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Neuropharmacology 63 (2012) 1227e1237
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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
Review
Selective kinase inhibitors as tools for neuroscience research
Kirsty J. Martin a, J. Simon C. Arthur b, *
a
b
Life-Physical Sciences Interface Laboratory, School of Engineering and Physical Sciences, Heriot Watt University, Edinburgh EH14 4AS, UK
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2012
Received in revised form
6 July 2012
Accepted 11 July 2012
Signal transduction cascades, including the MAPK, PI3 kinase, Ca2þ and PKC pathways, play important
roles in neurons downstream of multiple signals including neurotrophins and neurotransmitters. Small
molecule kinase inhibitors that block these pathways provide a powerful way of studying the in vivo or
cellular roles of these signaling systems. Over the last 15 years there has been a major effort by the
pharmaceutical industry to develop kinase inhibitors as potential drugs for a variety of diseases including
cancer and auto-immunity. As a result of this there are now many compounds available that can be used
as research tools. One major drawback is however that many of these compounds are not truly selective
for a single kinase, and therefore the possibility that their cellular effects may be due to an off target
activity must be considered. This problem has been brought into sharp relief by modern in vitro screening
methods that allow an inhibitor to be screened against a significant proportion of the kinome. In this
review we discuss the advantages and problems with the use of kinase inhibitors as research tools and
describe some of the available compounds that target pathways important to neurons.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Kinase inhibitor
Small molecule
Signaling
1. Introduction
Signal transduction is the process by which a cell translates an
external signal into a cellular response. Typically this process starts
with the binding of an external ligand, such as a growth factor or
neurotransmitter, to its appropriate receptor. This in turn activates
a complex set of signaling cascades that initiate the required
response in the cell. Understanding the regulation of these cascades
and identifying the key enzymes downstream of specific receptors
has been a major challenge in understanding the behavior of cells.
One important component of intracellular signaling pathways
are protein kinases, of which approximately 500 exist in mammalian genomes (Manning et al., 2002a, 2002b). Unraveling the
functions of individual kinases in signaling pathways, however, has
proved to be a complex issue. While many techniques have been
used to study kinase function, most suffer from potential drawbacks that make it difficult to rely solely on one method in these
investigations. Over-expression of protein kinases, or constitutively
active mutants, is a method that has been used extensively in the
past. It is now clear, however, that the over-expression of kinases
can result in the loss of specificity in signaling pathways: when
present at artificially high levels in the cell some kinases will start
* Corresponding author. Tel.: þ44 (0)1382 384003.
E-mail address: [email protected] (J.S.C. Arthur).
0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuropharm.2012.07.024
to phosphorylate proteins that are not their normal physiological
substrates. Over-expression of dominant negative mutations of
a kinase has provided a way of inhibiting the endogenous enzyme,
however this may also interfere with the functions of upstream
enzymes and thereby block the activation of parallel pathways. The
over-expression of RSK2, for instance, can inhibit the upstream
kinases ERK1 and 2 (Arthur et al., 2004). Even without such unintended effects, over-expression relies on transfection and expression of the product over a period of 12 h or more and so may change
the normal growth and development processes of the cell. The
unnaturally high concentration of protein resulting from overexpression could also disrupt the native subcellular localization of
the kinase, allowing access to potential substrates that it would not
normally encounter. An additional issue is that both these
approaches require the use of a cell culture system that is easy to
transfect. More recently, siRNA and gene targeting methods (such
as knockout or knockin mice or cell lines) have provided powerful
ways of studying protein function, although these approaches also
have drawbacks. It is sometimes difficult to obtain sufficient siRNA
knockdown to fully block the action of a kinase. This is a particular
issue with signaling networks as they allow for some potential
amplification of signal. Thus 90% inhibition of an upstream
component may not be sufficient to have a significant effect on
downstream events. In the PI3 kinase signaling pathway, for
example, reduction of PDK1 levels by 90% is not sufficient to inhibit
the phosphorylation of its downstream substrates (Lawlor et al.,
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K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237
2002). In addition, both mouse knockouts and siRNA can suffer
from compensation by closely related enzymes and may require
multiple kinases to be targeted in one experiment. Mouse knockouts may also result in developmental phenotypes that can
complicate the study of the kinase in adult animals. Finally while
many advances have been made in the production of gene-targeted
mice, it remains time consuming and expensive.
Selective, small molecule protein kinase inhibitors provide
another powerful way of studying kinase function. They have two
major advantages. First they will act on the endogenous kinase and
do not require transfection of over-expression. Secondly they can
be used over short time scales, which circumvents the issue of
potential developmental or secondary effects that can arise when
a kinase is blocked for a long period of time. However, as discussed
below, there are issues associated with the use of kinase inhibitors.
