, which enables its sensitive quantification with
HPLC (Figure S. 1). ATB is expected to give two essential polar interactions with the ATP
pocket of CK2. The carboxylate moiety gives an ion-ion interaction with the ammonium ion of
4
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10.1002/cmdc.201700457
ChemMedChem
Lys68 and nitrogen of the thiazole ring gives a canonical hydrogen bond to the hinge region of
CK2 [24]. On the other hand, the exocyclic amino group connected to the thiazole ring of ATB
fragment was expected to point out from the ATP pocket of CK2 and thus appared as a suitable
functional group for connecting the linker and peptoid moiety. Nevertheless, compared to the
biligand inhibitors previously designed by us [17,19,25] the expected position of 2-amino group of
thiazole is located farther away from the binding site of the substrate protein. This positioning
of interacting units presumed the application of a longer linker between the ATP competitive
fragment and the peptoid. Linker optimization is an important step of the successful
construction of the biligand inhibitors to achieve efficient simultaneous binding of both
fragments of the inhibitor to the target protein. This is especially critical if a novel structural
scaffold is introduced into the design [26]
.
In the present study, a conjugate of ATB and a peptide analogue was adapted for cellular studies
(Table 1). First, the oligo-aspartate moiety present in previously reported compounds[18,19] was
replaced with the corresponding carboxylate-rich peptoid chain comprising N-
carboxymethylglycine (iminodiacetic acid, Ida) residues. Such modification was necessary to
increase the stability of the conjugates in cellular milieu[27] and for their conversion into cell
plasma membrane permeable per-acetoxymethyl ester prodrugs.[17] Second, the length of the
linker connecting ATB moiety to the oligo-Ida peptoid was optimized for high affinity of the
inhibitor. A series of conjugates (1...6, Table 1) was synthesized with one Ida residue attached
to ATB via a hydrophobic linker with varying length made of succinic acid and ω-amino-n-
alkanoic acid residues. All constructed conjugates in this series (1...6) bound to CK2α with
higher affinity than ATB ( Kd,ATB = 48 7) nM.[23] However, Kd-values displayed bell-shaped
dependence on the length of the linker suggesting that while longer linker was more favourable,
negative charge close to ATB moiety additionally contributed to the binding energy. Based on
these data a linker comprising of commercially available succinic acid and 12-aminododecanoic
acid residues connected via N-carboxymethylated amide bond was developed. The Kd-value of
ARC-769 comprising one Ida residue was about 2-fold smaller than that of ARC-759, which
demonstrated the positive effect of the additional carboxymethyl group in the linker region of
the conjugate on the binding affinity.
Table 1. Optimization of the length of the linker and peptide-mimicking oligo-Ida peptoid moiety of the biligand inhibitor of
CK2. Kd values are presented as average SD, n = 3.
O-
O
O
O
-O
O
H
N
N
O-
S
X
n
N
O
ATB
linker
[Ida]n
Compound
code
Mw/
(g/mol)
X
n
Kd/nM
#
ARC-767
ARC-761
435
493 -NH-CH2-CO-
-
1
1
10
28
2
7
1
2
ARC-762
ARC-755
ARC-758
ARC-759
521 -NH-(CH2)3-CO-
549 -NH-(CH2)5-CO-
577 -NH-(CH2)7-CO-
619 -NH-(CH2)10-CO-
1
1
1
1
40
27
13
9
6
4
1
3
4
5
6
1
ARC-773
ARC-769
576 -N(CH2COO-)-(CH2)11-CO-
691 -N(CH2COO-)-(CH2)11-CO-
0
1
8
4
2
1
7
8
5
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ChemMedChem
ARC-772
ARC-771
921 -N(CH2COO-)-(CH2)11-CO-
1151 -N(CH2COO-)-(CH2)11-CO-
3
5
0.3 0.1
0.04 0.01
9
10
As previously reported for other biligand inhibitors of CK2,[17] affinity increased with the
number of negatively charged Ida-residues in the peptoid chain. Approximately 10-fold
decrease in the Kd values per two additional Ida residues was established, reaching 40 pM
affinity for conjugate with 5 Ida residues (ARC-771). However, for cellular experiments in the
present study, the conjugate ARC-772 comprising 3 Ida residues was considered structurally
optimal because of its sufficient affinity for cellular studies,[28] moderate molecular weight and
complexity. In order to enable attachment of various transporters to ARC-772, a sulfhydryl
group was added to the C-terminus of the peptoid [Scheme S. 1.E, compound 11 (ARC-772-
SH)].
2.2. Selectivity of ARC-772
The inhibition selectivity of ARC-772 was tested at 100 nM concentration of the compound in
a full commercial panel of 140 PKs to discover the PK off-targets of the inhibitor (Table 2).
The good selectivity of ARC-772 is highlighted by the values of the Gini coefficient (0.752)
and hit rate (0.05), the latter value demonstrates that among the tested PKs only 5% of the
enzymes (including CK2, 3 PKs of the DYRK family, BRSK1, BRSK2, and TAO1) were
inhibited more than 50%.
Table 2. Inhibition selectivity of ARC-772 presented in the percentage scale of residual activities of PKs
Res.
Activity
%
Res.
Activity
%
Res.
Activity
%
Res.
Activity
%
Protein
kinase
SD
(n=2)
Protein
kinase
SD
(n=2)
Protein
kinase
SD
(n=2)
Protein
kinase
SD
(n=2)
CK2
1.78
0.18 SIK2
91.3
2.6 p38d MAPK
97.2
7.5 PKD1
104.0
4.1
DYRK2
DYRK1A
5.0
9.4
1.2 PIM3
1.4 SGK1
91.3
91.5
8.3 MKK2
5.9 Aurora A
97
97.9
31 PKBb
6.2 PKCa
104.1
104
6.3
22
DYRK3
18.0
0.4 ABL
91.7
4.0 JAK3
98
27 HIPK3
104.4
8.6
BRSK1
BRSK2
TAO1
IKKe
18.2
24.6
47
1.3 YES1
1.3 PIM2
11 PKA
92.0
92.0
92.0
92.1
92
0.6 MST4
3.2 MINK1
8.4 CHK1
7.7 ULK2
14 MARK3
98.3
98.4
98.4
98.6
98.7
0.9 MST2
3.0 ROCK 2
0.4 TGFBR1
3.1 Src
104.6
105
105.0
106.2
106.3
4.6
13
5.4
3.0
2.7
68.4
72.0
4.5 CAMK1
4.0 MNK1
ERK5
3.6 EPH-A2
HIPK2
TAK1
74
75
15 MAPKAP-K3
12 PRAK
92.4
92.6
2.5 PKCz
1.6 MEKK1
98.8
99.1
6.9 Aurora B
6.2 OSR1
106
106.6
26
1.8
AMPK (hum)
CK1δ
76.0
78.2
1.0 TTBK1
5.2 TTBK2
93
93.1
11 PIM1
7.1 PAK5
99.1
99.3
0.2 S6K1
4.0 DAPK1
107.1
108.3
1.3
2.8
MKK6
78.7
5.2 STK33
93.4
6.3 RIPK2
CDK2-
99.7
5.6 ERK2
108
19
IRR
MLK3
81
81.3
15 ERK8
4.9 NUAK1
94
94
11 Cyclin A
16 ULK1
99.9
100.0
9.7 MLK1
0.9 IGF-1R
p38b
108.4
108.5
4.3
3.8
CLK2
82.9
85
85
86.2
87.4
2.0 PINK
14 IKKb
11 GCK
2.3 PAK6
8.8 ZAP70
94
94.1
