Potent and Selective CK2 Kinase Inhibitors with Effects on Wnt Pathway Signaling in Vivo

Article abstract of DOI:10.1021/acsmedchemlett.5b00452

The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC₅₀ = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.

Full text of DOI:10.1021/acsmedchemlett.5b00452

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Letter
Potent and selective CK2 kinase inhibitors
with effects on Wnt pathway signaling in vivo
James E Dowling, Marat Alimzhanov, Larry Bao, Claudio Chuaqui, Christopher
Denz, Emma L Jenkins, Nicholas Larsen, Paul D Lyne, Timothy Pontz, Qing Ye,
Geoffrey Allan Holdgate, Lindsay Snow, Nichole O'Connell, and Andrew D. Ferguson
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00452 • Publication Date (Web): 20 Jan 2016
Downloaded from http://pubs.acs.org on January 25, 2016
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Potent and selective CK2 kinase inhibitors with effects on
Wnt pathway signaling in vivo
James E. Dowlinga*, Marat Alimzhanova, Larry Baoa, Claudio Chuaquia, Christopher R. Denza, Emma Jenkinsa,
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Nicholas A. Larsena, Paul D. Lynea, Timothy Pontza, Qing Yea, Geoff A. Holdgateb, Lindsay Snowb, Nichole
O’Connellc and Andrew D. Fergusonc
a AstraZeneca R&D, Oncology Innovative Medicines Unit, 35 Gatehouse Drive, Waltham, MA 02451, USA
b AstraZeneca R&D, Structure & Biophysics, Discovery Sciences, Darwin Building, 310 Cambridge Science Park, Milton
Road, Cambridge, CB4 0WG UK
c AstraZeneca R&D, Structure & Biophysics, Discovery Sciences 35 Gatehouse Drive, Waltham, MA 02451 USA
KEYWORDS: CK2 kinase, pyrazolo[1,5ꢀa]pyrimidine, Wnt, βꢀcatenin
ABSTRACT: The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in
embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling
components, such as Axin, APC and ßꢀcatenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported
link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for
proof of concept studies in vivo. Starting from a pyrazolo[1,5ꢀa]pyrimidine lead (2), we identified compound 7h, a potent CK2 inꢀ
hibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling
(IC50 = 50 nM) in DLDꢀ1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an
intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monꢀ
otherapy in HCTꢀ116 and SWꢀ620 xenografts
The serine/threonine protein kinase CK2 is a constitutively
active heterotetrameric complex composed of two catalytic (α
or α’) and two regulatory (β) subunits,1 which has emerged as
an attractive drug discovery target in oncology.2 Researchers
from Cylene have recently advanced CXꢀ4945, a selective,
orally available inhibitor of CK2 into the clinic for treatment
of patients with solid tumors and haematological malignanꢀ
cies.3
Among its diverse functions, CK2 interacts with and reguꢀ
lates multiple components of the Wnt pathway, an evolutionꢀ
arilyꢀconserved signaling network that regulates embryonic
Figure 1. Early pyrazolo[1,5ꢀa]pyrimidine leads (1 & 2).