While it is possible to take steps to reduce the impact of these
problems, in an ideal situation the results from inhibitors should be
complemented by studies using a different technique.
The first highly selective kinase inhibitors were described in the
1990’s and since then have had a major impact on our understanding of several kinases. PD090859, for instance, was the first
inhibitor described that blocked the classical MEK/ERK cascade
(Alessi et al., 1995) and it has since been a key reagent in the study
of this pathway, as illustrated by the in excess of 2000 citations to
the initial paper describing this compound. Not all kinase inhibitors, however, have been as useful. Many of the initial compounds
described, especially those based on Staurosporine, have subsequently proved to be non-selective and target a large proportion of
the kinome (Davies et al., 2000; Fabian et al., 2005). Predictions
based on the use of these unselective compounds must therefore be
treated with extreme caution.
Over the last 10e15 years there has been a remarkable increase
in the number and selectivity of kinase inhibitors described, fueled
by the interest of the pharmaceutical industry in developing these
compounds as drugs for a range of diseases including cancer and
auto-immunity (for examples see Arslan et al., 2006; BonillaHernan et al., 2011; Cohen, 2009b; Ghoreschi et al., 2009; Zhong
and Bowen, 2011). Several kinase inhibitors have now been
approved as anticancer drugs, while many more compounds have
entered clinical trials (Zhang et al., 2009). A major goal of this
research has often been to develop compounds that are extremely
selective for a specific kinase known to be important in disease, in
the hope that it will provide an effective treatment with few side
effects.
In parallel with this work, systems have been developed for the
high throughput screening of inhibitors against kinase panels (Bain
et al., 2003, 2007; Fabian et al., 2005; Fedorov et al., 2007; Karaman
et al., 2008). As a result it is now relatively easy to obtain data on
the in vitro specificity of a compound against a significant proportion of the kinome either through academic facilities or via
commercial inhibitor screening companies. In addition screening
data has been published for many commonly used kinases inhibitors (Anastassiadis et al., 2011; Bain et al., 2003, 2007; Davis et al.,
2011; Fabian et al., 2005; Fedorov et al., 2007; Karaman et al., 2008).
As a result of these developments, many more kinase inhibitors
are available for use in basic research and it is also possible to get an
accurate assessment of their selectivity and therefore predict
potential off target effects in cells. Despite these advances however
some fundamental issues remain. Selective inhibitors are still only
available for a small proportion of mammalian kinases, and of those
compounds described few are truly selective for a single kinase;
most will inhibit at least several kinases in vitro. In addition to off
target effects on other kinases, inhibitors can also affect other types
of proteins. For instance H89 and KN-93, originally described as
inhibitors for PKA and CaMKII respectively, have since been shown
to affect the activity of ion channels (Rezazadeh et al., 2006; Ledoux
et al., 1999; Li et al., 1992; Murray, 2008). An awareness of these
problems allows approaches to be taken that minimize the possibility of obtaining misleading results (Cohen, 2009a). For instance,
it is important to use the inhibitor at the lowest concentration that
can be demonstrated to completely block the phosphorylation of
a known substrate of the target kinase, as use of the compound at
concentrations significantly higher than this is much more likely to
result in an off target activity. It is important to consider however
that more than one kinase can act upon the same substrate site at
once e for instance the transcription factor CREB is phosphorylated
at serine 133 by both PKA and MSK. The possibility of alternative
kinases acting on a substrate should therefore also be borne in
mind when planning or interpreting these experiments.
Where possible, the effects of structurally different inhibitors
directed against the same kinase should be compared. Alternatively
an inactive analogue of the inhibitor can be used as a control. For
some kinases it may also be possible to rescue the effect of an
inhibitor by introducing a mutated kinase with decreased affinity
for the inhibitor. As most kinase inhibitors are ATP-competitive this
can be attempted by mutating residues in the ATP binding pocket to
modify it so that ATP binding is unaffected but the binding of the
inhibitor is sterically blocked. While this may not always be
possible, this has been successfully achieved for some kinase/
inhibitor combinations, for instance p38/SB203580 and CaMKK/
STO-609 (Eyers et al., 1999; Tokumitsu et al., 2003). An alternative to this is the ‘chemical genetic’ approach. In this, the ATP
binding site of the kinase is mutated to allow the binding of bulky
ATP competitive inhibitors, normally derivatives of PP1, which
cannot access the ATP binding site of most unmodified kinases
(Bishop et al., 2000a, 2000b). Finally if the inhibitor is to be used in
animal models, the pharmacokinetic properties of the compound
must also be considered as many kinase inhibitors are rapidly
broken down by the liver. The ability of the inhibitors to cross the
blood brain barrier is also of relevance to neuroscience research.
