Protein Kinases CK1 and CK2 As New PDF

Protein Kinases CK1 and CK2 as New
Targets for Neurodegenerative Diseases
Daniel I. Perez, Carmen Gil, and Ana Martinez

Instituto de Quimica Medica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
Published online 23 June 2010 in Wiley Online Library (wileyonlinelibrary.com).

DOI 10.1002/med.20207
.
Abstract: Following the discovery of the human kinome, protein kinases have become the second most
important group of drug targets as they can be modulated by small ligand molecules. Moreover, orally
active protein kinase inhibitors have recently reached the market and there are many more in clinical
trials. The lack of treatments for neurodegenerative diseases has increased human and nancial efforts
in the search for new therapeutic targets that could provide new effective drug candidates. The impor-
tance of kinases in the molecular pathway of neuronal survival is under study, but different key
pathways have been described. New roles for the old casein kinases 1 and 2, currently known as protein
kinases CK1 and CK2, have recently been discovered in the molecular pathology of different neuro-
degenerative disorders, such as Alzheimers and Parkinsons diseases and amyotrophic lateral sclerosis.
The search for specic inhibitors of these enzymes has become an important challenge for the treatment
of these devastating diseases. The role of these two kinases in the molecular pathology of different
neurodegenerative diseases together with different chemical families that are able to more or less
specically inhibit CK1 and CK2 are discussed in this review. & 2010 Wiley Periodicals, Inc. Med Res Rev, 31,
No. 6, 924954, 2011
Key words: casein kinases; CK1 inhibitors; CK2 inhibitors; Alzheimers disease; Parkinsons disease
1. INTRODUCTION
Protein kinases catalyze key phosphorylation pathways that regulate most aspects of cell life,
whereas abnormal phosphorylation is a cause or a consequence of disease. The human
kinome codies nearly 500 different kinases, which are either Ser/Thr or Tyr specic. As they
can be modulated by small ligand molecules, protein kinases have now become the second
most important group of drug targets, after G-protein-coupled receptors.1 The growing
interest in developing orally active protein kinase inhibitors has recently culminated in the
approval of the rst drugs for clinical use, while a substantial number of candidates are still
Contract grant sponsor: MICINN; Contract grant numbers: PET2008__0245; SAF 2009-13015-C02-01; Contract grant sponsor:
ISCIII; Contract grant number: RD07/0060/0015; Contract grant sponsor: NOSCIRA.
Correspondence to: Ana Martinez, Instituto de Qu| mica Medica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain,
E-mail: amartinez@iqm.csic.es
Medicinal Research Reviews, 31, No. 6, 924--954, 2011

& 2010 Wiley Periodicals, Inc.
CK1 AND CK2 AS TARGETS FOR NEURODEGENERATION K 925
in clinical trials. All the approved compounds target the ATP binding site on the kinase,
despite the fact that this site is highly conserved across the entire protein kinase family.2
Nevertheless, as revealed by structural and mutational studies, the highly conserved ATP
binding site is surrounded by specic structural elements. These structural differences
amongst the protein kinase family enable the design and development of quite selective
ligands targeting the ATP site, using different strategies, such as allosteric or covalent kinase
inhibition, including the use of specic antibodies.3
The challenge now is to develop specic kinase inhibitors for other therapeutic indica-
tions where an urgent need for effective treatment exists.4 The overlap between molecular
pathways, involved in cancer and neurodegeneration,5 opens the door for the use of protein
kinase inhibitors as novel therapeutic approaches for neurodegenerative diseases.68 In fact,
the rst glycogen synthase kinase 3 (GSK-3) inhibitor is currently in phase II clinical trials for
Alzheimers disease (AD).9
Neurodegenerative diseases are estimated to contribute to as much as 35% of the disease
burden in the seven major pharmaceutical markets (United States, Japan, France, Germany,
Italy, Spain, and United Kingdom) as measured in terms of disability-adjusted life years.10
They comprise several severe and currently incurable pathologies, such as AD, Parkinsons
disease (PD), and Amyotrophic Lateral Sclerosis (ALS). All of them are characterized by the
loss of neurons in particular regions of the nervous system, an unknown etiology and, as a
consequence, a lack of effective drug therapy.11 However, there are some approved palliative
treatments that temporarily increase the patients quality of life. From the molecular point of
view, neurodegenerative diseases share, in addition to neuronal death, some other common
features, such as decits in neurotransmitter systems and protein misfolding and aggregation.
The clinical symptoms of these different diseases are related to the area of the CNS in which
the neuronal death occurs. AD is the main disease with cognition decits, PD is most
common pathology with debilitating tremors, and ALS is the most prevalent among the
neuromuscular diseases.
In this review, we present the therapeutic potential of the well-known casein kinases,
currently known as protein kinases CK1 and CK2 and their inhibitors, as new targets for
drug discovery programs in the search for a cure for neurodegenerative diseases.
A. From Casein Kinases to Protein Kinases CK1 and CK2

Protein kinases CK1 and CK2 are two structurally different enzymes that, such as other
protein kinases, were originally named according to the substrate (casein).
Casein kinases are multifunctional serine/threonine phosphotransferases that are ubi-
quitously expressed in eukaryotic organisms and yeast, being CK2 one of the rst two
protein kinases isolated.12
In 1971, Waddy and Mackinlay puried protein kinases from soluble extracts of
lactating bovine mammary glands.13 They were called casein kinase 1 and casein kinase 2,
according to the prole obtained by chromatography on diethylaminoethylcellulose.14
A third type of protein kinase related to the two earlier enzymes has been found in several rat
tissues, in particular in the liver and brain.15 The function of this casein kinase, named G-CK,
is not clear, and it seems to be present only in the Golgi apparatus. This G-CK is similar to
the casein kinase of lactating mammary glands and is responsible for the phosphorylation of
casein in the Golgi apparatus of the above-mentioned gland.16 However, it should be noted
that casein does not seem to be a physiological substrate for casein kinases 1 and 2. For this
reason, many researchers avoid reference to casein because it could be misleading about the
physiological role of this substrate. The names CK1 and CK2 were established in 1995.17
Medicinal Research Reviews DOI 10.1002/med
926 K PEREZ, GIL, AND MARTINEZ
In the 1980s, CK1 and CK2 were also puried from the cytosol fraction of hepatome
ascites cells based on their chromatographic differences regarding phosvitin-Sepharose.18 It
was then shown that the amino acid composition of CK2 resembled that of cAMP- and
cGMP-dependent protein kinases, but was considerably different from that of CK1. Indeed,
in the human kinome,19 CK1 represents a unique group within the superfamily of Ser/Thr-
specic protein kinases, while CK2 occupies a small branch adjacent to, but separate from,
the large CMGC subfamily, which includes other cyclin-dependent kinases, mitogen-acti-
vated protein kinases, and glycogen synthase kinase-3.
As CK1 and CK2 are two completely different enzymes in terms of their structure,
physiology, pharmacological properties, and mainly in their involvement in neurodegen-
erative diseases, they are described separately in the sections below.
B. Protein Kinase CK1 and its Role in Neurodegenerative Disorders