development and the regeneration of intestinal epithelial cells.4
Treatment of DLDꢀ1(APC mutant) cells with 2 inhibits βꢀ
Certain cancers, including colorectal carcinoma (CRC), arise
catenin phosphorylation and decreases Wntꢀmediated gene
due to gene mutations among constituents of the Wnt pathway,
transcription as shown in a Luciferase reporter assay in APC
including the CK2 substrates dishevelled (Dvl), APC and ß–
mutant DLDꢀ1 cells (IC50 = 0.06 µM).11 In an acute dose
catenin.5 Inhibition of CK2, either by RNA knockdown or
pharmacokinetic/pharmacodynamic (PK/PD) study, treatment
with small molecules, decreases βꢀcatenin–Tcfꢀmediated tranꢀ
of DLDꢀ1/AKT1 overꢀexpressing murine xenografts with 2
scriptional of Wnt target genes such as survivin and leads to
(10 mg/kg, PO) resulted in the 20% inhibition of Wntꢀ
cell death and apoptosis in a range of CRC lines.6,7 In addition,
mediated luciferase gene transcription at 8 hours, and coincidꢀ
elevated levels of CK2 activity have been reported in CRC
ed with an unbound drug concentration at the level of the Wnt
tissue samples and expression levels correlate with poor progꢀ
reporter IC50. When tested in disease model studies using a
nosis in CRC patients.8,9 Taken together, these data illustrate
murine DLDꢀ1(APCmut) xenograft, compound 2 showed limꢀ
the potential utility of CK2 inhibitors in CRC and other canꢀ
ited tumor growth inhibition.10
cers characterized by aberrant Wnt pathway activity.
Our medicinal chemistry strategy subsequently focused on
We sought to identify a potent, selective inhibitor of CK2
achieving potent inhibition of the Wnt pathway, as demonꢀ
kinase for hypothesis testing in vivo using preclinical models
strated using a Wnt luciferase reporter assay in DLDꢀ1 cells,11
of CRC. An early probe from our previouslyꢀdescribed series
while seeking to improve physicochemical properties, enhance
of ATPꢀcompetitive pyrazolo[1,5ꢀa]pyrimidineꢀderived inhibiꢀ
target coverage, and potentially deliver increased in vivo effiꢀ
tors of CK2 (1, 2; Figure 1) was used to assess the link beꢀ
cacy in preclinical models characterized by aberrant Wnt sigꢀ
tween CK2 inhibition and Wnt signaling.10
naling.
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To aid our design efforts we obtained the Xꢀray crystalꢀ
Scheme 1a Synthesis of compounds 7aꢀq.
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2
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5
6
7
8
lograhic structure of human CK2α at 2.55 Å resolution in
complex with compound 2.12 The inhibitor occupies the ATPꢀ
binding cleft and is anchored to the hinge region via a pair of
hydrogen bonds. The C7 aminocyclopropane group is directed
toward solvent and forms a hydrogen bond with the mainꢀ
chain carbonyl oxygen of V116 while the N1 position of the
pyrazolopyrimidine core interacts with the amide NH of the
same residue. An additional interaction is observed between
an ordered water molecule and the C5 NH of the inhibitor. The
orthoꢀmethyl substituent enforces an energetically disfavored
cisoid configuration of the acetamide that permits coordination
of the carbonyl group with a nearby water molecule. This
bound water, adjacent to the gatekeeper residue F113, also
interacts with the cyano group of the pyrazolo[1,5ꢀ
a]pyrimidine core and the mainꢀchain amide of D175. In addiꢀ
tion, the solved structure suggested that substitution of the
orthoꢀ position (R1 in Figure 1, Scheme 1 and Table 1) with
polar functionality could enable additional energetically favorꢀ
able interactions with the protein.
R1
R1
F
AcHN
AcHN
AcHN
a
b
NO2
NO2
NH2
3
5
4
N
R1
R1
9
N
Cl
AcHN
AcHN
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N
N
N
N
HN
N
NH
N
N
NH
6
g, h
N
N
N
HN
HN
7a-c, f,g, j,k (steps c, d or e)
7h,i,l-q (steps c, d or e then f)
7d,e
7f
(from
)
aReagents and conditions: (a) R,R'NH, Cs2CO3, DMF, 80 °C; (b) H2, Pd/C,
MeOH, 25 °C; (c) 6, 5 mol% Xantphos, 10 mol% Pd2(dba)3, Cs2CO3,DMA,
150 °C in a microwave; (d) 6, KF, NMP or DMSO, 150 °C; (e) 6, 20 mol%
tBuXphos, 10 mol% Pd2(dba)3, Cs2CO3, NMP/Dioxane, 100 °C; (f) TFA,
CH2Cl2, 25 °C; (g) 7f, pyridine, MsCl, 0 °C; (h) R,R'NH, MeCN, 65 °C.