Given the number of kinases within the human genome and the
rapid increase in the number of kinase inhibitors available, it is not
possible to list all the available compounds. The remainder of this
article, therefore, will concentrate on some of the small molecule
inhibitors reported for several of the pathways relevant to neuronal
signaling (Fig. 1, Table 1). Neurons are exquisitely sensitive cells that
can respond to action potentials as well as ligand binding to
receptors on the cell surface. These processes rely on a number of
second messengers, including Ca2þ ions and cAMP. The kinases that
mediate responses downstream of these messengers will therefore
be the focus of this article. Inhibitors of Trks will also be discussed.
Trk receptors are receptor tyrosine kinases directly activated by
molecules known as neurotrophins, and are important activators of
MAPK signaling in neurons. Inhibitors of the PI3 Kinase and mTor
pathways have been extensively reviewed recently and will
therefore not be further considered here. For examples of recent
reviews see Feldman and Shokat (2010), Hixon et al. (2010), Knight
(2010), Liu et al. (2012), Workman et al. (2010).
2. Trk inhibitors
Neurotrophins are important mediators of neuronal survival,
differentiation and function. There are four mammalian neurotrophins e Nerve Growth Factor (NGF), Brain-Derived Growth
Factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4 (NT4,
also known as NT5). These act on cells via the three members of the
Trk family (Trk A, B and C) or via an alternative receptor known as
the p75 neurotrophin receptor (p75NTR). Different neurotrophins
affect the various Trks in a specific fashion; NGF acts via TrkA, BDNF
via TrkB and NT4 via TrkC, while NT3 can bind to either TrkB or
K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237
GW441756
GST-TrkA
AZ-SHN722
STO-609
PIP2
CaMKK
Ca2+
release
CaMKI
CaMKIV
CaMKII
Trk
PI-3K
PLC
mTOR
IP3
DAG
Kn93
Kn62
PKC
1229
Akt
Raf
MEKK2/3
BIX02188
BIX02189
MEK5
ERK5
PD184352
PD0325901
MKKK
MEK1/2
MEK3/6
P38 (/ )
ERK1/2
XMD8-98
SB203580
VX745
BI-D1870
SL0101
RSK
MSK
SB-747651A
Fig. 1. Inhibitors of Trk dependent signaling pathways. Neurotrophins activate the Trk tyrosine receptor kinases to activate downstream signaling cascades, including the MAPK,
Ca2þ and PI3 kinase signaling networks. Some of the small molecule inhibitors against these pathways are indicated, and information of specificity in required concentrations are
given in Table 1.
TrkC. The p75NTR, on the other hand, is sensitive to all four. Neurotrophin signaling via the Trk receptors has demonstrable effects
on neuron survival and development (Huang and Reichardt, 2001;
Lu and Figurov, 1997; Reichardt, 2006; Segal, 2003; Webster and
Pirrung, 2008) and long term potentiation (LTP) of synapses
(Gooney and Lynch, 2001; Gubellini et al., 2005; Minichiello, 2009),
while p75 activation seems to have opposing effects, promoting cell
death signals (Nykjaer et al., 2005) and long term depression (LTD)
(Rosch et al., 2005). Neurotrophins are produced as precursor
molecules that are proteolytically cleaved to produce the mature
neurotrophin. The balance between Trk and p75NTR signaling may
be regulated by rates of proneurotrophin cleavage, as the proneurotrophin form has been reported to bind preferentially to
p75NTR (Volosin et al., 2006).
Neurotrophins function by activating the cytoplasmic tyrosine
kinase domain of their respective Trk and thus a specific Trk kinase
Table 1
Selected small molecule kinase inhibitors.