94.2
94.4
94.8
26 CSK
100.2
100.6
101
101
101.8
2.9 MAPK
2.2 PKBa
22 MARK2
17 ERK1
9.2 DDR2
109
110
110.4
110
22
12
0.8
15
GSK3b
MKK1
IRAK1
TTK
2.6 MARK1
4.0 MAP4K3
3.3 MELK
8.5 CK1γ2
111.9
4.6
CHK2
NEK6
87.5
87.5
3.0 RSK1
7.3 MARK4
95.0
95.2
4.6 p38g MAPK
6.3 LKB1
101.8
101.9
3.5 TESK1
0.1 SYK
113
113.3
11
8.1
TrkA
VEG-FR
IR
87.6
87.9
88.5
88.8
6.4 FGF-R1
6.7 PHK
5.8 TBK1
4.4 EPH-A4
95.5
96
95.9
96
9.6 p38a MAPK
11 NEK2a
9.5 WNK1
16 BRK
102.0
102
102.1
102
8.7 CAMKKb
11 EPH-B3
5.4 EPH-B2
18 PDK1
113.6
113.7
114.3
114
4.8
1.6
5.9
28
MPSK1
CDK9-
PLK1
MST3
TLK1
PAK4
89.2
89.3
89.5
89.7
2.3 IRAK4
4.4 PDGFRA
7.0 PKCγ
5.8 JNK3
96.0
96.0
96.0
96.1
3.8 Cyclin T1
8.5 JNK2
8.0 BTK
103
102.6
102.6
103
10 MSK1
4.7 SIK3
8.9 PAK2
14 EPH-B4
115
115
117.6
117.8
16
12
6.8
9.5
0.8 EIF2AK3
MAPKAP-K2
SRPK1
90
90.4
10 EPH-B1
1.6 ASK1
96.1
96
1.0 EF2K
14 HIPK1
103.0
103.7
0.3 HER4
7.0 TSSK1
119
119.0
18
3.1
6
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ChemMedChem
JNK1
90.7
91
8.8 PRK2
18 Lck
96.3
97.1
9.2 RSK2
104
10 TIE2
122.1
142
7.2
94
MAP4K5
4.8 MNK2
104.0
5.1 SmMLCK
DYRK2, DYRK1A, and DYRK3 were inhibited by ARC-772 despite the substantially different
protein-binding sites of PKs of the DYRK-family and the resulting different consensus
sequences, compared to these of CK2α.[29,30] Several ATP-competitive inhibitors of CK2 have
been reported to inhibit also PKs of the DYRK-family.[31,32] Therefore, the remarkable
inhibitory potency of ARC-772 towards PKs of the DYRK family may be caused by efficient
binding of the ATB moiety of the biligand inhibitor with no counter-productive effect of the
rest of the conjugate to association. DYRK kinases, similarly to CK2, play an important role in
the regulation of cell proliferation and apoptosis.[33–38] Thereby, CK2 as well as PKs of the
DYRK family (with the exception of DYRK2) are anti-apoptotic PKs and inhibition of any of
them induces the activity of caspases. Thus, inhibitors targeting both CK2 and anti-apoptotic
DYRK kinase may lead to a synergistic physiological effect.
The residual activity of BRSK1 and BRSK2 after inhibition with ARC-772 (c = 100 nM) was
below 25%. These two kinases belong to the CAMK Ser/Thr protein kinase group and do not
possess substrate consensus sequences comprising acidic amino acid residues Asp and Glu,
therefore their inhibition by ARC-772 was not predictable. BRSKs play an important role in
the establishment of neuronal polarity,[39] their concomitant effect (together with CK2) on
apoptosis has not been described.
2.3. Intracellular delivery
ARC-772 possesses good affinity and selective inhibition of CK2 but the negatively charged
peptoid-moiety of the compound retards cellular uptake of the compound. Five different
technologies were tested for the delivery of ARC-772 into cells (Figure 2). The efficiency of
uptake was compared by determining the intracellular concentration of the compound after 12
h incubation. The ATB moiety of ARC-772 is fluorescent, its fluorescence intensity (FI) is
enhanced at pH 2 (compared to FI at pH 7, Figure S. 1). HPLC measurements were carried out
on the RP C18 column, using ACN gradient in water (containing 0.1% TFA, pH 2) that enabled
good resolution of ARC-772. A FI detector (ex 322 nm, em 406 nm) was used for the sensitive
analysis of the compound (Figure S. 2). The limit of quantification (LoQ) of the developed RP-
HPLC-FL method is 5.1 fmol. The limit of quantifiable intracellular concentration depends on
the particular assay conditions. As an example, in case of analysing 1 000 000 HeLa cells with
an average cell volume of 2.1 pL in three replicates and with 100% of recovery, the established
LoQ for intracellular concentration ARC-772 was 4 nM.
7
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10.1002/cmdc.201700457
ChemMedChem
Figure 2. Scheme of delivery systems used for the transport of ARC-772 into cells.
ARC-772 without any transporter penetrated the cell plasma membrane poorly: (1.0 0.2) μM
intracellular concentration was determined after incubation of HeLa cells for 12 h with 5 μM
compound and incubation with 1 μM solution led to (0.20 0.07) μM cellular concentration of
the compound (Figure 3). We compared the transport efficiency of the following internalisation
techniques to facilitate the uptake of ARC-772 (Table S. 2): conjugation of folate, RPARPAR,
or (L-Arg)9 moiety via a disulfide bridge; masking the negatively charged carboxyl groups with
esters; encapsulating ARC-772 in micelles of the transfection reagent TurboFect.
Figure 3. Comparison of the efficiency of different internalization methods on basis of the achieved intracellular concentration
in HeLa cells. Incubation solution containing the compound at 5 μM or 1 μM concentration was applied on the cells for 12
hours (n = 3, average SD).
Two of the applied delivering techniques included receptor-mediated endocytosis: first, folate
receptor-mediated delivery of the folate conjugated compound (ARC-778) and, second,
neuropilin-1 receptor (NRP-1) mediated delivery of the compound conjugated with RPARPAR
8
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10.1002/cmdc.201700457
ChemMedChem
peptide (ARC-791). Although folate[40–42] as well as NRP-1 receptors[43,44] are overexpressed in
cancerous cells, here the receptor-mediated transport resulted in only low micromolar
intracellular concentration of ARC-772-SH (Figure 3) – (1.0 0.3) μM for ARC-RPARPAR
conjugate and (2.3 0.7) μM for ARC-folate conjugate if the extracellular concentration of the
conjugates was 5 μM. In case of 1 μM extracellular concentrations of the conjugates
intracellular ARC-772-SH concentrations of (0.4 0.2) μM and (0.60
0.09) μM were
achieved, respectively. There are 1 x 106…3 x 106 folate receptors per cell[45], each capable of
transporting ~3 molecules of folate per day[42]. Thus, in HeLa cells [volume 2.1 pL, calculated
by applying the measured diameter (16 μm) of a spherical cell], the estimated intracellular
concentration of a folate receptor-transported compound after 12 h incubation would be
approximately 1…3 μM that is in good accord with the determined concentration of the
compound.