Introduction of an Nꢀmethylpiperazinyl substituent (7a)
enhanced solubility, but reduced enzymatic and cellular potenꢀ
cy relative to 2. However, the acyclic 1,2ꢀdiaminoethyl subꢀ
stituent of 7b imparted high solubility in addition to potent
enzymatic (CK2 IC50 <3 nM) and cellular activity (Wnt DLDꢀ
1 Luciferase IC50 = 75 nM). Further substitution of the termiꢀ
nal amino group, as in N,Nꢀdiethyl analog 7c, reduced the
biochemical and cellular potency. Weakly basic or nonꢀbasic
groups at the terminal position (7d-7f) contributed to a reducꢀ
tion in Wnt reporter activity. Extension of the linker segment
in 7b by an additional methylene unit, producing analog 7g,
results in a greater than 10ꢀfold reduction in enzyme activity
and suggested that optimal positioning of the charged dimeꢀ
thylamino group was achieved with a three atom linker. Pharꢀ
macokinetics for 7b in the rat following oral administration of
10 mg/kg dose are characterized by low exposure (AUC =
0.36 ꢀM•h) and high clearance (CL = 65 mL/min/kg). In vitro
metabolite identification studies using rat hepatocytes identiꢀ
fied a series of products derived from 7b that likely arise via
oxidative demethylation of the side chain nitrogen atoms. Inꢀ
cubations in the presence of ABT in rat and human microꢀ
somes give less extensive metabolite formation and suggest a
predominantly oxidative mechanism of clearance for 7b. We
subsequently synthesized the diꢀdemethylated analog 7h,
which exhibits improved cellular activity (Wnt DLDꢀ1 Lucifꢀ
erase IC50 = 50 nM), high solubility and reduced intrinsic
clearance in rat hepatocytes and human microsomes relative to
7b. In addition, 7h showed greatly reduced activity (IC50 >100
ꢀM) compared to 7b (IC50 = 4 ꢀM) in our hERG ion channel
assay. Interestingly, rigidification of the linker segment
through the use of 3ꢀaminopyrrolidinoꢀ (7l, m) and 3ꢀ
aminopiperidinoꢀ substituents (7n, o), despite contributing to
an overall increase in lipophilicity, afforded compounds with
reduced turnover in rat hepatocyte and human microsome
preparations but led to a reduction in Wnt reporter assay poꢀ
Figure 2. Xꢀray crystallographic structure of human CK2α in
complex with compound 2 determined at 2.55 Å resolution (PDB
accession code: 5H8B).12, 13 Water molecules are shown as red
spheres. Hydrogen bonds to the inhibitor are shown as black
dashed lines.14
Analogs of 2 were synthesized by adapting the convergent
approach described in our earlier work (Scheme 1).10 Treatꢀ
ment of Nꢀ(2ꢀfluoroꢀ5ꢀnitroꢀphenyl)acetamide (3) with secꢀ
ondary amines, followed by reduction of the nitro group in the
resulting products (4), afforded anilines of general structure 5.
Palladiumꢀcatalyzed or KFꢀpromoted coupling of these interꢀ
mediates (5) with 5ꢀchloroꢀ7ꢀ(cyclopropylamino) pyrazoꢀ
lo[1,5ꢀa]pyrimidineꢀ3ꢀcarbonitrile (6) provided the desired
analogs (Table 1) either directly (7a-c, f, g, j, k), via the
methansulfonyl derivative of 7f or, as in the case of 7h-i, l and
q, following TFA deprotection of the corresponding Boc deꢀ
rivatives (Scheme 1)
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tency (Table 1). Substitution adjacent to the primary amine, as
in 7p and 7q, led to further improvement in the in vitro metaꢀ
bolic stability while preserving cellular activity.