Compound
Suggested
concentration (mM)
PD184352
2
PD0325901
0.2
GW441756
BMS-SHN753
AZ-SHN722
Bi-D1870
SL0101
SB-747651A
BIX02188
BIX02189
XMD8-98
SB203580
BIRB796
VX745
JNK-IN-8
Kn-92, Kn-63
STO 609
Akt-1-1,2
PI103
GDC-0941
H-89
KT5720
5
0.5
0.5
10
100
10
1
5
0.05
1
3
10
25
1
0.5
0.5
10
Target
Major off target effects
Selectivity profiling
MEK1/2 (blocks ERK1/2
activation)
MEK1/2 (blocks ERK1/2
activation)
Trk
Trk
Trk
RSK
RSK
MSK1
MEK5
e
Bain et al. (2007)
e
Bain et al. (2007)
Multiple
Multiple
Multiple
PLK
AuroraB. Pim1/3
RSK, PKA, PKB
Martin et al. (2011)
Martin et al. (2011)
Martin et al. (2011)
Bain et al. (2007)
Bain et al. (2007)
Naqvi et al. (2012)
DCAMKL
PLK4
LRRK2
Q. Yang et al. (2010)
ERK5
p38a/b
p38a/b
p38a/b
JNK
CaMK
CaMKK
Akt
PI3Kinase
PI3Kinase
PKA
PKA
P38g/d, JNK
RIPK2, CK1, NLK
PRKX
Blocks interaction with Ca2þ/calmodulin
MNK1, CK2, AMPK, PIM1/3
Acts via blocking Akt activation
mTOR
Bain et al. (2007)
Kuma et al. (2005)
Davis et al. (2011)
Zhang et al. (2012)
Anastassiadis et al. (2011)
Bain et al. (2007)
Bain et al. (2007)
Bain et al. (2007)
RSK, MSK, ROCK
RSK, MSK, PDK1, PHK
Bain et al. (2007)
Davies et al. (2000)
1230
K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237
inhibitor should allow neurotrophic signaling to be completely
blocked. There is a dual interest in Trk inhibitor development.
A specific inhibitor of the receptors would be an invaluable research
tool for the study of neurotrophin action, but the elevated Trk
kinase activity associated with a number of cancers (particularly
pancreatic cancer and neuroblastoma, Thiele et al., 2009; Wang
et al., 2009) and chronic pain (Wang et al., 2009) provides a more
urgent, therapeutic demand for Trk inhibitors. The high degree of
homology between the kinase domains of the 3 Trk receptors
means that development of inhibitors specific to one family
member alone is likely to be difficult.
The intrinsic kinase activity of the neurotrophin receptors was
found to be susceptible to the Staurosporine analogue K252a
(Koizumi et al., 1988; Tapley et al., 1992), a compound that is still
commonly used in neurotrophin signaling studies (Bozdagi et al.,
2008; Lemtiri-Chlieh and Levine, 2010; Shi et al., 2009; Taylor
et al., 2011) in spite of its lack of specificity toward Trk (Davies
et al., 2000). The K252a derivative CEP-701 also inhibits Trk
activity (Miknyoczki et al., 1999), but it has been shown to have
greater specificity toward the kinases FLT3 and Jak2 than toward
Trks (Hexner et al., 2008; Levis et al., 2002). Although CEP-701
clinical trials have been initiated for the treatment of some conditions (Minturn et al., 2011) the broad target spectra of this inhibitor
makes it impractical for the study of Trk function. A number of
pharmaceutical companies have pursued the development of novel
compounds designed to specifically inhibit Trk kinase activity (Han
et al., 2006; Kim et al., 2008; Thiele et al., 2009; Wang et al., 2009;
Wood et al., 2004; Zage et al., 2011). Little information is available
concerning the specificity of these compounds, but those reports
that have been published show that several of them also inhibit
other receptor tyrosine kinases, including Tie1 and Flt3.
The kinase inhibition profile of three of these recently developed compounds has been investigated (Martin et al., 2011). These
compounds come from three separate programs: an oxindole
compound, GSK-TrkA, developed by GlaxoSmithKlein (Wood et al.,
2004); a pyrimidine based molecule, referred to as AZ-SHN722,
developed by AstraZeneca (Han et al., 2006); and the thiazolecontaining compound developed by Bristol-Myers Squib (Kim
et al., 2008) dubbed BMS-SHN753. In a direct comparison with
K252a these new compounds had higher affinities for Trk and
exhibited far fewer off-target effects in in vitro kinase assays.
GSK-TrkA was the most selective for the Trk kinase, only
inhibiting a further 7 out of 105 tested kinases by more than 90%.