CK1 is a Ser/Thr kinase that is ubiquitously expressed in eukaryotic organisms.20 So far, at
least seven isoforms (a, b, g1-3, d, and e) and their various splice variants have been
characterized in different organisms.21,22 They have been reported to act as monomeric,
constitutive enzymes. All CK1 isoforms are highly homologous within their kinase domains,
but differ signicantly in the length and primary structure of their N-terminal and C-terminal
non-catalytic domains.23,24 They exclusively use ATP as a phosphate donor and, in general,
they are cofactor independent.25 Autophosphorylation of carboxy-terminal residues inhibits
the activity of a, d, and e isoforms and helps to regulate their catalytic activity.2628 Members
of the CK1 family are constitutively active and can be isolated as active enzymes from many
different cell types, in several subcellular compartments, including the plasma membrane,
cytosol, and nucleus.
Glutamate, the major excitatory neurotransmitter in the brain, plays a critical role in
multiple brain functions, such as the activity of neurons in the striatum. This brain region is
involved in movement- and motivation-related behaviors. These neuron activities are
dependent on strong glutamatergic innervation from the cortex. Recently, it was reported
that all major CK1 isoforms are expressed in the striatum and the cortex, and that CK1
regulates glutamatergic synaptic transmission mediated by NMDA receptors. Given the
central role of NMDA receptors in several physiological and pathological processes in the
striatum and throughout the CNS, CK1 may be an important regulatory enzyme in normal
states, such as movement- and motivation-related behaviors.29
In addition, CK1 is involved in diverse biological functions,23 such as membrane
transport,30 regulation of DNA repair,31 cellular morphology,32 modulation of the Wnt/b-
catenin and Hedgehog pathway during development,33 and regulation of circadian
rhythms.34,35 In mammals, CK1d and e phosphorylate PERIOD proteins, one of the key
components of the circadian clock, promote their degradation.36 Thereby, the overexpression
of CK1d and/or e should result in the acceleration of the mammalian clock in the brain and
periphery.23
During recent years, several studies have highlighted the importance of CK1 in neuro-
degenerative diseases, especially in tauopathies, such as AD.
AD is a progressive neurodegenerative disorder involving specic neurons of the hip-
pocampus, neocortex, and other regions of the brain. Markers of end-stage disease include
brillar lesions, which accumulate hyperphosphorylated tau protein that is polymerized into
laments, and granulovacuolar lesions, which appear primarily within the hippocampus.37
Results from a distribution study of the three CK1 isoforms (CK1a, CK1d, and CK1e) in
AD and control brains showed that in addition to colocalizing with elements of the brillar
pathology, CK1 was found within the matrix of granulovacuolar degeneration bodies.38
The colocalization patterns revealed that the CK1a isoform was preferentially associated
with neurobrillary lesions, while the CK1d isoform staining pattern was dominated by
colocalization with granulovacuolar degeneration bodies.39
Furthermore, levels of all CK1 isoforms are elevated in the CA1 region of the AD
hippocampus relative to controls, with one isoform, CK1d, being elevated 430-fold. Sub-
sequent studies conrmed a sharp upregulation of CK1d mRNA in AD brain but not in
peripheral organs, the upregulation in the brain being directly correlated with the degree of
regional pathology.40 Because Alzheimer-like phosphoepitopes of tau can be generated by
CK1, the CK1d isoform may play an important role in this fundamental aspect of AD
pathology. Moreover, b-amyloid (bA) protein stimulates CK1 activities, contributing to
abnormal protein phosphorylation in AD.41
Microtubule fractions are used to determine which of the microtubule-associated kinases
most readily impacts the phosphorylation of tau at AD-like sites; one of these kinases is
CK1.42 Specically, CK1d phosphorylates tau at residues Ser202/Thr205 and Ser396/Ser404
in human embryonic kidney 293 cells. Colocalization of CK1d and its substrate tau in
different neurodegenerative diseases as well as co-immunoprecipitation assays, showing the
direct association of tau and CK1d in situ, suggest a clear function for this CK1 isoform in
the abnormal processing of tau.43 It is worth mentioning that the sites that are directly
phosphorylated by CK1d on tau protein are those involved in its binding to tubulin,
indicating the important role of CK1 in tau aggregation.44 A recent study demonstrated that
CK1d and GSK-3 phosphorylate at least 15 sites that are also phosphorylated in the paired
helicoidal laments (PHF) of tau from an Alzheimer brain.45 A combination of CK1d and
GSK-3 activities could account for more than three-quarters of the Ser/Thr phosphorylation
sites identied in PHF tau, indicating that CK1d may play a role, together with GSK-3, in
the pathogenesis of AD.
AD is also associated with the accumulation of the neurotoxic peptide bA, which is
produced by sequential cleavage of the amyloid precursor protein (APP) by the aspartyl
protease b-secretase and the presenilin-dependent protease g-secretase. An in silico analysis
showed that APP, b- and g-secretase subunits contain, in their intracellular regions, multiple
CK1 consensus phosphorylation sites, many of which are conserved among human, rat, and
mouse species. Overexpression of constitutively active CK1e, one of the CK1 isoforms
expressed in the brain, leads to an increase in bA production.46 Conversely, three structurally
dissimilar CK1-specic inhibitors signicantly reduced endogenous (bA) peptide production,
which demonstrated that CK1 inhibitors act during g-secretase cleavage without affecting
Notch cleavage.
All these results indicate that CK1 is a clear therapeutic target for disease modication of
AD neurodegeneration. In particular, the CK1d and CK1e isoforms seem to be the most
interesting to modulate pharmacologically and target with their specic inhibitors as a
therapeutic strategy.47
Furthermore, new evidence of CK1 involvement in other neurodegenerative diseases has
increased its value as a potential drug target.
The TAR DNA-binding protein of 43 kDa (TDP-43) was identied as a major disease-
associated protein in fronto temporal lobar degeneration (FTLD) with ubiquitin-positive
inclusions and in ALS.48 Hyperphosphorylated cytoplasmic and intranuclear inclusions of
TDP-43 have been found in brains of patients with these neurodegenerative diseases. It was
reported that 29 different sites on the recombinant TDP-43 are phosphorylated by CK1.49
Therefore, CK1 plays not only an important role in tauopathies (e.g. AD or FTLD) but also
in motor neuron degenerative diseases, such as ALS.
PD is a common progressive bradykinetic disorder characterized by the presence of
severe pars compacta nigral cell loss, and by the accumulation of aggregated a-synuclein in
specic brain stem, spinal cord, and cortical regions. As in other neurodegenerative diseases,
age is the main risk factor. Susceptibility genes including a-synuclein, leucine rich repeat
kinase 2, and glucocerebrosidase have shown that genetic predisposition is another important
causal factor. Moreover, a history of severe head injury, encephalitis, toxin exposure, or
hypertension and cerebrovascular disease might be important as secondary causes. Although
PD is still an incurable progressive disease, some palliative treatments, such as dopamine
replacement therapy, substantially improve life quality and functional capacity.50
In addition, a-synuclein was found to be the major constituent of Lewy bodies, one of
the main disease hallmarks.51 It has been demonstrated that a-synuclein is constitutively
phosphorylated at Ser129 by CK1 within its C-terminus and/or CK2. Results showed that
the function of a-synuclein is regulated by phosphorylation/dephosphorylation, showing a
key role of CK1 and/or CK2 in PD.52 Furthermore, it is well known that mutations in the
parkin gene, which cause autosomal recessive juvenile-onset Parkinsonism and parkin dys-
function may have important implications in the pathogenesis of sporadic PD. Although its
precise function remains largely unknown, parkin seems to have a neuroprotective function.
Several studies suggest that changes in parkins solubility are the main mechanism of
parkin inactivation in PD. Different pathogenic parkin point mutations decrease parkin
solubility and promote its aggregation.53 It has been reported that an array of oxidative
stressors,54 as well as direct post-translational parkin modications (including dopamine
modication)55 or S-nitrosylation,56,57 contribute to changes in parkins solubility, causing
its dysfunction in PD.
Recent studies have shown that phosphorylation of parkin by both CK1 and cyclin-
dependent kinase 5 (CDK5) decreases parkin solubility, leading to its aggregation and
inactivation.58 In addition, enhanced parkin phosphorylation is detected in distinct brain
areas of individuals with sporadic PD. This correlates with an increase in the levels of p25,
which is a CDK5 activator. These ndings reveal CK1, CDK5, and their inhibitors to be
novel combinatorial therapeutic targets for treating PD.
C. Protein Kinase CK1 Inhibitors

As the involvement of CK1 in physiology and pathology has only recently been identied,
there has been little work regarding inhibitors of this protein kinase. However, different
chemical families of ATP-competitive inhibitors of both synthetic and natural origin
have been discovered and/or designed to target CK1 more or less specically (Table I).
Different methodologies, including structure-based virtual screening approaches59 and
crystallographic complex studies, have been applied to study CK1 inhibitors and their
specic interactions.60
1. Heterocyclic Compounds
The rst CK1 inhibitor, described in the micromolar range (IC50 5 9.5 mM), was the com-
pound (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide) known as CKI-7.61 It showed
inhibition without specicity toward the CK1d and CK1e isoforms, but did not inhibit
CK1g. Although CKI-7 inhibited CK1 competitively with ATP, some kinase specicity was
found and it did not inhibit CK2, PKC, PKA, or CaM kinase II. Its potential for treatment
of AD has recently been identied and a decrease in bA peptide formation due to the action
of CKI-7 in the g-secretase cleavage pathway has been described.46
The oxoindole heterocyclic compound IC261 showed inhibitory activity against three
CK1 isoforms (CK1a, IC50 5 16 mM; CK1d and CK1e, IC50 5 1.0 mM) without inhibiting
other protein kinases, such as PKA, p34cdc2, and p55fyn. Competition with ATP was also
shown in its binding mode. One advantage of IC261 compared to CKI-7 relates to its ability
to cross the cell membranes.60 This compound is able to inhibit CK1d/e-mediated phos-
Table I. Summary of Kinase Inhibition and Biological Prole for Described CK1 Inhibitors
Comp. Chemical structure CK1 inhibition Kinase selectivity Observations Ref.
CKI-7 SO2NH(CH2 )2 NH 2 CK1 No inhibition against CK2, Reduced b-amyloid 61

N (IC50 5 9.5 mM)a PKC, PKA and CamK II formation produced in 60
Tested in a panel of 70 N2A cells 46
kinasesb Poor ability to penetrate 74
Cl
through cell membrane due
to its charge at
physiological conditions
No toxicity observed in cells
after 24 h at 50 mM
concentration
IC261 OMe CK1d/e No inhibition against PKA, Reduced b-amyloid 60
(IC50 5 1 mM)a p34cdc2 and p55fyn formation produced in 46