The uptake efficiency of the compound ARC-783 (concentration in the incubation solution of
5 μM), conjugate of ARC-772 and transport peptide (L-Arg)9, was low: (2.1 0.3) μM
intracellular concentration of ARC-772-SH was achieved. Both inefficient endosomatic uptake
mechanism and masking of the positively charged guanidinium groups of (L-Arg)9 with the
negatively charged peptoid of ARC-772 could reduce positive effect of the transport peptide.[46]
Commercially available transfection reagent TurboFect did not substantially enhance the
uptake of ARC-772 after 12 h incubation – leading to (3.1 0.8) μM intracellular concentration
of ARC-772 in case of its 5 μM extracellular concentration and (0.5 0.2) μM intracellular
concentration in case of 1 μM extracellular concentration of the compound.
The treatment of HeLa cells with solution of ARC-775, a per-esterified prodrug of ARC-772
led to the highest intracellular concentration (more than 100 μM) of ARC-772. The uptake
mechanism of the prodrug, permethyl ester of a derivative of a carboxylate-rich peptoid, is not
known. In case of peptide derivatives often direct penetration and transport via human peptide
transporter 1 (PEPT1) have been attributed to the prodrug approach.[47,48] In case of peptoid
derivatives uptake mechanisms may be different. Either way, ARC-772 is not trapped in
endosomes and quickly finds its target protein CK2. Upon uptake, the ester is hydrolysed in an
hour to be fully activated (hydrolysed to ARC-772 by esterases) for its interaction with CK2.[17]
The experiments were also performed on PPC-1 cells, where the technology of conjugation of
RPARPAR has been applied excessively. As statistically similar results were obtained (Figure
S. 3), in further experiments with ARC-775 only HeLa cells were used.
Cellular uptake intensity of ARC-775 was further investigated upon 1 h, 5 h, and 12 h
incubation period with the prodrug (Figure 4). At 5 h time point, approximately 20-fold higher
concentration of ARC-772 was registered in cells, compared to the concentration of its prodrug
ARC-775 in the incubation solution at tested concentrations of 0.5 μM, 1.0 μM, and 5.0 μM of
the latter compound. Reduction of the intracellular concentration of ARC-772 at 12 h time point
may point to the extracellular degradation of the prodrug ARC-775 and recycling of ARC-772.
Revival of apoptotic processes may also lead to reduction of ARC-772 concentration in cells.
However, decrease of the intracellular concentration at 12 h time point was not significant in
case of 5.0 μM extracellular concentration, pointing to excretion of certain, not proportional
amount of ARC-772.
9
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ChemMedChem
Incubation time:
140
120
100
80
1 h
5 h
12 h
20
10
0
M
M
M
M
M
µ
µ
µ
µ
µ
5
1
5
.
1
5
.
0
.
0
0
0
Extracellular concentration
Figure 4. Intracellular concentration of ARC-772 in HeLa cells after treatment of the cells with various concentrations of ARC-
775 for 1 h, 5 h, or 12 h (n = 3, average SD).
2.4. Cellular efficiency
Intracellular concentration of a compound gives valuable insight of its potential as a drug.
Nevertheless, the cellular efficiency of the compound should be demonstrated with different
assays. Here, we proved that upon cell membrane penetration, the prodrug ARC-775 was
hydrolyzed by esterases into its active form, compound ARC-772 that efficiently inhibited CK2.
The functionality of ARC-772 as an inhibitor was demonstrated in three different assays:
induction of changes in phosphorylation level of a CK2 substrate – Cdc37; activation of
caspase-3 (CASP3), as detected by the accelerated cleavage of FRET probe GFP-DEVD-RFP;
and suppression of ADP-induced platelet aggregation (Figure 5). Experiments were performed
mostly with HeLa cells in which high expression level of two catalytic (α or α′) and the
regulatory (β) subunits was established long ago.[49]
10
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10.1002/cmdc.201700457
ChemMedChem
Figure 5. Upon penetration of cell plasma membrane, the prodrug ARC-775 is cleaved by esterases into its active form ARC-
772. The efficiency of the inhibitor in the cellular milieu is demonstrated by three assays (written in bold).
2.4.1. Cdc37 phosphorylation
HeLa cells treated with ARC-775 or the control compound CX-4945 demonstrated efficient
inhibition of CK2-catalyzed phosphorylation of co-chaperon protein Cdc37 at Ser13. The
average pCdc37/Cdc37 ratio of ARC-775 (5 μM) treated cells compared to that of control was
(44 8)% after 5 h and (36 7)% after 12 h incubation, CX-4945 revealed phosphorylation
level of (72 10)% after 5 h and (55 8)% after 12 h incubation (Figure 6). As the main
function of co-chaperon Cdc37 is the recruiting of proteins to heat shock protein 90 (Hsp90)
and the folding of proteins for their proper physiological function, diminished phosphorylation
of Cdc37 affects many processes in cell (Figure 1.A).[50–52] In conclusion, the results support
the use of oligoanionic biligand inhibitors in the form of acetoxymethyl ester prodrug for
targeting CK2 in cells to affect the phosphorylation of Cdc37.
Control
5 µ
M
5 µM
ARC-775
CX-4945
100
75
50
25
0
Incubation time/h
Figure 6. Average pCdc37/Cdc37 ratio results of HeLa cells (n = 3, average SD).
2.4.2. Activation of CASP3
Secondly, we studied the effect of treatment of cells with compounds of interest (COIs) on the
activity of CASP3. While CX-4945 and ARC-775 apparently affect the activity of CASP3 via
CK2 inhibition, the effect of Staurosporine on CASP3 activity is mediated by reduced activity
of many PKs.[53,54] CK2 participates in caspase activity regulation in in multiple ways.[10,14]
First, the phosphorylation of the targets of CASP3 prevents the cleavage of pro-survival
proteins by CASP3. Secondly, phosphorylation of procaspase-3 protects it from cleavage by
CASP8 and CASP9, which, in turn, prevents the activation of CASP3. Thirdly, phosphorylation
of CASP3 directly inactivates it (Figure 1.B).
The activation of CASP3 upon treatment of cells with PK inhibitors is clearly seen in case of
cancerous HeLa and noncancerous CHO cell lines (Figure 7.A, Figure S. 4). The maximal
activity of CASP3 was achieved at 4 h treatment of HeLa cells with Staurosporine, while CX-
4945 and ARC-775 revealed slower effect on CASP3 activity – it took 8 h and 12 h to achieve
maximal activity of CASP3, accordingly (Figure 7.C). Differently, in case of noncancerous
CHO cells, the CASP3 activation kinetics was slower for all tested compounds (Figure 7.D).
For Staurosporine treated cells, the plateau was reached after 12 h and for CX-4945 and ARC-
775 treated cells, at these concentrations, a clear plateau was not reached even after 16 h. Data
points after 16 h were diffusive and were not used for analysis. In case of Staurosporine, the
time-scale of the induction of apoptosis is dependent on the origin of particular apoptotic
11
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10.1002/cmdc.201700457
ChemMedChem
pathway – the caspase dependent pathway is quicker (3 h) and caspase independent pathway
slower (>12 h).[53] In case of CK2 inhibitors CX-4945 and ARC-775, apoptosis is induced by
the caspase-dependent pathway, nevertheless, CASP3 was slowly activated. The effect of ARC-
775 is additionally delayed by the time period needed for de-esterification by esterases in cells
(approximately an hour)[17], but still the reasoning for retarded effect of ARC-775 (compared
to the effect of Staurosporine) is not clear and needs further investigation.
Comparison of the results with cancerous HeLa cells and noncancerous CHO cells proves the
commonly known fact – the viability of cancerous cells is more dependent on the activity of
CK2.[12,55] It follows that ARC-775 more selectively activates CASP3 of cancerous HeLa cells
than CX-4945 and Staurosporine – the difference in the EC50 values determined for these
compounds in HeLa and CHO cells is 53-, 5.5-, and 3-fold, respectively (Figure 7.B).