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2
3
4
Table 1. Optimization of the orthoꢀsubstituent improves cellular potency, solubility and metabolic stability.
pAKTS129
b
b
5
6
7
8
Wnt DLDꢀ1
Sol.
Hu PPB Hu Mics CLint Rat Heps CLint
CK2α
Cmpd
R1
IC50 (µM)a
0.066
IC50 (µM)a pH 7.4 (µM) (% free) (µL/min/mg)
(µL/min/106)
26
IC50 (µM)a
H
0.009
0.60
15
8
5
20
10
1
2
Me
<0.003
0.002
0.27
0.060
10
5
7
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4ꢀmethylpiperazinꢀ1ꢀyl 0.041
1.1
0.075
0.39
570
18
26
23
<4
9
7a
7b
7c
<0.003
0.009
0.001
0.019
>1000
>1000
32
27
44
<0.003
0.005
0.30
275
18
135
>300
7d
0.013
0.14
ND
0.76
0.32
241
2
2
7
ND
21
ND
17
7e
7f
<0.003
0.006
ND
0.73
>1000
>1000
22
39
<4
7
15
8
7g
7h
<0.003
0.004
0.043
<0.003
ND
0.032
>1000
30
5
2
7i
0.006
0.016
0.38
>800
29
9
2.5
7j
0.004
0.031
0.07
0.51
0.69
>900
590
11
31
19
<3
<1
<1
7k
7l
<0.003
<0.003
0.07
0.96
812
32
<3
2.5
7m
<0.003
0.005
0.008
0.08
0.18
0.65
704
22
20
<3
<3
<1
<1
7n
7o
>1000
<0.003
<0.003
0.004
0.004
0.40
0.67
825
895
33
36
<3
<3
<1
<1
7p
7q
aMean value of two experiments. Deviations were within < ± 25%; bIntrinsic clearance (CLint) determined from human liver microꢀ
some incubations (ꢀL/min/mg) or rat hepatocyte incubations (µL/min/106 cells); ND = not determined
To confirm our initial design hypothesis, we determined
the Xꢀray crystallographic structures of CK2α with 7b and 7h
(Figure 3).12 Both inhibitors are bound in the ATPꢀbinding site
in a manner analogous to that of 2. However, while the
sidechain of 7b positions the terminal dimethylamino group
for an electrostatic interaction with D175, the unsubstituted
amine of 7h is able to directly coordinate an ordered water
molecule and the sideꢀchain carbonyl group of N161.
A number of compounds in this series possess halfꢀ
maximum inhibitory potency below the lower limit of detecꢀ
tion (IC50 <3 nM) in our enzymatic assay, which measures the
inhibition of recombinant human fullꢀlength CK2α mediated
phosphorylation of a synthetic peptide substrate at Km ATP
concentration. To better understand the contribution of the
sideꢀchain interactions on ligand binding we developed a Surꢀ
face Plasmon Resonance (SPR) assay to determine the binding
affinities of 2, 7b and 7h.14 The results, (Table 2) indicate that
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2 and 7h possess similar affinity and are approximately 10ꢀ The ability of our compounds to inhibit the Wnt pathway, as
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fold more potent than 7b. Comparison of 7b and 7h indicate
that the dimethylamino sideꢀchain of 7b is disfavored comꢀ
pared to the primary amine of 7h.
measured using the DLDꢀ1 Topflash reporter assay, correlates
well with inhibition of a direct CK2 substrate, pAKTS129, in
cells.16 These data are consistent with the finding that CK2αꢀ
dependent upꢀregulation of βꢀcatenin driven transcriptional
activity requires phosphorylation of AKT.17 In addition, Wnt
pathway inhibition correlates with antiproliferative effects in
DLDꢀ1 (APC mutant) cells (Figure 5). Similar levels of activiꢀ
ty are observed in other CRC cell lines with constitutively
activated Wnt signaling that is driven either by βꢀcatenin
(HCTꢀ116) or APC mutations (SW620) (Table 3).