Tested in a panel of 70 N2A cells 62
MeO OMe kinasesb Non-charged molecule, highly 74
active in cells
O
No toxicity observed after 3 h
N Inhibited CK1d in intact
H
murine cells SV3T3
PF-670462 N CK1d Tested in a panel of 45 kinases Slows down circadian clock 66
N N N
(IC50 5 14 nM) Inhibition of EGFR, HGK, in vitro 67
CK1e p38 and PKACa Induces phase delay in vivo in 68
2 HCl
N (IC50 5 7.7 nM)c a rodent model of
F circadian rhythm
Rapidly metabolized in rats
PF-4800567 O CK1d Tested against a panel of 50 No effect on circadian clock 68

(IC50 5 0.71 mM)a kinases in vitro
N N CK1e Inhibition of EGFR Showed a small phase delay
CK1 AND CK2 AS TARGETS FOR NEURODEGENERATION
N
N (IC50 5 32 nM)a in vivo
K
NH2 O
Cl
929
Table I. Continued
930
D4476 O CK1 Tested in a panel of 45 kinases In H4IIE hepatome cells 72

K
O (IC50 5 0.2 mM)d Inhibition of ALK5d and inhibited the 46

p38a MAP kinase phosphorylation of 74
N Tested in a panel of 70 FOXO1a
N kinasesb Reduced b-amyloid
N H formation produced in
CONH 2
N2A cells
Showed limited cellular
permeability
Slight toxicity observed in
cells after 24 h at 50 mM
PEREZ, GIL, AND MARTINEZ
concentration
Recommended for inhibiting
CK1 isoforms in cell-based

assays
Polyhalogen- Br H CK1 (IC50 5 2.2 mM)a CK2 (IC50 5 0.91 mM)a 76

ated benzi- Br N G-CK (IC50440 mM)a
midazole 9 S
Br N
H
Br
(R)-DRF053 CK1d/e CDK1 (IC50 5 0.22 mM)e Low antiproliferative effect in 24

(IC50 5 14 nM)e CDK5 (IC50 5 0.08 mM)e human neuroblastoma SH-
HN
N
GSK-3b (IC50 5 4.1 mM)e SY5Y and in HEK293
N N
Inhibitory effect on amyloid
HN N N b-40 in N2A-APP695 cells
OH
1,4-Diamino NH 2 O CK1d CK2 (IC50 5 18 mM)b Cytotoxic prole on human 59

anthra (IC50 5 0.33 mM)b HIPK2 (IC50 5 3.3 mM)b ovarian carcinoma cell line
quinone CK1g1 (IC50 5 34 mM)b DYRK1a (IC50 5 3.6 mM) (2008)
CK1a (IC50 5 4 mM)b PIM1 (IC50 5 24.7 mM)d
NH 2 O
Fgr (IC50 5 24 mM)
1-Hydroxy-4- OH O CK1d Cytotoxic prole on human 59
amino (IC50 5 0.66 mM)b ovarian carcinoma cell line
anthra CK1g1 (IC50 5 26 mM)b (2008)
quinone CK1a(IC50 5 4 mM)b
NH 2 O
la-4 Br CK1e (IC50 5 25 nM)a Cellular circadian assay in 86

Mper-1-luc Rat 1
broblasts (P2C4):
S ECDt11 h 5 0.12 mM
N NH2
N O
H
Id H 2N CK1e Cellular circadian assay in 87

(Ki 5 110 nM)a Mper-1-luc Rat 1
S

broblasts (P2C4):
S NH 2
ECDt11 h430 mM
N O
H
Im CK1e Cellular circadian assay in 88,89

S
(Ki 5 0.064 mM)a Mper-1-luc Rat 1
NH2 broblasts (P2C4):
H 3C(H 2C)4 N
H ECDt11 h 5 4.55 mM
N O
H
5 N CK1e Cellular circadian assay in 90

F
N (IC50 5 1633 nM)a Mper-1-luc Rat 1
N N
N
CK1d broblasts (P2C4):
Me N (IC50 5 4156 nM)a ECDt11 h 5 5 nM
N
Bis-indole CK1 CDK1 (IC50 5 1.5 mM)e Cytotoxic prole on 5 human 91

K
indirubine (IC50 5 0.65 mM)e CDK5 (IC50 5 0.59 mM)e tumour cell lines (Huh7,
Caco2, HCT116, PC3 and
931
Table I. Continued
932
H 2N GSK-3 (IC50 5 0.08 mM)e NCI)

K
DYRK1A (IC50 5 2.5 mM)e No toxicity observed in

O
normal skin broblasts
NH
after 48 h at 25 mM
N concentration
H O
Isoxazole-15 F CK1d p38a (IC50 5 2.52 mM)d Low cytotoxic effect (38% cell 92
(IC50 5 0.033 mM)d death) in human immortal
N trophoblast hybrid cell line
O (ACI-M88) at 3.32 mM
OMe
H
MeO N
PEREZ, GIL, AND MARTINEZ
Me
O N Me
Imidazole-17 O CK1d (IC50 5 4 nM)d Tested in a panel of 76 kinases Cytotoxic effect (87% cell 92

NH at 0.1 mM death) in human immortal
CK1e Inhibition of p38a trophoblast hybrid cell line
MeO OMe N
H (IC50 5 73 nM)d (IC50 5 0.019 mM) (ACI-M88) at 0.38 mM
N
SMe
N
Imidazole-18 O CK1d Tested in a panel of 76 kinases Cytotoxic effect (77% cell 92

NH (IC50 5 5 nM)d at 0.1 mM death) in human immortal
Inhibition of p38a trophoblast hybrid cell line
MeO OMe N
H CK1e (IC50 5 0.041 mM) (ACI-M88) at 0.26 mM
N O
S (IC50 5 447 nM)d
N Me
F
Meridianin E H 2N CK1 CK2 (IC504100 mM)e Cytotoxic effects in different 94
N
(IC50 5 0.40 mM)e CDK1 (IC50 5 0.18 mM)e cell lines (Hep2, U937,
N
OH CDK5 (IC50 5 0.15 mM)e LMM3, PTP)
PKA (IC50 5 0.09 mM)e
PKG (IC50 5 0.60 mM)e
N GSK-3b (IC50 5 2.5 mM)e
H
(-)-Mataireni H O CK1 PKCa (IC50 5 10 mM) Inhibits HIV replication in 95
MeO
sol (IC50 5 10 mM)a No inhibition of CK2, PKA H9 lymphocyte cells 96
O
HO
H
OMe
OH
Lamellarin 3 HO CK1d/e CDK1 (IC50 5 0.53 mM)e Inhibition of SH-SY5Y cells

O (IC50 5 0.41 mM)e CDK5 (IC50 5 0.60 mM)e IC50 5 0.05 mM
N
MeO PIM1 (IC50 5 0.15 mM)e
O
GSK-3b (IC50 5 0.58 mM)e
MeO
DYRK1A (IC50 5 0.06 mM)e
HO
MeO OH

Lamellarin 6 HO CK1d/e CDK1 (IC50 5 0.10 mM)e Inhibition of SH-SY5Y cells 97
O (IC50 5 0.8 mM)e CDK5 (IC50 5 0.03 mM)e IC50 5 0.11 mM
N
MeO PIM1 (IC50 5 0.33 mM)e
O
GSK-3b (IC50 5 0.13 mM)e
HO
DYRK1A (IC50 5 0.09 mM)e
MeO
MeO OH
Hymenialdi- H2 N CK1 CDK1 (IC50 5 22 nM)e In vivo inhibitory effects on 98