These included ERK8, MLK2, MST2 and NUAK, none of which have
major established links to neurological function. The receptor
tyrosine kinases VEGF-R and FGF-R1 are also affected, demanding
great care in the design of any experiments using this molecule as
the ligands activating these kinases also play roles in some
neuronal systems (During and Cao, 2006; Grothe and Timmer,
2007; Guillemot and Zimmer, 2011; Lambrechts and Carmeliet,
2006). The major disadvantage of GSK-TrkA comes with the high
concentration required for full TrkB inhibition in a cellular system
(5 mM). AZ-SHN722 demonstrated a similar overall selectivity to
GSK-Trk, inhibiting an additional 9 out of 105 kinases by 90% or
more in the in vitro assays. These included the kinases AuroraA, Pim
and AMPK. Of these AMPK has been shown to have an important
role in the neural response to ischemia, hypoxia and glucose
deprivation (Cheung and Hart, 2008; McCullough et al., 2005;
Zhang et al., 2010). BMS-SHN753 was the most selective for tyrosine kinases e of the13 kinases that were inhibited either more
strongly or to a similar degree as TrkA, 6 were tyrosine kinases e
however it did inhibit several Ser/Thr kinases, including CDC like
kinase 2, AuroraA and ERK8, as strongly as it inhibited Trk. It is
noteworthy that these three molecules have different profiles of
off-target effects, providing an ideal example of how the parallel
use molecules of different chemical descent, rather than relying on
a single compound, could give extra confidence in the results of
inhibitor experiment. Other Trk inhibitors have been described as
part of various drug development programs. Little information,
however, is available on the properties of these newer compounds.
Wang et al. described an elegant cell based assay system making
use of stable over-expression of Trk receptors for screening Trk
inhibitors and were able to show that another compound,
GW441756, can also inhibit the activity of all 3 receptors, although
the IC50 was approximately 10 fold higher than that of K252a
(Wang et al., 2008). GW441756 is from the same chemical series as
the GSK-Trk molecule mentioned previously, and like GSK-Trk has
a significant number of off target activities (Wood et al., 2004;
Martin et al., 2011). Screening data has also been reported for
another compound developed by AstraZeneca, AZ-23 (Thress et al.,
2009). This compound is related to AZ-SHN722, however although
it showed improved selectivity it did have a small number of off
target activities, including FGFR and Flt3. AZ-23 has been suggested
as a potential therapeutic for neuroblastoma and in mice this
compound was able to inhibit BDNF driven neuroblastoma cell
proliferation (Zage et al., 2011).
3. MAPK pathway inhibitors
The classical MAPK (ERK1/2) pathway is activated in neurons in
response to many signals, including neurotrophins and neurotransmitters. The pathway consists of the upstream kinase Raf,
which activates MEK1/2, which in turn activate ERK1/2. This
pathway is constitutively activated in many cancers, and has
therefore been the target of multiple drug development programs.
The ERK1/2 pathway was also one of the first signaling pathways
for which selective inhibitors became available. PD90859 was first
described in 1995 as an inhibitor of MEK1/2 (Alessi et al., 1995). In
cells it not only inhibits MEK1/2, but also blocks their activation,
making it a very effective inhibitor of the ERK1/2 pathway.
PD98059 is also very selective with little off target activity (Davies
et al., 2000). This selectivity may be because, unlike most kinase
inhibitors, PD98059 does not bind to the ATP binding site. While
PD98059 has been a very useful research tool, it has limited
aqueous solubility and therefore high concentrations are required
for inhibition of ERK1/2 activation in cells. Improved MEK1/2
inhibitors quickly followed the initial description of PD98059.
U0126 is more soluble and will inhibit the ERK1/2 pathway at
10 mM in cells (Favata et al., 1998), and another MEK1/2 inhibitor
SL327 has been shown to cross the blood brain barrier (Selcher
et al., 1999). More recently some very potent and selective MEK1/
2 inhibitors have been described including PD184352, PD0325901,
AZD6244 and GSK1120212 (Gilmartin et al., 2011; Sebolt-Leopold,
2008; Sebolt-Leopold et al., 1999; Yeh et al., 2007). Of these, the
compounds from Pfizer have been most extensively used as
research tools. PD184352 (CI-1040) will inhibit ERK1/2 activation at
2 mM in cells while PD0325901 is effective at 0.2 mM (Bain et al.,
2003, 2007). Both PD184352 and PD0325901 entered clinical
trials for the treatment of cancer that were terminated due to
insufficient clinical improvement and/or adverse neurological
effects (Boasberg et al., 2011). Trials using AZD6244 and
GSK1120212 as cancer therapies are still ongoing.
At present, few off target activities have been ascribed to MEK1/
2 inhibitors. However the use of most of the above compounds at
a 5e10 fold higher concentrations than required to block MEK1/2 in
cells can also inhibit MEK5, the upstream activator of ERK5 (Bain
et al., 2007). ERK5 has been proposed to have a role in neurons,
for example in retrograde transport, and therefore selective ERK5
inhibitors are also of interest to this field (Ginty and Segal, 2002;
Obara and Nakahata, 2010). There are two compounds, BIX02188
K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237
and BIX02189, that have been reported to selectively inhibit MEK5
(Tatake et al., 2008), while more recently a selective inhibitor of
ERK5, XMD8-98 has also been reported (P. Yang et al., 2010). XMD898 does however have some off target activity, including against
LRRK2 (Deng et al., 2011; P. Yang et al., 2010), a kinase important in
Parkinson’s disease, making the use of XMD8-98 in parallel with
either BIX02188 or BIX02189 desirable for the study of ERK5 in
neurons.