A
B
60
50
40
30
20
10
0
EC50/μM
~ 16
36
ARC-775
CX-4945
O
a
2
H
C
Staurosporine 0.09 0.01
ARC-775_CHO
CX-4945_CHO
Staurosporine_CHO
ARC-775_HeLa
CX-4945_HeLa
ARC-775
CX-4945
0.3 0.1
6.5 1.1
L
e
H
Staurosporine 0.03 0.01
Staurosporine_HeLa
-8
-6
-4
-2
0
-10
log(Cinh/M)
C
D
ARC-775 (5 µ
CX-4945 (50 µ
Staurosporine (1 µ
Control
M)
M
ARC-775 (5 µ
M)
)
CX-4945 (50 µ
M
)
M)
60
50
40
30
20
10
0
Staurosporine (1 µM)
Control
60
50
40
30
20
10
0
2
4
6
8
10 12 14 16
2
4
6
8
10 12 14 16
-10
-10
t/h
t/h
Figure 7. A) Dose-response curves of ARC-775, CX-4945, and Staurosporine in case of cancerous HeLa cells and non-
cancerous CHO cells. B) Corresponding EC50 values SD, n = 2…4. C) Kinetics of CASP3 activity in HeLa cells. D) Kinetics
of CASP3 activity in CHO cells.
2.4.3. Inhibition of platelet aggregation
Finally, the effect of three CK2 inhibitors and the control compound Forskolin on the ADP-
induced aggregation of human platelets was studied. CK2 inhibitors act through lowering the
expression of P-selectin, inhibition of glycoprotein IIb/IIIa[6] and downstream targets of the PI
3-kinase pathways (PDK1, Akt, GSK3β, Figure 1.C)[11]. Forskolin was applied as the control
compound, as its presence strongly inhibits platelet aggregation by activating adenylyl cyclase,
production of cAMP, and, in consequence, activation of PKA.[56,57] The platelets were pre-
incubated with 10 μM COIs for 30 min or 60 min; thereafter, aggregation was initiated by the
addition of ADP. Activation of platelets by ADP results in the inhibition of PKA pathway and
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10.1002/cmdc.201700457
ChemMedChem
utilization of the intrinsically active CK2-catalyzed pathways, thus grounding the choice of
compound initiating the aggregation process for studying the effect of particular COIs. The
optimal concentration of ADP was determined to be 25 μM – the lowest concentration that
consistently induced significant aggregation and sufficient measurement window with low
signal to noise ratio (Figure 8.A). The addition of ADP induced aggregation promptly, reaching
a plateau within 5…6 min.
A
B
0.4
0.3
0.2
0.1
0.0
100
75
50
25
0
*
*
*
*
*
*
*
*
*
*
30 min
60 min
0
2
4
6
8
0
2
4
6
8
1
1
1
1
1
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
Platelets
t/min
25 µM
M
ADP
COI
50 µ
25 µ
12.5 µ
M
ADP + Platelets
6.25 µ
0 µ ADP + Platelets
Buffer
M ADP + Platelets
-
-
10 µ
M
ADP + Platelets
M
M
ADP + Platelets
C
D
100
100
50
*
*
*
*
*
*
*
50
0
*
*
*
*
*
*
*
*
*
0
*
*
*
*
-50
-100
-150
-50
-100
30 min
60 min
30 min
60 min
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
Platelets
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
Platelets
+
-
10 µ
M
ADP
COI
+
-
25 µM
M
ADP
COI
-
-
10 µ
M
-
10 µ
Figure 8. ADP-induced aggregation of platelets. A) Initiation of platelet aggregation by 6.25…50 μM ADP. As negative
controls, buffer and non-treated platelets were used (n = 3, average SD). B) and C) The platelets were pre-incubated for 30
min or 60 min with 10 μM compound of interest (COI): ARC-775, ARC-1859, CX-4945, or Forskolin (n = 3, average SD).
The aggregation of platelets was initiated with 25 μM ADP. Assays B and C were performed with different samples of platelets
on different days. D) Same as C, but the aggregation of platelets was initiated with 10 μM ADP. * p < 0.05 compared to active
platelets (Platelets + ADP).
The results of experiments with the first sample of human platelets (Figure 8.B) and the second
sample of platelets (Figure 8.C and D, Figure S. 5, performed on a different day) demonstrated
good efficiency of ARC-775 in inhibition of ADP-induced platelet aggregation. In the first case,
30 min pre-incubation with ARC-775 resulted in (66 6)% of maximal aggregation (MA)
compared to the control (active platelets). After 60 min pre-incubation with ARC-775, (33
5)% of MA was achieved (Figure 8.B). The increased effect in inhibition of aggregation over
time clearly indicated the improved effect of the inhibitor after increased proportion of de-
esterified ARC-775. In case of the second sample of platelets, the effect of ARC-775 was even
more pronounced compared to the first sample (Figure 8.C). While the platelets pre-treated for
30 min with ARC-775 showed effectively suppressed aggregation [MA = (16 3)%], the
platelets pre-treated for 60 min demonstrated even negative aggregation percent [MA = (-56
11)%] indicating a shift from the basal level of aggregation.
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ChemMedChem
The control compounds ARC-1859 and CX-4945 diminished the ADP-induced platelet
aggregation in case of both samples, but did not show significantly different results for
experiments performed on two different days (p < 0.05). Pre-treatment of the first sample of
platelets with Forskolin resulted in significant suppression of aggregation, whereas the
corresponding MA did not statistically differ (p < 0.05) from the active platelets (Figure 8.B).
Interestingly, in case of the second sample of platelets, the effect of Forskolin was more
pronounced after 30 min pre-incubation as compared to 60 min pre-incubation (Figure 8.C).
Aggregation of the second sample of platelets was in parallel initiated with 10 μM ADP to
compare these results with the experiments performed by Ampofo et al.[6] With the exception
of Forskolin, all COIs demonstrated similar or slightly better results with 10 μM ADP compared
to the results with 25 μM ADP (Figure 8.C). In our experiments, treatment of platelets with 10
μM CX-4945 for 30 min resulted in (54 9)% of MA (Figure 8.D). In literature, pre-treatment
with 10 μM CX-4945 for 30 min resulted in approximately 20% of MA after addition of 10 μM
ADP.[6] Notably, the latter assays were performed with washed platelets. Unwashed platelets
(as in our assays), however, include additional plasma proteins that may bind to COIs and, as a
result, reduce their effect on platelet aggregation.
To conclude, these results demonstrate for the first time the effective suppression of platelet
aggregation as a result of their treatment with biligand inhibitors of CK2, ARC-1859 and ARC-
775. As the role of CK2 in the development of arterial thrombosis has been proven and platelets
represent an important target for therapeutic mediation,[58] the tested compounds hence
represent a novel class of biologically active compounds that may be developed into promising
drug candidates.
3. Conclusions
Biligand inhibitors of CK2 were constructed by conjugating 4-(2-amino-1,3-thiazol-5-
yl)benzoic acid (ATB) and carboxylate-rich peptoids. ARC-772, a conjugate with optimized
linker between CK2-interacting structural fragments that possesses subnanomolar affinity
towards CK2 (Kd = 0.3 nM), moderate molecular weight, and relatively low structural
complexity was chosen for cellular studies. ARC-772 revealed good inhibitory selectivity
towards CK2α in a panel of 140 PKs as indicated by the value of Gini coefficient of 0.752 and
hit rate of 0.05.