sine N (IC50 5 35 nM)e CDK2 (IC50 5 40 nM)e the phosphorylation of tau
HN
O CDK5 (IC50 5 28 nM)e protein
GSK-3b IC50 5 10 nM)e
Br
HN
NH
O
a
Final ATP concentration of10 mM. bFinal ATP concentration of 20 mM. cFinal ATP concentration of 5 mM. dFinal ATP concentration of100 mM. eFinal ATP concentration of15 mM.
933 K
phorylation of p53 in intact SV3T3 murine cells.62 Furthermore, it has been shown that this
compound inhibits induced tumor growth in a xenotransplantation model.63 Compound
IC261 is also able to reduce the formation of bA peptide acting during the g-secretase
cleavage46 and still reduces AKt phosphorylation,64 which is a very important pathway in
processes, such as cell metabolism, cell cycle control, and cancer.65
Two imidazole derivatives have also been reported to be potent CK1 inhibitors. The rst
is PF-670462, chemically named 4-[3-cyclohexyl-5-(4-uoro-phenyl)-3H-imidazol-4-yl]-
pyrimidin-2-ylamine. It is a potent CK1e inhibitor (IC50 5 7.772.2 nM), being 1.8-fold less
active than the CK1d isoform (IC50 5 14 nM). PF-670462 has a selective kinase prole,
presenting some inhibitory activity against EGFR, HGK, p38, and PKACa. Several studies
have shown its ability to induce phase delays in circadian rhythms in rats, in which it is
rapidly metabolized,66 and in monkeys.67
The circadian clock links our daily cycles of sleep and activity to the external environ-
ment. Deregulation of this clock is involved in different human disorders, such as depression,
metabolic and seasonal affective disorders.68 CK1d and CK1e are essential components of
the biological clock, phosphorylating PER proteins, and thus regulating their turnover and
nuclear entry in oscillator cells of the suprachiasmatic nucleus.69 A potential pharmacological
use of these specic compounds could be for therapy of cognitive decits in shift workers,
mood changes in bipolar disorders, and phase advances in the sleepwake cycle in elderly
people. PF-670462 was also used to attenuate the locomotor stimulant response to me-
thamphetamine, showing that CK1e is essential for the locomotor response to this drug and
blocking Darpp-32 phosphorylation, a key molecule in complex signaling pathways, such as
dopamine and glutamate signaling.70
A potent ATP-competitive CK1e inhibitor, developed by Pzer, was the compound
PF-4800567, with an IC50 5 32 nM and a 22-fold selectivity toward CK1d (IC50 5 711 nM).
This compound showed greater selectivity toward GSK-3b, PKA, PKC, and p38 kinases
than PF-670462, although it also inhibits the EGFR kinase.68 One of the main advantages of
this compound is its selectivity toward CK1e. Due to the high degree of sequence identity
between CK1d and CK1e, it was difcult to identify which kinase was responsible for the
biological effect detected when a dual inhibitor was employed. For example, a signicant
phase delay in animal models was observed in the circadian clock rhythm, when the CK1d-
and CK1e-specic inhibitor PF-670462 was used. The effects detected were negligible when
the selective CK1e inhibitor PF-4800567 was tested under the same experimental conditions
in animal models. With this selective CK1e inhibitor, it was demonstrated that the major
enzyme acting as a mediator of circadian timing was CK1d.
The second compound reported to bear an imidazolic scaffold is D4476, a CK1 inhibitor
with an IC50 5 0.2 mM. It was earlier identied as an inhibitor of activin receptor-like kinase 5
(ALK 5).71 This compound inhibited CK1 in an ATP-competitive way, while it did not show
any inhibitory activity against other protein kinases, such as GSK-3b, CK2, PKA, CDK2,
and ERK2.72,73 D4476 is able to inhibit p38 and reduce the formation of b-amyloid peptide
acting during the g-secretase cleavage, with slight toxicity observed in cells after 24 hr in-
cubation at 50 mM.46 Recently, the kinase prole study was extended to a larger panel of
more than 70 kinases, showing that D4476 inhibited CK1d 2030-fold more potently than
PKD1 or p38a, while no other protein kinase in the panel was inhibited to a signicant
extent.74 Based on this high specicity, the use of D4476 is recommended for inhibiting CK1
isoforms in cell-based assays.
Halogenated benzimidazoles were earlier reported to be ATP-competitive CK1 and CK2
inhibitors.75 The optimization step in the development of these compounds showed that
4,5,6,7-tetrabromobenzimidazole derivatives substituted at position 2, mainly Cl, Br, or
S, had an inhibitory activity (IC50 value) of between 0.490.93 mM against CK2 and
no specicity toward CK1 (IC50 between 18.22.2 mM). Polyhalogenated benzimidazole 9 was
the compound that showed the most potent activity toward CK1 with an IC50
of 2.2. mM.76
Following the [615] fused heterocycle series, analogues of roscovitine were synthesized.
Roscovitine77,78 (CYC202 developed by Cyclacel Pharmaceuticals) is currently being in-
vestigated in clinical trials for cancer,79 renal and kidney diseases,80 and it is also being used
in preclinical studies for AD, stroke, and inammation.81 Some 2,6,9-trisubstituted purines
were synthesized to inhibit protein kinases implicated in AD, specically GSK-3, CDK5,
and CK1. In this series of new derivatives, one of the most potent CK1 inhibitors known to
date is the compound (R)-DRF053.24 It inhibited CK1d/e at a concentration of 14 nM,
while its activity against CDK5, GSK-3, and CDK1 had an IC50 value of 80 nM, 4.1 mM,
and 0.22 mM, respectively. Docking studies using the CK1d isoform revealed two diffe-
rent binding modes. In both cases, the typical adenine ligandreceptor interaction based on
the hydrogen bond between purine Nitrogen 6 as a donor and Nitrogen 7 as an acceptor
in the binding cavity was found. Different orientations of the 3-pyridinylphenylamino
scaffold are possible. The rst is characterized by a mixed hydrogen bond stacking
interaction between pyridine and the side chain of Arg16 of the glycine loop. In this binding
mode, the pyridine nitrogen forms a hydrogen bond with the guanidinium moiety while
the phenyl ring is oriented parallel to Arg guanidinium, stabilized by a cation-p interaction.
In the second binding mode, the phenylamino bond is rotated 601 and orientates the
3-pyridinylphenylamino group toward the C-terminal lobe where different interactions
take place (hydrophobic Pro87 and stacking with Phe95). The improved afnity of 3- and
4-pyridinyl derivatives toward CK1 compared to roscovitine compounds could be
explained by the favorable interaction between the pyridine ring and Arg13. In a cell model,
an inhibitory effect on the production of b-amyloid was observed.24 The CDK/CK1 dual
inhibition present in these compounds could be used for cancer treatment, as CDKs are
involved in cell hyperproliferation and CK1 exhibits anti-apoptotic features and modulates
the activity and/or interaction of tumor suppressors and oncogenes,82,83 or for AD treatment,
where CK1 is implicated in different pathological pathways, such as b-amyloid and tau
protein abnormalities.
As mentioned at the beginning of this section, the use of a structure-based virtual
screening technique, using different docking protocols and browsing an in-house molecular
database, led to the discovery of the amino-anthraquinone scaffold as a CK1 inhibitor. As no
crystal structure of human CK1d is available, a homology modeling approach was also
carried out to obtain a 3D model of the catalytic subunit of CK1d. Among all the isoforms of
CK1, CK1d was selected due to its role in different neurodegenerative diseases. Two deri-
vatives of the anthraquinone family were discovered, surprisingly, given the low activity of
emodin84 and 1,4-diamino-5,8-dihydroxyanthraquinone (DAA)85 (anthraquinone analogues)
against CK1. One of these active compounds was the 1,4-diamino anthraquinone, which was
5-fold more potent (IC50 5 0.33 mM) compared to IC261, in an ATP-competitive manner.59
According to the homology model, the compound is stabilized by interactions between the
amino group (at position 1) with the backbone carbonyl of Glu83 in the hinge region,
interaction of one of the carbonyl groups with the backbone moiety of Leu85 between the
amino group in the 4-position, and the carboxylic group of Asp149, and also by hydrophobic
interactions (Ile15, Ile23, Ala36, Leu135, and Ile147). This compound also showed selectivity
against a panel of different protein kinases (PKA, CSK, Lyn, Syk, and GST-ALK). The
other CK1d inhibitor discovered by structure-based virtual screening was the 1-hydroxy-4-
amino anthraquinone (IC50 5 0.6 mM). In addition, the preliminary prole of 1,4-diamino
anthraquinone on the human ovarian carcinoma cell line (2008) and on its cisplatin-resistant
clone (C13) showed an IC50 of 14.4 mM and 87.9 mM, respectively, while 1-hydroxy-4-amino
anthraquinone was more potent against the cisplatin resistant cell line (IC50 5 8 mM).
This anthraquinone scaffold represents a versatile structure for the design of new and specic
inhibitors of protein kinases.
Within the patent literature, the heterocyclic series developed by Aventis (currently
Sano-Aventis) have been described as CK1e inhibitors for treating CNS diseases focused on
mood and sleep disorders. These heterocyclic compounds belong to a series of substituted
pyrrolopyridine Ia-4,86 thienopyrrole or pyrrolothiazole Id,87 indole Im,88,89 and related
analogues. More recently, in 2009, Sano-Aventis reported that imidazo[1,2-b]pyridazine 5
and its derivatives are CK1e and/or CK1d inhibitors with the potential to prevent tau
hyperphosphorylation.90
Very recently, various bis-indole indirubins have been reported as CK1 inhibitors with
IC50 values in the submicromolar range, similar to those for the inhibition of CDK1 and
GSK-3.91 The average bioactivity efciency found for these compounds in different tumoral
cell lines was similar to the standard reference roscovitine. Although this chemical scaffold
could represent a new lead for further optimization against CK1, more medicinal chemistry
development is needed to obtain candidates for neurodegenerative diseases targeting CK1.
Additional imidazole/isoxazole derivatives have been reported to specically inhibit CK1 in
an isoform-specic manner.92 Their mode of inhibition of CK1d and p38a kinase was
identied, revealing clues that could explain the selectivity of isoxazole-15 toward CK1d. In
principle, its acrylamide moiety could function as a Michael acceptor, addressing a chemi-
cally reactive amino acid residue (such as cysteine), thereby forming a covalent bond to the
protein. However, a cysteine residue capable of reacting through this mechanism has not
been found either in the Hydrophobic Region II (HRII) of p38a or in the HRII of CK1d.
Moreover, imidazoles 17 and 18 are highly potent dual-specic inhibitors of CK1d and p38a
with IC50 values in the single digit nanomolar range and strong isoform specicity toward
CK1d in an ATP-dependent way, providing further evidence against Michael reactivity.
These two recently described inhibitors could be used to discover new functional properties
of protein kinases.
2. Natural Marine Compounds