ERK1/2 activates several downstream kinases including the
p90RSK and MSK families. Mutations in RSK2 result in Coffin
Lowery syndrome demonstrating that this kinase family plays
a role in plasticity and behavior (Anjum and Blenis, 2008; Jacquot
et al., 2002; Poirier et al., 2007; Romeo et al., 2011). Early reports
used Ro 31-2880 or H89 to inhibit RSK, however both these
compounds are very non-selective and target a significant number
of other kinases. Ro 31-2880 for instance inhibits PKC while H89
inhibits PKA (Davies et al., 2000). Several newer compounds for
RSK, such as BI-D1870, fmk and SL0101 have now been reported,
and screening has shown these to be reasonably selective for RSK
(Bain et al., 2007; Cohen et al., 2005; Sapkota et al., 2007; Smith
et al., 2007).
MSK1 and 2 are two kinases closely related to RSK, and are
similarly activated by the ERK1/2 pathway in response to neurotrophins (Arthur et al., 2004). MSKs are required for efficient
phosphorylation of CREB in response to neurotrophins (Arthur
et al., 2004), and additionally MSK knockouts have effects in
several behavioral paradigms (Brami-Cherrier et al., 2005;
Chandramohan et al., 2008; Chwang et al., 2007). Like RSK, MSKs
can be inhibited by Ro 31-2280 and H89, however as discussed
above neither of these compounds are very selective. Recently
a more selective MSK inhibitor, SB-747651A, has been described,
however while this compound is an improvement over Ro 31-2880
and H89 for the study of MSKs, it can also inhibit other AGC kinases
including RSK, Akt and to a lesser extent PKA (Naqvi et al., 2012).
Parallel experiments with more selective inhibitors of RSK and Akt
would therefore need to be carried out to distinguish between
inhibition of MSKs and these other kinases.
Inhibitors of the two other major MAPK families, p38 and JNK
have also been described. The p38 MAPK family has been a target
of considerable interest for its potential therapeutic roles in
autoimmune conditions (Pettus and Wurz, 2008), neurodegeneration (particularly Alzheimer’s disease, Munoz and Ammit,
2010) and cancer (Gaundar and Bendall, 2010). The p38 MAPKs
have been further implicated in the inhibition of ES cell differentiation into neurons (Aouadi et al., 2006), modulation of neuronal
excitability (Poolos et al., 2006) and regulation of synaptic plasticity (Zhong et al., 2008). Many highly selective p38 inhibitors
have now been described (reviewed in Cohen, 2009b; Genovese,
2009; Zhang et al., 2007), providing effective tools for the
further understanding of the roles of this important kinase family.
Chemical inhibition of JNK, however, has proved more problematic, in spite of a long standing interest in the family as potential
regulators of both diabetes (Kaneto, 2005) and oncogenesis
(Heasley and Han, 2006). There is also considerable genetic
evidence that JNKs have crucial roles to play in the cells of both
the immune (Constant et al., 2000; Conze et al., 2002; Dong et al.,
1998a) and nervous systems (Bruckner et al., 2001; Hunot et al.,
2004; Yang et al., 1997), SP 600125 and AS 601245 have both
been reported as JNK inhibitors but both have been shown to
exhibit poor selectivity in kinase screens, limiting their usefulness
as research tools (Bain et al., 2007). Recently JNK-IN-8, a new
compound which has a much improved specificity for JNK, has
been described (Zhang et al., 2012), and it is hoped that this
compound will provide further valuable insights into the functions
of JNK.
1231
4. CaMK inhibitors
Ca2þ is a critical 2nd messenger in neurons and plays an integral
part in neuronal development and plasticity (Wayman et al., 2008).
An important action of Ca2þ is the activation of calmodulin
dependent protein kinases (CaMKs). Both the CaMKII and CaMKI/IV
pathways have been strongly implicated in regulating synaptic
development and plasticity, and defects in learning and memory
have been reported in mouse models gene targeted for proteins in
these pathways (reviewed in Wayman et al., 2008). CaMKII is
directly activated by binding to Ca2þ/calmodulin complexes. It then
undergoes autophosphorylation which allows it to maintain its
activity after the dissociation of Ca2þ/calmodulin. The activation of
CaMKI and CaMKIV also requires Ca2þ/calmodulin binding to
initiate a conformational change that exposes the activation loop of
the kinase and allows it to be phosphorylated. This phosphorylation
is carried out by CaMKKa or b, which are also activated by interaction with Ca2þ/calmodulin (Wayman et al., 2011).