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Table 3. Selected growth inhibition data.
Cpd
HCTꢀ116
DLDꢀ1
SW620
GI50 (ꢀM)a GI50 (ꢀM)a GI50 (ꢀM)a
0.7
ND
0.17
0.08
0.05
0.7
0.1
0.03
0.005
1
2
7b
7h
0.08
0.03
0.01
aMean value of at least three experiments; ND = not deterꢀ
mined.
Figure 3. Xꢀray crystallographic structure of human CK2α in
complex with compounds 7b (A) and 7h (B) determined at 2.00 Å
and 2.15 Å resolution (PDB accession codes: 5H8G and
5H8E).12,13 Water molecules are shown as red spheres and hydroꢀ
gen bonds to the inhibitor are shown as black dashed lines.
To further strengthen the mechanistic data, we subsequentꢀ
ly demonstrated that 7h induced a concentrationꢀdependent
decrease in the active form of βꢀcatenin in Wnt3a expressing
mouse fibroblast Lꢀcells,14 an in vitro system in which pathꢀ
way upꢀregulation is triggered by constitutive Wnt3a expresꢀ
sion (Figure S1). In addition, the mouse L S/L line contains a
TCF4 driven luciferase construct and the degree of active βꢀ
catenin inhibition seen with 7h correlates with the potency of
the compound in the corresponding Wnt3a L S/L reporter asꢀ
say (IC50 = 0.05 ꢀM).14
Table 2. CK2α SPR Data.
Compound
KD (pM)
4.91 ± 1.64
48.2 ± 5.78
6.33 ± 0.054
2
7b
7h
Kinase selectivity profiling of 7h at a concentration of 0.1
ꢁM against a panel of 402 kinases revealed a high degree of
selectivity (Figure 4).15 The limited offꢀtarget activity (12
kinases with > 50 % inhibition) was restricted to members of
the CMGC family, including isoforms of the dualꢀspecificity
tyrosineꢀregulated kinases (Dyrk) and CAMK kinases such as
the deathꢀassociated protein kinases (Dapk) and homeodomain
interacting protein kinases (Hipk). Moderate inhibition (74%)
of bone morphogenetic protein receptor typeꢀ1B kinase
(BMPR1b) was also observed. Biochemical IC50
determinations ([ATP] = Km) revealed that 7h exhibits
moderateꢀtoꢀweak activity against the Hipk and Dyrk isoforms
(IC50s = 0.04 ꢀ 1.3 ꢀM), and is active in the 10ꢀ20 nM range
against Dapk2 and Dapk3.14
Based on the in vitro biomarker, pathway and growth inhiꢀ
bition data of 7h, characterization of this compound in vivo
was undertaken. Oral dosing of 7h resulted in low bioavailaꢀ
bility and limited the unbound drug concentration to levels
expected to be subꢀtherapeutic. However, intraperitoneal (IP)
or intravenous (IV) dosing regimens delivered sustained free
drug concentrations above efficacious levels.
Figure 4. Kinase selectivity profile for 7h when tested at a
concentration of 0.1 ꢁM against a panel of 402 kinases
.
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tant/ model and 74% TGI in a SW620 (APC mutant) model at
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a 30 mg/kg dose given weekly for 3 cycles. Reversible doseꢀ
proportional body weight loss was observed in both experiꢀ
ments; in the SW620 study , mean body weight changes obꢀ
served on day 25 in treated animals ranged from ꢀ0.9 to ꢀ6.8%.
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Figure 5. Relationship between pAKTS129 depletion and Wnt
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Topflash reporter inhibition in DLDꢀ1 cells (A). Relationship
between pAKTS129 depletion and growth inhibition in DLDꢀ1
cells (B).