Marine organisms represent a very promising source of new bioactive molecules in the
pharmaceutical eld.93 Currently, different molecules obtained from marine organisms are
either being commercialized, are in clinical trials, or are being used as lead compounds for
new derivatives. Various marine compounds have been reported for the inhibition of CK1.
Meridianins were isolated from the marine ascidian Aplidium meridinum, acting as inhibitors
of different protein kinases (CDK1, CDK5, PKA, PKG, GSK-3) at a low micromolar range.
Their structure is composed of brominated 3-(2-aminopyrimidine)-indoles. These com-
pounds prevent cell proliferation and induce apoptosis. Meriadinin E was also tested in a
panel of 25 kinases, with inhibitory activity against most of them at a micromolar range,
except for Erk1, Erk2, MAPKK, and CK2. The IC50 value in terms of CK1 is 0.4 mM,94 thus
representing an interesting but very unspecic scaffold. In addition, it showed cytotoxic
effects in different cell lines (Hep2, U937, LMM3, and PTP).
()-Matairenisol was rst reported to inhibit HIV replication in H9 lymphocyte cells.95 It
was also tested in a small protein kinase panel comprising CK1, CK2, PKCa, and PKA,
among others, showing no inhibitory activity for the enzymes except for PKCa and CK1, for
which IC50 values of approximately 10 mM were found.96
Lamellarins are a family of pyrrole alkaloids that can be isolated from marine in-
vertebrates. These compounds showed antitumor activity and inhibited several protein
kinases relevant to cancer (CDKs, DYRK1A, CK1, GSK-3, and PIM-1).97 Lamellarins 3 and
6 also showed an IC50 value for CK1de of 0.41 and 0.8 mM, respectively. Different analogue
programs are currently underway in order to investigate the molecular mechanisms of action
of lamellarins in different protein kinases and to design specic inhibitors for CK1.
Hymenialdisine, isolated from a Caribbean sponge, was discovered by a random
screening while trying to discover new CDK inhibitors. Hymenialdisine is a potent inhibitor
of CK1 with an inhibitory activity of 35 nM in an ATP-competitive manner. It is very
unspecic and inhibits other protein kinases implicated in Alzheimers disease, such as
GSK-3b, CDKs, and presenilin-2.98
D. Protein Kinase CK2 and its Role in Neurodegenerative Diseases

Protein kinase CK2 is highly conserved and is found in all eukaryotic cells investigated to
date.99 The enzyme is important for cell proliferation and is biomedically relevant because
overexpression of its catalytic subunit can cause lymphoma.
The CK2 holoenzyme forms a heterotetrameric complex composed of two catalytic
(CK2a and CK2a0 ) and two regulatory (CK2b) subunits (i.e. a2b2 complex). The assembly of
CK2 complexes containing the catalytic subunits requires the earlier formation of dimers of
the regulatory CK2b subunit, which can take place in the absence of the catalytic subunits.100
The catalytic subunit (CK2a) with a molecular weight between 39 and 45 kDa is distantly
related to the CMGC subfamily of kinases, such as the CDK kinases, and alone is en-
zymatically active. The CK2b subunit (MW 5 26 kDa) is able to stimulate the activity of
CK2 in the tetramer.
There are some peculiarities associated with protein kinase CK2, which are lacking in
most other protein kinases:101 (i) the enzyme is constitutively active, (ii) it can use ATP and
GTP for phosphorylation, and (iii) it is elevated in most tumors investigated and in rapidly
proliferating tissues. With the elucidation of the structure of the catalytic subunit, it was
possible to explain why the enzyme is constitutively active and why it can bind GTP. More
than 300 substrates of CK2 have been reported to date.102 Most of them are proteins that
play a role in cell signaling.103 These substrates can be classied in different groups according
to their function. At least 87 proteins have been implicated in signaling functions, 60 in
transcriptional factors, 50 of them affecting the structure of DNA/RNA functions, 38 are
viral proteins, and 14 are related to the cytoskeleton.102
Until recently, CK2 was believed to be particularly required for cell cycle progression in
non-neural cells. In the light of recent ndings, four essential features suggest potentially
important roles for this enzyme in specic neural functions: (1) CK2 is much more abundant
in the brain than in any other tissue; (2) there seem to be a myriad of substrates for CK2 in
both synaptic and nuclear compartments that have clear implications in development,
neuritogenesis, synaptic transmission, synaptic plasticity, information storage, and survival;
(3) CK2 seems to be associated with mechanisms underlying long-term potentiation in the
hippocampus; and (4) neurotrophins stimulate the activity of CK2 in the hippocampus.104
Overexpression of CK2 has been linked to several pathological conditions, ranging from
cardiovascular pathologies105 and cancer progression106 to infectious diseases107,108 and
neurodegeneration.109 The role of CK2 in tumorigenesis has been extensively studied,110 and
the potential of its inhibitors in cancer therapy has been the subject of a number of pub-
lications and patents.110114
In fact, in 2009, CX-4945 from Cylene Pharmaceuticals entered phase I development in
patients with advanced solid tumors, Castlemans disease, or multiple myeloma.115 The data
obtained may help to further elucidate the role of CK2 in neurodegenerative diseases.
Because not all aspects of what has been published on CK2 can be covered in this review,
we will focus on the possibility of using CK2 inhibitors as potential neurodegenerative drugs,
as extensive description of the drug ability of CK2 has recently been reported.116
CK2 was one of the rst protein kinases identied in AD with abnormal protein
phosphorylation.117 The function and distribution of CK2 in AD is altered and precedes tau
accumulation in the formation of tangles.118 CK2 activity increases due to the presence of
b-amyloid, and thus may accelerate tau phosphorylation.41 Furthermore, although the neu-
robrillary tangles stain very strongly with an anti-CK2 antibody, CK2 was demonstrated to
be an externally deposited component of neurobrillary tangles.119 These data indicate that
CK2 may be involved in the pathology of AD in a fashion that is not yet well understood.120
Apolipoprotein-E (apoE) plays an important role in neuronal lipid transport and is
thought to stabilize microtubules by preventing tau hyperphosphorylation.121,122 It was
recently shown that CK2 readily phosphorylated puried human apoE as well as re-
combinant forms of human apoE3 and apoE4. Additionally, it was found that apoE potently
activated CK2. As a result, an increase in CK2b subunit autophosphorylation and phos-
phorylation of tau when the latter was added to the kinase reaction mixtures was observed.
Other proteins, such as apolipoprotein A-I and albumin, did not effectively activate CK2.
The phosphorylation of apoE by CK2 as well as the activation of CK2 by apoE may be
relevant in vivo where apoE, CK2, and tau are colocalized on neuronal microtubules.123
In AD, the pathological mechanism by which b-amyloid causes neuronal dysfunction
and death remains largely unknown. Recent ndings indicate that soluble intracellular oli-
gomeric b-amyloid (oAb) species may play a critical role in AD pathology due to its toxicity.
It is known that oAb interferes with fast axonal transport (FAT).124 The FAT system is
responsible for moving proteins and vesicles from the neurons cell body where they are
made, down the long axon, to the functional areas where they are needed and back again. It
depends on motor proteins that attach to the cargo (a vesicle or protein) and carry it along a
track made of microtubules. Real-time analysis showed bidirectional axonal transport in-
hibition as a consequence of endogenous CK2 activation, probably due to oAb.124 This CK2
overactivation causes the motor protein to drop its cargo. Conversely, inhibition of FAT by
oAb was prevented by two specic pharmacological inhibitors of CK2 as well as by com-
petition with a CK2 substrate peptide. In an earlier study, it was shown that tau tangles halt
transport to the neuron periphery through other regulatory enzymes by causing the motor
protein to release the microtubule track. It was found that the CK2 activated by amyloid also
works as a primer for one of the enzymes activated by tau tangles, GSK-3.125 Furthermore,
experimental results indicate that acute exposure to oAb42 peptides disrupts synaptic
transmission by altering the activity of CK2, which causes a reduction in synaptic vesicle
pools during synaptic stimulation. This effect occurs only when the oAb42 peptides are
present in the presynaptic compartment, but oAb40 did not alter synaptic transmission. Of
particular note, the inhibitory effect of oAb42 peptide on synaptic transmission was pre-
vented by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, a CK2 inhibitor.126 These
data prove that pharmacological modulation of CK2 activity may represent a promising
target for therapeutic intervention in AD.
Several pieces of evidence have identied CK2 as one of the players in the pathogenesis
of PD. Studies carried out on brains of PD patients have recently shown that CK2 subunits
are present in Lewy bodies, one of the main hallmarks of this neurodegenerative disease.
Immunohistochemical studies on these brains harboring Lewy bodies revealed positive
staining for CK2b but not for CK2a.127 In addition, CK2b subunits were colocalized with
a-synuclein in most Lewy bodies. These ndings suggest that CK2b subunits may play a role
in the formation of intracytoplasmic inclusions in human a-synucleinopathies. This could
take place either through phosphorylation events or by a separate mechanism linked to the
b subunit itself. a-Synuclein is the major component of pathological inclusions that char-
acterize neurodegenerative disorders, such as PD, dementia with Lewy body disease, and
multiple system atrophy. Various studies have been performed using novel phospho-specic
antibodies to assess the presence and regulation of phosphorylated Ser87 and Ser129 in
a-synuclein in human brain samples and in a transgenic mouse model of a-synucleinopathies.
It was reported that cellular toxicity, including proteasomal dysfunction, increased CK2
activity, which boosted Ser129 a-synuclein phosphorylation and provided an explanation for
the presence of this protein in pathological inclusions.128 Moreover, CK2 is also responsible
for the phosphorylation of synphilin-1, a protein that is present in Lewy bodies and binds to
a-synuclein. The interaction between synphilin-1 and a-synuclein is markedly dependent on
phosphorylation and is stopped by CK2 inhibition.129 Thus, inhibition of CK2 activity by
5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) blocks the binding between these two
proteins and signicantly reduces the percentage of cells that contain cytoplasmic inclusions.
This fact demonstrates that CK2-mediated phosphorylation of synphilin-1 rather than that
of a-synuclein is critical in modulating their tendency to aggregate into inclusions, and that
pharmacological modulation can prevent and/or alleviate the toxic effect.
Inhibitors of CK2 have recently shown promising applications related to neuronal cells.
As an example, heme oxygenase (HO) enzymes catalyze the rate-limiting step of heme
breakdown, and may accelerate oxidative injury to neurons exposed to heme or hemoglobin.
One hypothesis postulated that CK2, PKC, and PI3K inhibitors could reduce both HO
activity and neuronal vulnerability to haemoglobin in murine cortical cultures.130 HO
activity and neuronal lactate deshydrogenase activities were measured. Both endpoints were
signicantly reduced by the two CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and
2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), showing the protective
neuronal effect of CK2 inhibitors.
However, much work is needed to determine whether CK2 activity should be decreased
or enhanced in relation to its therapeutic potential for neurological disorders. Moreover, the
mouse brain proteins associated with CK2a have recently been identied. Several of the
identied proteins are relevant to neurological disorders, such as schizophrenia, neuro-
blastoma, AD and PD, Huntington disease, and Zellweger syndrome.131 Alongside an earlier
mentioned data is the association of CK2 with the development of neuritogenesis and
synaptic plasticity. Activation of CK2 occurred in a dosedependent manner in response to
brain-derived factor release,132 some of its properties being abolished when specic CK2
inhibitors were used.133 In addition, it has been suggested that CK2 plays an important
role in the induction of long-term potentiation (LTP) through selective regulation of
synaptic NMDA receptors. Its activity was reduced by the use of specic inhibitors, such as
DRB or TBB.134
E. Protein Kinase CK2 Inhibitors