KN-62 and KN-93 were initially described as small molecule
inhibitors of CaMKII, however subsequent studies demonstrated
that they also inhibit CaMKI and CaMKIV (Mochizuki et al., 1993;
Sumi et al., 1991; Tokumitsu et al., 1990 Enslen et al., 1994). These
compounds act by blocking the interaction between CaMK and
Ca2þ/calmodulin, and therefore are less likely to inhibit other
kinases in cells. Both CaMKII and CaMKIV, however, may exhibit
some Ca2þ independent activity in cells which could be less
sensitive to KN-62 and KN-93. Additionally these compounds have
been shown to affect voltage-gated Ca2þ or Kþ ion channels, which
may contribute to their overall cellular effects (Ledoux et al., 1999;
Li et al., 1992; Rezazadeh et al., 2006).
For CaMKI and CaMKIV an alternative approach is to inhibit the
upstream kinase, CaMKK. STO-609 was initially described as
a specific CaMKK inhibitor (Tokumitsu et al., 2002), however kinase
profiling has shown that it also has off target activity. In addition to
CaMKKs, STO-609 effectively inhibits MNK1, CK2, AMPK, PIM2,
PIM3, HIPK2, ERK8 and DYRK (Hawley et al., 2005). This can be
controlled for to some extent by expressing CaMKK mutants that
are insensitive to STO-609 (Tokumitsu et al., 2003). A further
complication of CaMKK inhibition is that CaMKI and CaMKIV are
not the only substrates of CaMKK; for instance under certain
circumstances CaMKK can activate AMPK, a kinase that acts as a key
sensor of cellular energy status (Hawley et al., 2005) as well as
being a point of crosstalk between Ca2þ signaling and the PKA, PI-3
kinase and MAPK signaling pathways (Soderling, 1999).
While KN-93, KN-62 and STO-609 are useful tools for the study
of CaMKs, results obtained with these compounds should ideally be
backed up by independent methods. One possibility for CaMKII is
the use of peptide inhibitors fused to tags that allow them to cross
the cell membrane. Inhibitory peptides based on either the autoinhibitory domain of CaMKII or an endogenous inhibitory protein
for CaMKII, CaMKIIN, have been described (reviewed in Wayman
et al., 2011), and these could be used in conjunction with the
known highly-basic peptide tags such as ‘Ant’ and ‘Tat’ that confer
cell permeability on peptides. The Ant sequence, however, has been
reported to bind to calmodulin and is therefore best avoided in
CaMK studies (Buard et al., 2010).
5. PKA inhibitors
The cyclic-AMP activated protein kinase (PKA) is a serine/threonine protein kinase that responds to the elevated levels of cyclicAMP (cAMP) that result from adenylate cyclase activity stimulated
by, for example, the activation of G-protein coupled receptors
(GPCRs). In neurons a large proportion of metabotropic neurotransmitter receptors (including those for dopamine, serotonin,
1232
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acetylcholine, GABA and glutamate) belong to the GPCR family and
thus have the potential to act via PKA. PKA is a tetramer of 2
regulatory (R) and 2 catalytic (C) subunits that is activated by
dissociation of the R and C subunits (Beavo et al., 1975), providing
several potential approaches to its inhibition. Small molecule ATP
competitive inhibitors of PKA, such as H89 and KT5720, have been
described, although it is now apparent that both these compounds
have many off target effects. KT5720 was the first small molecule
targeted to PKA, identified in the late 1980s (Chijiwa et al., 1990;
Engh et al., 1996). Its specificity was initially evaluated against the
small selection of kinases that had been well characterized at the
time. The same is also true for the second PKA inhibitor, H89, which
was distinct from its predecessor H7 in its selectivity of PKA over
PKC (Kase et al., 1987). The recent use of kinase panels, however,
has allowed the identification ‘off target’ effects for both
compounds (Anastassiadis et al., 2011; Davies et al., 2000). KT5720
was found to inhibit several other protein kinases, and is therefore
unlikely to act as a selective PKA inhibitor in cells. Observation of
particular concern were that KT5720 also inhibited both RSK and
MSK1, kinases with substrate specificities overlapping with that of
PKA, and that it inhibits the kinases PDK1 and PHK more potently
even than PKA (Davies et al., 2000). H89 can also inhibit other
kinases in addition to PKA; recent studies showed that both MSK1
and ROCKII are inhibited by H89 to a similar degree to PKA, and
several other kinases, including RSK, were also inhibited, though at
a higher IC50 than that reported for PKA (Davies et al., 2000;
Leemhuis et al., 2002). These nonspecific effects are particularly
troubling as both MSK1 and ROCKII can act in parallel to PKA. MSK1
phosphorylates the transcription factor CREB at the same site as
PKA (Gonzalez and Montminy, 1989; Wiggin et al., 2002), while
PKA phosphorylation of the small GTPase RhoA reduces its affinity
for the kinase ROCKII/ROKa (Dong et al., 1998b). Given that MSK1
and PKA can both regulate gene expression via CREB, this effect
introduces a major element of uncertainty into conclusions drawn
from the use of H89 or KT5720 alone. These problems are particularly relevant in neurons: MSK1 has been shown to be a key player
in the MAPK response to neurotrophic signals (Arthur et al., 2004)
and has been linked to the regulation of memory and plasticity
(Chandramohan et al., 2008; Chwang et al., 2007). This problem is
compounded by the potential crosstalk between PKA and MSK1;
MSK1 is activated by ERK1/2, and PKA can promote ERK1/2 activation in neurons (Impey et al., 1998). ROCKII also functions in
neurons and can regulate neurite outgrowth (Duffy et al., 2009).