Figure 6. Inhibition of substrate (AKTS129) and downstream
marker (active βꢀcatenin) phosphorylation in SW620 xenoꢀ
grafts by 7h (10, 30 mg/kg, IV). Individual lanes correspond
to vehicle or compound treated animals and are numbered.
(A); Inhibition of Wnt/Tcf4 Topflash luciferase reporter acꢀ
tivity in DLDꢀ1 xenografts following treatment with 7h (10
mg/kg, IV) (B).
The ability of compound 7h to inhibit substrate and downꢀ
stream marker phosphorylation in an APC mutant CRC model
was evaluated in SW620 tumorꢀbearing murine xenografts.
Administration of 7h induced doseꢀdependent modulation of
the downstream markers pAKTS129 and βꢀcatenin, as deterꢀ
mined by Western blot analysis of tumor cell lysates (Figure
6A).14 Similarly, DLDꢀ1 TOPflash luciferase (APC mutant)
xenografts were utilized to assess the effect of the compound
on Wntꢀassociated gene transcription. In this model, treatment
with a single dose (10 mg/kg, IV) of 7h led to 40ꢀ50% inhibiꢀ
tion of Tcf4ꢀluciferase signal at the 8h timepoint with suppresꢀ
sion of this signal, and AKTS129 phosphorylation (not shown),
still evident at 24 hours, by which time no detectable drug
remained in plasma (Figure 6B).
In conclusion, we have identified a series of potent and
selective CK2 kinase inhibitors that decrease AKTS129 phosꢀ
phorylation in cells and whose antiproliferative effects correꢀ
late with inhibition of Wnt luciferase reporter gene transcripꢀ
tion. Using the in vivo probe 7h, we have demonstrated a reꢀ
duction in the downstream biomarkers pAKTS129 and βꢀcatenin
and a high level of activity as a monotherapy in HCTꢀ116 and
SWꢀ620 xenografts.18 Further studies, using 7h and related
analogs, are planned to more fully understand the dependence
of CRC on CK2ꢀmediated Wnt pathway inhibition and will
reported in future communications.
The durable substrate and pathway suppression observed
with 7h following its clearance from plasma may be due, in
part, to the high (pM) affinity of the compound (Table 2) and
its associated slow dissociation rate (kd = 0.00025 sꢀ1). These
and other data,18 suggested the potential for an intermittent
dosing schedule as a means to achieve activity in disease modꢀ
el studies while minimizing tolerability issues.
SUPPORTING INFORMATION AVAILABLE:
Experimental details and characterization data for key comꢀ
pounds, crystallographic and biophysical protocols; active βꢀ
Compound 7h showed a doseꢀdependent tumor growth
inhibition, achieving 94% TGI in a HCTꢀ116 (βꢀcatenin muꢀ
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13. Figure produced using Pymol. DeLano, W. L. The PyMOL
catenin Western blot protocol. This material is available free of
charge via the Internet at http://pubs.acs.org.
Molecular Graphics System; DeLano Scientific: San Carlos, CA,
USA, 2002. http://www.pymol.org.
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14. See supplementary material.
CORRESPONDING AUTHOR:
15. Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge;
C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen;
P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov;
Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.;
Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase
inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127–132.
16. Di Maira, G.; Brustolon, F.; Pinna, L.A.; Ruzzene, M.
Dephosphorylation and inactivation of Akt/PKB is counteracted by
protein kinase CK2 in HEK 293T cells. Cell. Mol. Life Sci. 2009, 66,
3363–3373.
17. Ponce, D. P.; Maturana,J. L.; Cabello,P.; Yefi, R.; Niechi, I.; Silva,
E.; Armisen, R.; Galindo, M.; Antonelli, M.; Tapia, J. C.
Phosphorylation of Akt/PKB by CK2 is necessary for the Aktꢀ
dependent upꢀregulation of βꢀcatenin transcriptional activity. J. Cell.