A number of different classes of chemical compounds have been reported to be CK2 in-
hibitors. Most of them target the highly conserved ATP-binding pocket, although there is
increased interest in identifying new inhibitors with different mechanisms of action.135,136
Resolution of the crystal structure of several inhibitors in complex with the CK2 catalytic
subunit has provided valuable information for the rational design of new derivatives with
improved inhibitory properties.137
Different classes of chemical compounds described as ATP-competitive inhibitors of
CK2, such as condensed polyphenolic compounds, tetrabromobenzimidazole/triazole deri-
vatives, and indoloquinazolines, have been reported (Fig. 1). The crystal structures of some
of them in complex with the catalytic a subunit of CK2 have been solved, providing new
clues for the rational design of more potent and specic CK2 inhibitors. This selectivity is
dictated by the shape and size of the hydrophobic cavity adjacent to the ATP binding site.
The CK2 hydrophobic cavity is smaller than in the majority of other protein kinases due to a
Comp. X Y CK2 inhibition
X
TBBz Br CH Ki=50 Mb
X N
Y TBBt Br N IC50=0.90 Mc
X N
H
X DMAT Br C-CH(CH3)2 Ki=40 nMb
TIBI I CH Ki=23nMb
Comp. X Y Z R CK2 inhibition
X Z MNA H CO NO2 OH Ki=0.78 Mb

Y
MNX H O NO2 OH Ki=0.80 Mb
R O OH DAA NH2 CO OH NH2 Ki=0.42 Mb
O O OH OH O
Me OH OH
HO HO O
OH O OH OH O OH
OH
Emodin Quinalizarin Quercetin OH
IC50=5.9 Mc IC50=0.11 Mb IC50=0.55 Ma
OMe
OH O MeO OMe
O
NH O OH O
HO O
N
OH O
N W16
Apigenin H IC50=30 M
IC50=0.80 Ma O
O N O
IQA Bn
IC50=0.39 Md O
a
: Final ATP concentration of 10 M
b
c
d
Figure 1. Casein kinase 2 inhibitors.
number of unique bulky apolar residues.138 Active CK2 mutants that are not sensitive to
specic inhibitors represent a valuable tool for validating the involvement of CK2 in cellular
functions. These functions are altered under treatment by an inhibitor.1,139
Apart from rational design based on the structural knowledge of the target enzyme,137
various strategies have been used to identify new CK2 inhibitors, such as the high-
throughput screening of chemically diverse libraries of inhibitors.136
F. ATP-Competitive CK2 Inhibitors

1. Halogenated Benzimidazole (Triazole) Derivatives
The nucleoside DRB, described in 1986,140 was the rst member of this class of compounds
able to inhibit CK2. Although additional halogenated benzimidazole nucleoside analogues
were also synthesized,75,141 only the halogenated parent bases, TBBt142 (4,5,6,7-tetra-
bromobenzotriazole) and TBBz143 (4,5,6,7-tetrabromobenzimidazole), showed higher
potency and selectivity.144 The IC50 of TBBt was around 1 mM, and after being tested in a
panel of 33 protein kinases, only CK2 was drastically inhibited.145 These compounds could
serve as precursors for the development of more effective inhibitors.
The crystal structure of the Zea mays protein kinase CK2a catalytic subunit in complex
with the specic inhibitor TBBt revealed the basis for the selectivity of TBBt for CK2. It
seems to be mainly dictated by the reduced size of a hydrophobic pocket, adjacent to and
partially overlapping the ATP binding site. In the majority of protein kinases, this ATP site is
too large to produce stable interactions with this inhibitor.146 Based on these results, the
inhibitory potency of TBBt could be improved by generating adducts in which N2 is replaced
by a carbon atom. This atom could be linked to a variety of polar functions, being able to
interact with polar side-chains of the kinase. The most efcient of these derivatives was the
2-dimethylamino derivative, DMAT, with a Ki value of 40 nM.147,148 However, testing in a
wide kinase panel (more than 70 kinases) showed that DMAT and TBB are less selective CK2
inhibitors than earlier thought. Further efforts have been made regarding the design of
specic CK2 inhibitors that are able to discriminate between CK2 and structurally related
kinases.149
A new series of iodinated benzimidazoles has recently been reported,150 showing that
4,5,6,7-tetraiodobenzimidazoles (TIBI) are more potent inhibitors of CK2 than their tetra-
brominated analogue.
2. Emodin and Related Compounds