As ARC-772 revealed low cell plasma membrane penetrative capability, 5 different strategies
were tested to enhance its cellular uptake. Esterification of the negatively charged carboxylate
groups (compound ARC-775) led to distinctively improved plasma membrane penetration
properties of the compound – more than 100 μM intracellular concentration was obtained (20-
fold increase from 5 μM concentration in incubation solution). Upon cell plasma membrane
penetration the per-estererified prodrug of ARC-772 was hydrolyzed by esterases into the active
form of the compound (ARC-772) that was trapped in the cytosol and efficiently inhibited CK2
in cells.
Three different assays were used to demonstrate the capability of ARC-772 to regulate the
activity of CK2 in cells and compare its inhibitory potency with that of the reference compound
CX-4945, the first CK2 inhibitor in clinical trials. First, inhibition of CK2 with ARC-772
resulted in lowered proportion of phosphorylated Cdc37 in HeLa cell lysate, at similar or
slightly lower level than in the presence of CX-4945. This change in phosphorylation balance
could result in aberrant folding of client proteins of Cdc37 and thus affect many processes in
cells. Next, the role of CK2 in the regulation of the activity of CASP3 was studied. Here, ARC-
772 revealed significantly better results compared to CX-4945 in terms of dose-response
relationship. Even more, a distinctive specificity between cancerous HeLa cells and non-
14
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10.1002/cmdc.201700457
ChemMedChem
cancerous CHO cells was achieved by ARC-772. Although the CK2-binding affinities of ARC-
772 and CX-4945 are very similar, ARC-772 possessed remarkable 20-fold lower EC50 value
for CASP3 activation in HeLa cells. Finally, this paper demonstrated for the first time that the
biligand inhibitor of CK2 ARC-772 effectively inhibited ADP-induced human platelet
aggregation.
In conclusion, the results presented in this study support the use of oligoanionic biligand
inhibitors for selective targeting of CK2 in cells. These inhibitors respresent a novel class of
biologically active compounds that may be developed into drug candidates and specific tools
for investigation of still unspecified signalling pathways regulated by CK2.
4. Materials and Methods
4.1. Chemical synthesis
4.1.1. Materials and methods
Chemicals were purchased from Acros, Alfa Aesar, Iris Biotech GmbH [2-chlorotritylchloride
(2CTC) resin and most of the chemicals for peptide synthesis], Fluka, Novabiochem
(cysteamine-4-Mmt resin), Fisher Scientific, and Sigma-Aldrich. HPLC-MS was performed
with Schimadzu LC Solution (Prominence) system connected to LCMS-2020 ESI-MS.
Purification of the samples was performed with RP-HPLC equipped with a Phenomenex Luna
C18 analytical column (5 μm, 250 × 4.6 mm) maintained at 40 °C, and photodiode array
detector SPD-M20A. Acetonitrile/water + 0.1% aqueous TFA gradient at a flow rate of 1
mL/min was used. ESI-HRMS of compounds were measured with Thermo Electron LTQ
Orbitrap and combined Varian 910-FT-ICR and Varian J-320 3Q spectrometers in positive ion
mode.
4.1.2. Synthesis of derivatives of ATB
Me-ATB, Me-ATB-Suc and tBu-ATB (Scheme S. 1.A) were synthesized in solution phase as
described elsewhere.[23,59] Other ATB conjugates were synthesized according to Scheme S.
1.B...1.E.
Compounds 7, 12, 13 and ATB-peptoid conjugates with C-terminal carboxylic acid (1...6,
8...10) were synthesized on 2CTC resin (1.1 mmol/g). The resin was loaded with the first
residue from a solution of the carboxylic acid (bromoacetic acid for compounds 1...6 and 8...10
or 12-bromododecanoic acid for 12 and 13, both 5 eq) and DIPEA (7.5 eq) in DCM at r.t. (4 h).
The synthesis of 11 was started from cysteamine-4-Mmt resin (1.1 mmol/g) that was loaded
with the first residue by treatment with the solution of bromoacetic acid (5 eq) and DIC (5 eq)
in DMF at r.t. (1 h).
The [Ida(tBu)]n peptoid chain of the conjugates (1...6, 8...11) was synthesized by submonomer
strategy of peptoid synthesis according to previously established protocols.[17]
Compounds 2...6 (Scheme S. 1.C) were prepared by first linking Fmoc-ω-aminoalkanoic acid
to the resin-bound peptoid by COMU mediated coupling (3 eq Fmoc-ω-aminoalkanoic acid,
2.9 eq COMU, 6 eq 2,4,6-collidine in DMF, r.t., 2...3 h), followed by removal of Fmoc by
standard procedure (20% piperidine/DMF, 5 min + 15 min treatment). Second, Me-ATB-Suc
was amidated to the resin-bound ω-aminoalkanoic acid-peptoid conjugate (1 eq Me-ATB-Suc,
0.95 eq COMU and 2 eq 2,4,6-collidine, r.t., 5 h).
Coupling of Me-ATB-Suc to resin-bound peptoid as described for synthesis of 2...6 did not
yield compound 1. Instead, compound 1 was made by sequential coupling of succinic acid (10
15
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10.1002/cmdc.201700457
ChemMedChem
eq succinic anhydride in DMF/pyridine 1/1 by volume, 9 h at r.t.) and Me-ATB (resin activated
with 3 eq COMU and 6 eq 2,4,6-collidine at r.t., 0.5 h, then 1 eq of Me-ATB and 4 eq of DIPEA,
r.t., 8 h) to the Ida(tBu)-2CTC resin (Scheme S. 1.B).
Compounds 12 and 13 were prepared by 6-step scheme starting from 2CTC-resin (Scheme S.
1.D). 12-bromododecanoic acid was esterified to 2CTC resin as described above. The resin was
treated with a solution of glycine tert-butyl ester hydrochloride (10 eq) and DIPEA (12.5 eq) in
DMF (0.4 mL) at 50 °C overnight (16 h). The resin was washed (DMF) and solution of succinic
anhydride (10 eq) in DMF/pyridine (1/1 by volume) was added. The resin was shaken at r.t. for
9 h and washed (DMF). Finally, the free resin-bound carboxylic acid group of succinic acid
residue was activated [3 eq COMU and 6 eq 2,4,6-collidine in DMF (synthesis of 12) or 3 eq
HBTU, 4 eq HOBt and 6 eq NMM in DMF (synthesis of 13); r.t. 0.5 h], the resin was filtered
and agitated with a solution of Me-ATB (1 eq) and DIPEA (4 eq) or tBu-ATB (1 eq) and NMM
(6 eq) in DMF at r.t. for 8 h. The resin was washed, vacuum-dried and the compounds were
liberated from the resin by treatment with a mixture of AcOH/TFE/DCM (1/1/3 by volume, 0.6
mL; TFE: trifluoroethanol; r.t., 3 h). The resin was filtered and washed with DCM (2 × 1 min).
The combined filtrates were evacuated, co-evaporated with hexane and toluene and purified by
RP-HPLC.