Physiol. 2011, 226: 1953–1959.
18. Dowling, J. E.; Alimzhanov, M.; Bao, L.; Chuaqui, C.; Denz, C.;
Ferguson, A.; Graff, C.; Liu, Z.ꢀY.; Lyne, P.; Racicot, V.; Wu, A.;
Wu, J.; Ye, Q.; Cooke, E. L. Discovery and characterization of
AZ968, a potent and selective inhibitor of CK2 kinase with effects
on AKT signaling in vivo. Poster presented at the 103rd Annual
Meeting of the American Association for Cancer Research, March
31ꢀApril 4, 2012. Chicago, IL. Abstract No. 3907.
*(J.E.D.) Eꢀmail: james.dowling@astrazeneca.com
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ACKNOWLEDGMENTS:
The authors wish to thank Mei Su and Helen Xiaomei Feng for
compound synthesis, Vicki Racicot and ZhongꢀYing Liu for in
vitro and in vivo biology support, respectively.
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ABBREVIATIONS USED:
heps: hepatocytes, CL: clearance, PK: pharmacokinetics, PD:
pharmacodynamics:, aq.: aqueous, sol.: solubility, PPB: plasma
protein binding, CLint: intrinsic clearance, IP: intraperitoneal, IV:
intravenous, Hu: human, ABT: aminobenzotriazole.
REFERENCES:
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structure of human protein kinase CK2: insights into basic properties
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enhanced βꢀcatenin–T cell factor/lymphoid enhancer binding factorꢀ
dependent transcription. PNAS, 2006, 41, 15079–15084.
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C.;Lin,Y.ꢀF.;Uen, Y.ꢀH. Overexpression of Nuclear Protein Kinase
CK2 α Catalytic Subunit (CK2α) as a Poor Prognosticator in Human
Colorectal Cancer. PLoS One, 2011; 6(2): e17193.
10. Dowling, J. E.; Bao, L.; Brassil, P.; Chen, H.; Chuaqui, C.; Cooke,
E. L.; Denz, C. R.; Larsen, N. A.; Lyne, P. D.; Peng, B.; Pontz, T.
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Zhang, T. Potent and selective inhibitors of CK2 kinase identified
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Hawkins, J. L.; Holmes, J. L.; Howard, T.; Huang, J.; Ioannidis, S.;
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Figure 1. Early pyrazolo[1,5-a]pyrimidine leads (1 & 2).
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Figure 2. X-ray crystallographic structure of human CK2α in complex with compound 2 determined at 2.55 Å
resolution (PDB accession code: 5H8B).12, 13 Water molecules are shown as red spheres. Hydrogen bonds
to the inhibitor are shown as black dashed lines.14
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Synthesis of compounds 7a-q.
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Figure 3. Xꢀray crystallographic structure of human CK2α in complex with compounds 7b (A) and 7h
(B)determined at 2.00 Å and 2.15 Å resolution (PDB accession codes: 2TBD and 3TBD).12,13 Water
molecules are shown as red spheres and hydroꢀgen bonds to the inhibitor are shown as black dashed
lines.14
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Figure 4. Kinase selectivity profile for 7h when tested at a concentration of 0.1 µM against a panel of 402
kinases.
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Figure 5. Relationship between pAKTS129 depletion and Wnt Topflash reporter inhibition in DLD-1 cells (A).
Relationship between pAKTS129 depletion and growth inhibition in DLD-1 cells (B).
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Figure 6. Inhibition of substrate (AKTS129) and downstream marker (active βꢀcatenin) phosphorylation in
SW620 xenografts by 7h (10, 30 mg/kg, IV). Individual lanes correspond to vehicle or compound treated
animals and are numbered. (A); Inhibition of Wnt/Tcf4 Topflash luciferase reporter activity in DLDꢀ1
xenografts following treatment with 7h (10 mg/kg, IV) (B).
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