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a natural product that can be isolated
from the rhizomes of Rheum palmatum. It was reported to inhibit a number of protein
kinases including CK2.151 The crystal structure of the catalytic subunit of Zea mays
CK2a with the nucleotide binding site-directed inhibitor emodin showed that it penetrated
deeper than ATP into the active site of CK2a, partially overlapping the position occupied
by ATP. Quite remarkable is the structural alteration undergone by the N-terminal lobe
of the kinase upon replacement of ATP with emodin. Hydrophobic interactions seemed
to be the driving force for the formation of the complex, although some polar contacts
that involved the hydroxyl 3 group of emodin were also possible.152 Based on the structural
data, the potency and selectivity of the emodin scaffold was improved by proper
derivatization.85 The cocrystallization protocol for inhibitors MNA (1,8-dihydroxy-4-ni-
troanthraquinone), MNX (1,8-dihydroxy-4-nitro-xanthen-9-one), and DAA (1,4-diamino-
5,8-dihydroxy-anthraquinone) showed three different binding modes to the active site of
CK2: (1) anchoring to the hinge region (DAA); (2) anchoring to Lys68 and Asp175, albeit
with different orientations (MNA and MNX); and (3) sitting in the middle of the cavity with
no strong polar interactions (emodin). These three binding modalities were correlated with a
gradual increase in the Ki value from 0.35 to 1.85 mM. Based on this information, a strategy
to improve the afnity to CK2 could involve the design of new molecules able to ll the entire
cavity and, therefore, to interact on both sides: the hinge region and the Lys68/Asp175
region.153
Quinalizarin (1,2,5,8- tetrahydroxy-anthraquinone) has been identied as a new CK2
inhibitor that is more potent and selective than emodin.154 Quinalizarin was discovered after
virtual screening of a database based on the crystal structure of the human CK2a subunit.
CK2 inhibition is competitive with respect to ATP, with a Ki value of 50 nM, and its
selectivity toward CK2 was tested on a panel of 75 protein kinases. The determination of the
crystal structure of a complex of quinalizarin and the CK2a subunit highlighted the relevance
of polar interactions in stabilizing binding.
3. Flavonoids
The Citrus avonoids, such as quercetin and apigenin, are in general broad specic inhibitors
of many classes of protein kinases. Even though they may be fairly potent, they are, there-
fore, likely to be poorly specic. In the case of CK2, they differ from anthraquinone and
uorenone derivatives in their relative promiscuity, and the irregular number and position of
their hydroxyl groups. Among the compounds tested, the most effective inhibitors of CK2
are setin (IC50 5 0.35 mM) and quercetin (IC50 5 0.55 mM), whose only difference lies in the
hydroxyl group at position 5 of quercetin. The modication of quercetin by the addition of a
6th hydroxyl group at position 50 to give myricetin (IC50 5 0.92 mM), or by elimination of two
hydroxyl groups at positions 3 and 30 to give apigenin (IC50 5 0.80 mM), is also relatively
ineffective. Apigenin, however, undergoes a substantial drop in inhibitory efciency if its
40 hydroxyl group is either eliminated to give the di-hydroxyavone chrysin (IC50 5 9.0 mM)
(implying a minimum number of three hydroxyl groups for efcient inhibition), or if two
hydroxyl groups are added at positions 20 and 3 to give the pentahydroxyl avone morin
(IC50 5 10.0 mM). While it is possible that a hydroxyl group at the 20 position is detrimental
per se, a hydroxyl group at position 3 is also found in setin and quercetin, which are the most
efcient avonoid inhibitors of CK2. The relative promiscuity of avonoid inhibitors sug-
gests that these compounds may adopt different orientations in the active site of CK2 and
that these are determined by the number and position of their hydroxyl groups. In contrast,
hydrophobic interactions, which are important for the binding of anthraquinone derivatives
and TBB, may play a marginal role with a number of avonoids, whose inhibitory potency,
in fact, is unaffected by the Val66Ala mutation.155
4. Derivatives of Indoloquinazolines
Virtual screening by high-throughput docking of a 3D database with nearly 400,000 com-
pounds in the Novartis collection, led to the identication of compound CGP029482, (5-oxo-
5,6-dihydroindolo-[1,2-a]quinazolin-7-yl)acetic acid, as a powerful CK2 inhibitor (IC50 5
0.08 mM).156 This indoloquinazoline derivative, later renamed IQA, almost completely sup-
presses CK2 activity in vitro, whereas it is ineffective or weakly effective on a panel of
44 protein kinases.84 Inhibition of CK2 by IQA is competitive with respect to ATP, and the
structure of the crystallographic complex has been determined.84
As earlier mentioned, the ATP binding site in CK2 is smaller than in the majority of
kinases due to a number of unique bulky apolar residues, in particular Val66 and Ile174,
whose bulky side chains contribute to the relative smallness of the hydrophobic pocket in
which TBB and emodin are situated.155 Consequently, the mutation of either Val66 or Ile174
to alanine signicantly decreases the inhibitory efciency of emodin and TBB. Both mutations
are even more detrimental to the inhibitory efciency of IQA, probably because the binding of
IQA is due to non-polar interaction with both Val66 and Ile174. In contrast, the Val66Ala
mutation has almost no effect on the inhibitory efciency of the avonoid quercetin.84
G. Non-ATP-Competitive CK2 Inhibitors

Although most described CK2 inhibitors target the highly conserved ATP-binding pocket,
new classes of molecules that interact in a different way with the surface areas of CK2a
outside its catalytic gorge region have been reported. By exploiting new mechanisms
of action, these compounds offer exciting opportunities for the design of more specic
inhibitors.135
1. Substrate Target Inhibitor
The cyclic peptide CIGB-300 (formerly P15-Tat) was identied by screening a random cyclic
peptide phage display library for pro-apoptotic compounds. It was able to interfere with CK2
phosphorylation by blocking substrate interaction.157 The biological effect of this peptide

binder has been tested in various tumor cell lines and animal models, as well as in cancer
patients. In fact, apart from inducing apoptosis in tumor cells and tumor regression, when it
was injected directly into solid tumors in mice, it was also able to reduce solid tumor growth
after systemic administration.158 Moreover, the synthetic CIGB-300 showed efcacy when it
was assayed in patients with cervical malignancies. These results provide an early proof-of-
principle of the clinical benet of using a CK2 inhibitor. In addition, this is the rst clinical
trial in which a drug has been used to target the acidic phosphorylation domain of the CK2
substrates.159
2. Allosteric Inhibitor
Using high-throughput screening of highly diverse chemical libraries, polyoxometalates
(POMs) were identied as nanomolar ATP non-competitive inhibitors of CK2.160 These were
discovered by an initial screening of the National Cancer Institute Diversity Set (1985
compounds) and the Mechanistic Diversity Set (879 compounds) using an in vitro kinase
assay (15 mM concentration) and by a secondary screening (1.5 mM concentration), using a
standard radioactive kinase assay with high ATP concentrations. Following this procedure,
POMs were identied as active hits. These inorganic CK2 inhibitors [P2Mo18O62]6 were
also quite selective for CK2 when they were tested on a panel of 29 kinases. Mechanistic
studies have shown that POMs bind outside the CK2a/CK2b interface and the ATP/peptide-
binding pocket. The effectiveness of POMs in terms of potency and selectivity, and their
original mode of interaction with CK2, led to promising chemotherapeutic agents whose
pharmacokinetic properties are now being improved.
3. CK2b-Targeted Inhibitor
Of the possible new targets for CK2 inhibitors, the CK2b subunit has emerged as a very
promising one. The regulatory (non-catalytic) CK2b subunit plays an important role in the
assembly of tetrameric CK2 complexes, in enhancing the catalytic activity and stability of
CK2 and in the modulation of the substrate selectivity of CK2.99
With the aim of discovering inhibitors targeting this subunit, a two-hybrid screening of a
combinatorial library of peptide aptamers was carried out. This led to the isolation of a
random-sequence aptamer (P1 peptide) that is a potent CK2b-interacting peptide in vitro and
in vivo (KD 5 0.4 mM). This aptamer also interacts with CK2b present in cell extracts from
different cell lines and is able to induce apoptosis in cells expressing wild-type p53.161
4. CK2a/CK2b Subunit Interaction Inhibitors