The attachment of the ATB-linker moiety to the peptoid part of 8 started from coupling 12-
bromoacetic acid to the resin-bound peptoid (5 eq acid and 5 eq DIC in DMF, r.t., 4 h) and the
rest of the conjugate was assembled using the same scheme (Scheme S. 1.E) as described for
compound 12. This strategy appeared unpromising for scale-up due to low yield. More
successful strategy was based on direct coupling of separately made compound 12 or 13 to
resin-bound peptoid in the following conditions: compound 12 (1 eq; synthesis of 10) or 13 (1
eq; synthesis of 9 and 11), COMU (1.1 eq) and 2,4,6-collidine (3 eq) in DMF, 5 min
preactivation and overnight coupling at r.t. to the resin-bound peptoid (2 eq; Scheme S. 1.E).
The attachment of ATB moiety to the resin was confirmed by the appearance of strong blue
fluorescence of the resin beads when illuminated under 350 nm. The removal of tBu-groups
and cleavage of ATB-peptoid conjugates from 2CTC-resin (compounds 1...6 and 8...10) was
achieved by treatment with a cocktail of TFA/H2O/TES (100/5/5 by volume; TES:
triethylsilane; r.t., 3...4 h). The same treatment of 13 yielded 7. Compound 11 was cleaved from
the resin by treatment with Reagent B (TFA/PheOH/H2O/TIPS 88/5/5/2 by volume; r.t., 1h).
After cleavage, the resins were washed with TFA. Solvents were removed in vacuum, the
residue was co-evaporated with H2O and washed with Et2O. The methyl ester of the Me-ATB-
fragment of the conjugates (1...6, 8 and 10) was hydrolysed in a solution of KOH (1 M in H2O,
r.t., 1 h), followed by neutralization with aqueous HCl (1 M).
14 (ARC-775, hexaacetoxymethyl prodrug of ARC-772) was prepared by per-esterification of
compound 9 following previously published acetoxymethylation protocol (Scheme S. 1.E).[17]
All products were purified by RP-HPLC and analysed by MS (Table S. 1).
4.1.3. Polymer-supported peptide synthesis
C(Npys)-Ahx-RPARPAR (16) and C(Npys)-R9-NH2 (17) were synthesized on 2CTC (1.1
mmol/g) and Rink-amide MBHA (0.61 mmol/g) resins, respectively, according to Fmoc solid-
phase peptide synthesis protocols. The following coupling conditions were applied: protected
amino acid (3 eq), COMU (2.9 eq), and 2,4,6-collidine (6 eq) in DMF, 1 min preactivation and
1 h coupling. Fmoc-Arg(Pbf) and Boc-Cys(Npys) were used as building blocks of Arg and N-
16
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10.1002/cmdc.201700457
ChemMedChem
terminal Cys residues, respectively. The Cys(Npys)-protected peptides were released from the
resin by treatment with TFA/H2O/TIPS (95/2.5/2.5 by volume, TIPS: triisopropylsilane, r.t., 3
h), purified by RP-HPLC and analysed by MS (Table S. 1).
4.1.4. Folate-NH-CH2-CH2-S-S-Pyr (15)
Folic acid was converted into NHS ester analogously to reported procedure.[60] Folic acid (49
mg, 0.11 mmol, 5 eq) was dissolved in anhydrous DMSO (1 mL) at 60 °C. The solution was
cooled to r.t. and DIC (6.25 eq) was added. After 30 min, a solution of NHS (6.25 eq) in DMSO
(0.1 mL) was pipetted into the mixture. After 3 h the solution was diluted with EtOAc (5 mL).
The light-yellow precipitate was washed with Et2O and air-dried. The obtained folic acid NHS
ester was dissolved in DMSO/TEA (10/1 by volume, 0.5 mL) and combined with cysteamine-
4-Mmt resin (1.1 mmol/g, 20 mg, 0.022 mmol, 1 eq), pre-swollen in DMSO. The resin was
agitated at r.t. overnight, washed with DMF (3 × 1 min), iPrOH (1 min) and DCM (3 × 1 min)
and vacuum-dried. The capture of folic acid was visible by intensification of the yellow colour
of the resin. Treatment with a solution of 2,2’-dithiodipyridine (AldrithiolTM-2, 5 eq) in a
mixture of TFA/TIPS/H2O (100/5/5 by volume, 0.4 mL; r.t., 0.5 h) cleaved the compound from
the resin as pyridyl disulphide 15 (Scheme S. 2).[61] The product was precipitated by pouring
the cleavage mixture into MTBE (40 mL). The yellow suspension was centrifuged and washed
with Et2O. The title compound was isolated by RP-HPLC and analysed by MS (Table S. 1).
4.1.5. ARC-S-S-transport vector conjugates (18, 19, 20)
Heterodimerization of transport vectors 15...17 with Spyr- or Npys-activated sulfhydryl group
and ARC-772-SH (11) was carried out according to published procedure (Scheme S. 2).[62] The
solution of 11 in aqueous ACN/0.1% AcOH (1/1 by volume) was added dropwise over 5 min
to a molar equivalent of 15, 16, or 17 in NH4OAc (1 M in H2O or in H2O/ACN 1/1 mixture to
increase solubility). After 15 min at r.t. the solvents were removed in vacuum, the conjugates
were purified by RP-HPLC and analysed by MS (Table S. 1).
4.2. Binding/displacement assay
The binding affinities of ligands were determined in the displacement assay, as described
earlier.[23,63–65] The assay was carried out in 4-component buffer [50 mM HEPES (Sigma), 150
mM NaCl (Riedel-de Haën), 0.005% Tween® 20 (Sigma), 5 mM DTT (Fluka), pH 7.5] in the
final volume of 20 μL on black 384-well polystyrene microplates with nonbinding surface
(Corning #4514). Experiments were run in 3…6 parallels. The concentrations of stock solutions
of ligands and probes were determined spectrophotometrically (NanoDrop 2000c, Thermo
Scientific) in 4-component buffer or DMSO (Sigma Aldrich) by the reference to molar
extinction coefficients determined previously: for compounds containing ATB-fragment ε322nm
= 23 000 M-1cm-1; for ARC-1504 ε647nm = 250 000 M-1cm-1, and for ARC-1530 ε558nm = 80 000
M-1cm-1.[18,23] For compound containing 4-(2-amino-1,3-thiazol-5-yl)benzoic acid and folate
moiety (ARC-778), the molar extinction coefficient was calculated combining the epsilon
values of ATB[23] and folate[66] 306nm = 32 000 M-1cm-1. Full length human recombinant protein
ε
His-CK2α-His was prepared as described previously.[67] The concentration of CK2α was
determined by titration of a fixed concentration (20 nM) of PromoFluor-647-labeled fluorescent
probe ARC-1504 or 5-TAMRA-labelled luminescent probe ARC-1530 (available from
̈
Kinasera OU, Estonia) with the solution of the enzyme (2-fold dilutions). Displacement
experiments with unlabelled compounds were performed by addition of the competing ligand
(3-fold dilutions) to either ARC-1504 (2 nM) in complex with CK2α (3 nM) or ARC-1530 (10
nM or 30 nM) in complex with CK2α (2 nM). CX-4945 (Synkinase) was applied as the
reference compound in all experiments. The microplates were incubated for 60 min at 30 °C.