The structural properties of the holoenzyme showed that the CK2a/CK2b interface is rela-
tively small,162 thus offering another attractive opportunity for the identication of small
molecules that modulate this interaction.
A conformationally constrained 11-mer peptide (Pc) was the rst small antagonist found
to bind to the CK2 interface by inhibition of the high-afnity subunit interaction. Moreover,
functional analysis showed that Pc could act as an antagonist of CK2b-dependent phos-
phorylation through the inhibition/disruption of the CK2 holoenzyme complex.163 Following
this discovery, other attempts to discover low molecular weight chemical inhibitors of the
CK2 subunit interaction led to the identication of W16 during screening of a podo-
phyllotoxine indolo-analogue library.164 W16 is the most potent analogue able to inhibit the
proteinprotein interaction between the two subunits in vitro, inducing selective disruption of
the CK2a/CK2b assembly and concomitant inhibition of CK2a activity.
H. Perspectives and Conclusions
Protein kinases have recently emerged as promising therapeutic targets, whilst enthusiasm
and optimism in the eld of kinase inhibitor development has increased, especially for a
number of peripheral disorders. However, kinase-targeted therapies for CNS diseases are not
as well developed, largely due to the specic challenges associated with CNS drug discovery
and the lack of opportunity for in vivo validation of these new targets. It is known that
several protein kinases are deregulated and/or implicated in the pathogenesis of CNS dis-
orders, although they have not been validated as therapeutic targets. This validation is
hampered by the absence of suitable small inhibitors that could be used to explore the in vivo
consequences of kinase modulation in the CNS. Nevertheless, these protein kinases represent
potentially attractive drug discovery targets and, at present, several therapeutic candidates
(targeting CNS protein kinases) are being tested in different preclinical and clinical stages.165
The most advanced is the GSK-3 inhibitor, NP-12 (phase II), which is being tested as a
disease-modifying treatment for AD.
Similar to the case of GSK-3, a well-known enzyme in the glycogen synthesis signaling
pathway that is now known to play a key role in AD pathogenesis (linking the amyloid and
tau pathology), new functions related to CNS pathologies are continuously being ascribed to
well-known kinases. In this way, new roles for the old casein kinases 1 and 2, currently
known as protein kinases CK1 and CK2, have recently been discovered to link the molecular
pathology of different neurodegenerative disorders, such as AD, PD, and ALS.
The CK1 family of serine/threonine protein kinases has the potential to be targeted for a
variety of CNS disorders, with the most available data related to neurodegenerative diseases,
such as AD. CK1d is increased in AD, being one of the main kinases involved in tau
phosphorylation and disrupting its binding to microtubules. The tau-based drug discovery
programs in AD therapy research have recently gained importance, with CK1 being an
important drug target. Moreover, recent discoveries regarding CK1 in PD pathology and in
TDP-43 phosphorylation are of utmost importance for further developments of future
therapies for PD and ALS, respectively. As a consequence, specic inhibitors of this enzyme
have increased its value as new therapeutic approaches for effective treatment of these
devastating diseases. Thus, CK1 inhibitors, especially the d and e isoforms, will be the focus
of attention in the coming years.
Intensive work has also been carried out in the search for specic CK2 inhibitors and
different approaches have been developed. However, more work is required to make a major
breakthrough in understanding the cellular functions of CK2, which will provide a molecular
interpretation for its involvement in neurodegenerative diseases. Moreover, the inherent
assumption of structural studiesthat Zea mays and human CK2a are almost identical at
the ATP sitewas challenged recently by the crystal structure of the catalytic subunit of CK2
from Homo sapiens (CK2a), in which a part of the ATP binding site deviated distinctly from
all known CK2a structures.166,167 This observation suggested that the co-crystal structures of
inhibitors with genuine human CK2a are worth solving in order to discover effective drugs
for human treatments.
One of the greatest challenges in any CNS-targeted drug discovery campaign is the
effective penetration of the bloodbrain barrier. The physicochemical properties of a drug,
such as its molecular weight, polar surface area, and lipophilicity, signicantly contribute
to this critical pharmacokinetic characteristic.168 These specic properties could form the
molecular basis of CNS failures for small molecule drugs approved and marketed for dif-
ferent disease indications involving peripheral tissues. Accordingly, future protein kinase
inhibitor design should consider CNS-relevant physicochemical properties, especially lower
MW and PSA, at the early ligand design stage. Moreover, an adequate balance between
hydrophilicity and lipophilicity should be pursued if the desired future drug therapy targets
the CNS and oral administration is required.
Protein kinase selectivity is also a common challenge in relation to inhibitors in the rst
steps of the drug discovery process and has traditionally been regarded as one of the lim-
itations in the kinase inhibitor research eld. It is currently being debated as to whether a
small molecule should be a neat and highly specic single protein kinase inhibitor or if the
new drug candidate should target relatively small related pathways in different protein
kinases.74 This point should also be reevaluated for CK1 and CK2 inhibitors due to the
increase in the number of kinases available for testing in recent years.
All these results open new avenues for further drug research and mean that two old
enzymes, CK1 and CK2, are interesting targets for developing effective treatments for
neurodegenerative diseases.
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Daniel I. Perez joined the Medicinal Chemistry Institute of the Spanish National Council for
Research (CSIC) in February 2009 as a postdoctoral researcher. He received his Ph.D. degree
from the Autonoma University of Madrid in November 2005. In mid 2003, he started working as
a researcher at the biopharmaceutical company NOSCIRA. During more than 3 years working
in the industry, he was mainly involved in different projects in the eld of medicinal chemistry. In
2007, he took a postdoctoral position at Delft University of Technology (The Netherlands)
where he worked in the biocatalysis eld. Currently, his research interests include the synthesis
of different protein kinase inhibitors (e.g. GSK-3, CK1) and the use of different computational
methods in order to nd new lead compounds for the treatment of neurodegenerative diseases.
Carmen Gil received her Ph.D. from Complutense University of Madrid in 2001, under the
supervision of Dr. A. Martinez. After a postdoctoral appointment at Bonn University (Dr.
Stefan Brase), she joined the Medicinal Chemistry Institute in 2004 (rst as an Associate
Researcher and since 2007 as a Staff Scientist), with responsibility for the design and synthesis
of biologically active organic molecules in the neurodegenerative eld.
Ana Martinez is a Research Professor in the Medicinal Chemistry Institute of the Spanish
National Council for Research (CSIC), having a background in Organic Chemistry. Her
interest is focused on neurodegenerative disorders, leading numerous research projects on the
medicinal chemistry and rational design of new drugs for Alzheimers and Parkinsons disease,
amyotrophic lateral sclerosis, spinal cord trauma, and multiple sclerosis. Active areas of
research involve GSK-3 inhibitors, BACE inhibitors, PDE 7 inhibitors, neurogenic drugs, and
CK 1 inhibitors among others. From February 2002 to January 2008, she worked at
NeuroPharma, currently named NOSCIRA, as R&D Director, where she provided strategic
leadership management and guidance in R&D activities, with two of her research compounds,
NP-12 and NP-61, being in clinical trials as disease modifying agents for Alzheimers disease.
She is the author of more than 150 scientic publications, more than 20 active patents in the
eld, and the editor of several books.

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Protein Kinases CK1 and CK2 as New

Targets for Neurodegenerative Diseases

Daniel I. Perez, Carmen Gil, and Ana Martinez

Published online 23 June 2010 in Wiley Online Library (wileyonlinelibrary.com).

Medicinal Research Reviews, 31, No. 6, 924--954, 2011

A. From Casein Kinases to Protein Kinases CK1 and CK2

B. Protein Kinase CK1 and its Role in Neurodegenerative Disorders

C. Protein Kinase CK1 Inhibitors

CKI-7 SO2NH(CH2 )2 NH 2 CK1 No inhibition against CK2, Reduced b-amyloid 61

Medicinal Research Reviews DOI 10.1002/med

PF-4800567 O CK1d Tested against a panel of 50 No effect on circadian clock 68

D4476 O CK1 Tested in a panel of 45 kinases In H4IIE hepatome cells 72

O (IC50 5 0.2 mM)d Inhibition of ALK5d and inhibited the 46

Medicinal Research Reviews DOI 10.1002/med

Polyhalogen- Br H CK1 (IC50 5 2.2 mM)a CK2 (IC50 5 0.91 mM)a 76

(R)-DRF053 CK1d/e CDK1 (IC50 5 0.22 mM)e Low antiproliferative effect in 24

1,4-Diamino NH 2 O CK1d CK2 (IC50 5 18 mM)b Cytotoxic prole on human 59

la-4 Br CK1e (IC50 5 25 nM)a Cellular circadian assay in 86

Id H 2N CK1e Cellular circadian assay in 87

Medicinal Research Reviews DOI 10.1002/med

Im CK1e Cellular circadian assay in 88,89

5 N CK1e Cellular circadian assay in 90

Bis-indole CK1 CDK1 (IC50 5 1.5 mM)e Cytotoxic prole on 5 human 91

H 2N GSK-3 (IC50 5 0.08 mM)e NCI)

DYRK1A (IC50 5 2.5 mM)e No toxicity observed in

Medicinal Research Reviews DOI 10.1002/med

Imidazole-18 O CK1d Tested in a panel of 76 kinases Cytotoxic effect (77% cell 92

Lamellarin 3 HO CK1d/e CDK1 (IC50 5 0.53 mM)e Inhibition of SH-SY5Y cells

Medicinal Research Reviews DOI 10.1002/med

Hymenialdi- H2 N CK1 CDK1 (IC50 5 22 nM)e In vivo inhibitory effects on 98

2. Natural Marine Compounds

D. Protein Kinase CK2 and its Role in Neurodegenerative Diseases

E. Protein Kinase CK2 Inhibitors

Comp. X Y CK2 inhibition

Comp. X Y Z R CK2 inhibition

X Z MNA H CO NO2 OH Ki=0.78 Mb

R O OH DAA NH2 CO OH NH2 Ki=0.42 Mb

Figure 1. Casein kinase 2 inhibitors.

F. ATP-Competitive CK2 Inhibitors

2. Emodin and Related Compounds

G. Non-ATP-Competitive CK2 Inhibitors

phosphorylation by blocking substrate interaction.157 The biological effect of this peptide

4. CK2a/CK2b Subunit Interaction Inhibitors

H. Perspectives and Conclusions

Medicinal Research Reviews DOI 10.1002/med

18. Nakajo S, Shinkawa K, Shimizu T, Nakaya K, Nakamura Y. Purication and characterization of

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

92. Peifer C, Abadleh M, Bischof J, Hauser D, Schattel V, Hirner H, Knippschild U, Laufer S.

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

Medicinal Research Reviews DOI 10.1002/med

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