17
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10.1002/cmdc.201700457
ChemMedChem
The fluorescence anisotropy (FA) and/or time-gated luminescence (TGL) changes relative to
the signal of the free fluorescent probe were registered on a PHERAstar platereader (BMG
Labtech). For FA measurements, optical modules suitable for detecting PromoFluor-647 [ex
590 (50) nm, em 675 (50) nm] and 5-TAMRA [ex 540 (20) nm, em 590 (20) nm] were applied;
for TGL assays with ARC-1530, optical module [ex 337(300…360) nm, em 590(50) nm], delay
time of 80 µs, and gate time of 400 µs were applied.[23] The obtained data were analysed as
previously described to yield the concentration of the catalytically active CK2α and dissociation
constant values for the complexes between the investigated compounds and CK2α.[23,63–65]
4.3. Selectivity profile
The selectivity panel was run on commercial basis by International Centre for Kinase Profiling
(University of Dundee) using radiometric filter-binding assay. 140 PKs applied for
measurements were of human origin. The ATP concentration applied for inhibition
measurements was at or below the Km,ATP value of the particular PK. The final total
concentration of ARC-772 used in the selectivity panel was 100 nM. Residual activities are
expressed as the percentage retained activity compared to the control without inhibitor. Gini
coefficients and hit rates (the percentage of PKs inhibited more than 50% by a given inhibitor)
were calculated from the selectivity data as described in Ref.[68]
.
4.4. Cellular assays
The following cell-lines were used in this work: human cervical cancer HeLa cells (a kind gift
from Beatson Institute for Cancer Research, Glasgow), Chinese hamster´s ovary CHO cells
(ATCC), prostate cancer PPC-1 cell line (a kind gift from Dr. Tambet Teesalu, Cancer Biology
Laboratory, University of Tartu). The cells were grown in the medium (DMEM or
DMEM/Ham’s F12, PAA Laboratories) supplemented with 10% FBS, 100 U/mL penicillin,
100 μg/mL streptomycin (PAA Laboratories). Cell cultures were grown and maintained at 37
°C in a humidified 5% CO2 containing air environment.
4.4.1. HPLC and Western blot analysis
The intracellular concentrations of compounds were determined as described elsewhere.[69]
Briefly, cells were seeded on 6-well culture plates (Thermo Scientific™ 130184) at an initial
density of 250 000 cells per well. After 1 day, ARC-compound of interest was diluted in serum-
and antibiotics-free medium [DMEM High glucose or folate free RPMI (PAA Laboratories); V
= 500 μL], added to the cells, and incubated for 1 h, 5 h, or 12 h. All assays were performed in
three replicates. The cells were detached from the surface by solution of 0.25% trypsin, 0.1%
EDTA (PAA Laboratories). After multiple washing steps with DPBS (PAA Laboratories), the
cell suspension was mixed with Trypan Blue (PAA Laboratories) and the number of cells,
viability, and size was determined with a cell counter (TC-10™, Bio-Rad). The cells were lysed
in cell lysis buffer (NP 40, Invitrogen) containing 1% of Triton-X (Sigma), protease inhibitor
cocktail (Sigma-Aldrich), 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (AppliChem).
The cell lysate was analysed by Western blot and/or HPLC.
4.4.1.1. HPLC analysis
For HPLC experiments, the internal standard ARC-769 (Table 1) was added to the lysate and
the proteins were precipitated with acetonitrile. Thereafter, the sample was centrifuged at 20
000 g, 4 °C for 30 min and the supernatant was dried in the rotational-vacuum-concentrator
(RVC 2-25, Christ). Dry sample was re-suspended in 100 μL of buffer (50 mM HEPES, 150
mM NaCl, 0.005% Tween® 20, pH 7.4) and analysed with HPLC. HPLC was equipped with
Kinetex XB-C18 (Phenomenex) analytical column (5 μm, 250 × 4.6 mm) maintained at 30 °C,
photodiode array detector SPD-M20A, fluorescence detector RF-20A xs, and autosampler SIL-
20A. Acetonitrile/water (0.1% TFA) gradient at a flow rate of 1 mL/min was used.
18
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10.1002/cmdc.201700457
ChemMedChem
Calibration solutions included the analyte and the internal standard. The ratio of the peak area
of analyte to that of the internal standard was used as the assay parameter. Peak area ratios were
plotted against corresponding concentration ratios, and standard calibration curves were
obtained from least-squares linear regression analysis of the data. The linearity of the method
was confirmed via evaluation of the calibration y-intercept and correlation coefficients. The
intracellular concentration was calculated by the measurement model:
ꢅ
ꢅ
ꢆ
ꢇ
ꢐ
ꢀꢁ_ꢂꢃꢄꢄ
=
ꢈꢉ ꢀꢋ ∙ ꢌ
,
(1)
ꢕ
ꢍꢎꢏꢊꢄ
ꢕ
ꢘ
ꢔ
ꢕ
ꢘ
ꢔ
ꢕ
ꢊ
ꢒꢓ
ꢒꢘ
ꢑ
ꢒꢓ
ꢖꢗ
ꢜ ꢝꢑ
ꢖꢗ
ꢜ
ꢙ∙ꢚꢛꢛꢛ
ꢒꢘ
ꢙ∙ꢚꢛꢛꢛ
where Cx-cell – intracellular concentration of ARC, Ax – peak area of the analyte, As – peak area
of the internal standard, b – intercept, a – slope, Cs – concentration of the internal standard,
Vfinal - final volume of the sample, Nca – number of living cells, dca – diameter of a live cell, Ncd
- number of dead cells, dcd – diameter of a dead cell.
The intracellular concentration of ARC-772-SH was determined with applying the same
calibration curve. The similarity of optical and physical characteristics of the compounds was
demonstrated by analysing a concentration series of ARC-772-SH and ARC-772 with HPLC
equipped with a fluorescence detector. F-test was applied for determining the equality of the
results of ARC-772-SH and ARC-772.
The calculations were performed with Microsoft Excel (2016).
4.4.1.2. Western blot analysis
Proteins of cell lysates were separated by gel-electrophoresis and transferred to Western blot
membranes. Proteins were identified by developing of the membranes with the following
primary antibodies: rabbit monoclonal antiphospho-Cdc37 (Ser13, Cell Signaling Technology
Cat# 13248), rabbit monoclonal anti-Cdc37 (Cell Signaling Technology Cat# 4793,
RRID:AB_10695539) and secondary antibody: goat anti-rabbit IgG alkaline phosphatase
conjugate (Thermo Fisher Scientific Cat# T2191, RRID:AB_11180336). Analysis of Western
blot images was performed with ImageJ (v 1.47) software and the calculations of band
intensities with Microsoft Excel (2016).
4.5. Caspase-3 activity assay
For determining the change in caspase-3 (CASP3) activity, a FRET biosensor (Casper3-GR,
Evrogen company) was used combined with the baculovirus-based BacMam transduction
system. The activation of CASP3 in cells leads to cleavage of the DEVD sequence between the
donor and acceptor fluorophores in the fusion protein and thus to elimination of FRET between
the fluorophores. The reaction resulted in decrease of the emission of TagRFP and increase in
the emission of TagGFP. Plasmid construction and generation of BacMam viruses was done as
described earlier.[70] The cells were incubated with the virus for 3…4 hours, detached with
0.25% trypsin/0.1% EDTA, and seeded on a black clear-bottom 96-well cell culture plates
(Ibidi, 89626) at the density of 50 000…100 000 cells/well in 90 µL of culture medium,
supplemented with sodium butyrate (Aldrich) at 10 mM final concentration for enhanced
protein expression. The cells were further incubated for 24 h to allow the expression of Casper3-
GR fusion protein. Before incubation with inhibitors, the cells were washed with DPBS.
All reactions were carried out in the final volume of 100 µL. 10 µL of the 10X concentrated
ligand solution was added to the wells containing 90 µL of serum- and antibiotics-free medium
and the image acquisition program was initiated. Autofocus was performed in the donor channel
(Tag-GFP) and the acceptor channel (Tag-RFP) used the same focus. At least 4 images per well
19
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10.1002/cmdc.201700457
ChemMedChem
were captured in both channels, using the fluorescence plate reader mode on Biotek microscope
Cytation5 at 37 °C, 5% CO2. The responses were measured every 30 min for the first 8 h, and
every 60 min for the next 16 h.
For excitation 465 nm LED cube was used for both channels, for donor (Tag-GFP) emission
525 nm cube and for acceptor (Tag-RFP) emission 593 nm cube were used. The change in
acceptor/donor emission ratios were calculated using the formula:
ꢨ
ꢨ
ꢨ
ꢨ
ꢩꢪꢕ
ꢩꢙꢩ
ꢩꢪꢕ
ꢛ
ꢛ
ꢫ
ꢫ
ꢈ
ꢩꢙꢩ
ꢨ
ꢀℎꢞꢟꢠꢡ ꢢꢟ ꢣꢤ ꢥꢞꢦꢢꢧ =
,
(2)
ꢩꢪꢕ
ꢛ
ꢨ
ꢩꢙꢩ
ꢛ
where I593_0, I525_0, I593_t, and I525_t refer to the mean pixel intensities of fluorescence emission
for Tag-RFP and Tag-GFP proteins before and after the ligand treatment, respectively. The
calculated values are plotted against time or concentration on the x-axis. Analysis of CASP3
activity images were performed with Gen5 software and the obtained data were analysed using
Aparecium software[71] running in Matlab environment, and GraphPad Prism (5.04) program.
For calculating the EC50 values, the average values of Change in FI ratio of 11...16 h data points
were used. The highest value of Change in FI ratio was unified for all data sets according to the
control compound Staurosporine.
4.6. Platelet aggregation
Platelet samples (4 BC platelet concentrate, platelet content 180…340×109, filtered) originating
from healthy volunteer anonymous donors, were obtained from Tartu University Hospital. A
general consent of every donor for use of his/her blood in scientific studies was preliminarily
obtained. The study was approved by the Research Ethics Committee of the University of Tartu
in accordance with The Code of Ethics of the World Medical Association (Declaration of
Helsinki; 251/M-1, PI Dr. Asko Uri and Dr. Darja Lavogina).
The measurement of platelet aggregation was performed by monitoring change in light
transmission at 590 nm using PheraStar microplate reader (BMG Labtech). The assay was
performed on transparent 96-well plates with flat bottom (Greiner, #655101). The pre-
o
incubation of platelets and incubations during the assay were performed at 30 C. Tyrode's
buffer [120 mM NaCl, 5 mM KCl (AppliChem), 3 mM MgCl2 (AppliChem), 2 mM CaCl2
(AppliChem), 45 mM glucose (Sigma), 25 mM HEPES pH 7.4] was used as the main assay
buffer. The final total volume in the assay was 120 µL. During the measurement, the following
stirring conditions were applied: 1 mm double orbital shaking, shaking time 9 s before each
cycle. Each measurement was performed as a triplicate; two independent experiments were
performed on two separate days.
Initially, the concentration of ADP (Sigma) suitable for induction of platelet aggregation was
identified as follows. 2-fold dilutions of ADP were prepared in the assay buffer and pipetted
together with blank solution (Tyrode’s buffer) onto the microplate. The measurement was
started in the kinetic mode. After 3 cycles (cycle time 27 s), the measurement was paused and
platelets or buffer were added to the indicated wells; aggregation of platelets was monitored for
30 min. Based on the results of this assay, we used final total concentration of 10 µM or 25 µM
ADP in the following measurements (25 µM was the lowest concentration that consistently
induced significant aggregation and sufficient measurement window with low signal to noise
ratio, 10 µM concentration was chosen as it has been used most often in platelet aggregation
assays).
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10.1002/cmdc.201700457
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In order to monitor the effect of CK2 inhibitors on aggregation, the following assay was
performed. The solutions of CK2 inhibitors and reference compound Forskolin (Tocris,
adenylyl cyclase activator) were prepared in the assay buffer and then pipetted together with
blank solution (Tyrode's buffer) onto the microplate. The measurement was started in the
kinetic mode. After 3 cycles [cycle time 37 s (first experiment) or 73 s (second experiment)],
the measurement was paused and platelets or buffer were added to the indicated wells; the
microplate was then incubated for the indicated amount of time (30 min or 60 min) and the
absorbance at 590 was constantly monitored. Finally, solution of ADP or buffer was added to
the indicated wells and the measurement was continued for another 30 min.
The final total concentration of inhibitors as well as Forskolin was 10 µM. The platelet count
in each well was calculated as >1.8×107 according to the initial number of provided platelets
and the dilution used. The data were analysed with GraphPad Prism 5.04. For the column
diagrams, the absorbance values at ~15 min following addition of ADP were used. Unpaired t-
test (two-tailed p-value, confidence level of 95%) was used for the analysis of statistical
significance of effects.
Abbreviations
ARC - conjugate of a heterocyclic fragment and a peptide (or a peptide analogue). ARC-772 –
biligand CK2 inhibitor comprising an ATB and oligo-Ida moieties linked with a succinic acid
and 12-aminododecanoic acid residues connected via N-carboxymethylated amide bond. ARC-
772-SH – ARC-772 with sulfhydryl group in the C-terminus of the peptoid. ARC-775 – per-
esterified prodrug of ARC-772. ATB – 4-(2-amino-1,3-thiazol-5-yl)benzoic acid. COI –
compound of interest. Ida – iminodiacetic acid. MA – maximum aggregation. PK – protein
kinases. RP-HPLC-FL – reverse phase HPLC with a fluorescence detector.
Acknowledgements
This work was funded by grants from from the Estonian Research Council (IUT20-17),
Estonian Science Foundation (ETF8230 and ETF8419), the Estonian Ministry of Education and
Sciences (SF0180121s08). HRMS measurements by Varian 910-FT-ICR-MS system were
supported by the Estonian national R & D infrastructure development program Measure 2.3
“Promotion of development activities and innovation” (Regulation No. 34) funded by the
Enterprise Estonia Foundation.
We are grateful to Dr. Kadri Ligi (Institute of Chemistry, University of Tartu, Estonia) for
assisting fluorescence spectra measurements, to prof. Tambet Teesalu (Cancer Biology
Laboratory, University of Tartu, Estonia) for consultation and for providing the PPC-1 cells,
and to prof. Ago Rinken (Institute of Chemistry, University of Tartu, Estonia) for proofreading
the paragraph of activation of CASP3.
Contributions
H.S. performed binding assays, Western blot analysis, purification, ESI-MS-analysis of
compounds, and determination of intracellular concentration by HPLC, and CASP3 activity
assays. H.S. and D.L. performed platelet aggregation assays. K.V. carried out the chemical
synthesis of the compounds. O.M. developed CASP3 activity method, D.L developed platelet
aggregation method. T.H. performed ESI-HRMS analysis. The study was designed by H.S.,
K.V., D.L., E.E., and A.U. All authors contributed to the writing of the manuscripts. The authors
declare no conflict of interest.
21
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10.1002/cmdc.201700457
ChemMedChem
Supplementary Information
Synthesis schemes; data of new compounds, MS and HPLC data; absorbance and fluorescence
spectra of ATB fragment, calibration data of HPLC method; intracellular concentration
measurement data of PPC-1 cells; example curves of CASP3 activity and platelet aggregation
measurements.
Keywords
Protein kinase CK2; inhibitors; apoptosis; platelet aggregation; prodrugs.
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