While a ROCKII inhibitor that does not significantly affect PKA or
MSK is available for comparison studies (Davies et al., 2000; Uehata
et al., 1997), the same is not true for MSK1 (Naqvi et al., 2012). A
further potential issue with H89 is that it has been shown to affect
the properties of b1- and b2-adrenergic receptors and certain ion
channels (reviewed in Lochner and Moolman, 2006; Murray, 2008).
Given the problems with ATP competitive kinase inhibitors for
PKA, alternative approaches for PKA inhibition should be considered in parallel with inhibitor studies. cAMP analogues such as RpcAMPS (de Wit et al., 1984; Dostmann, 1995) and its derivatives
(Gjertsen et al., 1995) block the cAMP binding sites of the R subunits
of PKA and so prevent dissociation of the complex and activation of
the C subunits in response to cAMP levels. Rp-cAMPS is unlikely to
target other kinases, but may affect other cAMP activated proteins.
For instance cAMP is known to activate EPACs in addition to PKA,
and while in vitro studies suggest that Rp-cAMPS can inhibit EPACs
it is less clear whether they effectively block EPACs in cells (Holz
et al., 2008).
Endogenous inhibitor proteins for PKA (PKIa and PKIb) have
been identified, and a peptide derived from this protein, often
referred to as PKI, has been shown to be a highly effective PKA
inhibitor (reviewed in Dalton and Dewey, 2006). Unlike H89 or
KT5720, PKI appears to show good selectivity for PKA and does not
inhibit other AGC kinases. On its own, PKI is not cell permeable. This
problem was overcome by the addition of a N-terminal myristoyl
group to a PKI that allows it to cross the cell membrane. The
resulting peptide is commercially available and has been used to
some effect to replicate results achieved with small molecule
inhibitors of PKA (Harris et al., 1997; Malki-Feldman and Jaffe,
2009; Salazar and Gonzalez, 2002). The use of PKI peptides does
require some care, however, as at high concentrations they also act
to inhibit the cGMP regulated kinase PKG (Glass et al., 1992), which
could be particularly relevant to behavioral phenotype analysis, as
cGMP signaling has been linked to behavioral responses in
a number of models (Engel et al., 2000; Gong et al., 2011; van der
Linden et al., 2008).
6. PKC inhibitors
The name protein kinase C (PKC) refers to a family of closely
related serine/threonine kinases that are activated downstream of
lipid signaling events, as is described in detail in Newton (2010),
Roffey et al. (2009). In brief, they are subdivided into three groups;
‘conventional’ cPKCs that require calcium and diacylglycerol (DAG)
binding for activation, the Phosphatidic-acid (PA)-lipid dependent
but calcium independent ‘novel’ nPKCs, and the ‘atypical’ aPKCs
whose regulation is achieved through recruitment to protein
complexes at sites of PI3K activity, where PDK1 can be brought to
bear upon the activation loop of the kinase domain. Individual PKC
isoforms can also be specifically targeted to the subcellular
compartment they are required to act in by interaction with RACK
(Receptors of Activated C-Kinases) proteins (Schechtman and
Mochly-Rosen, 2001).
PKC activation can occur following both GPCR and RTK signals, if
the correct scaffolds are assembled (as is neatly summarized in
Parker and Murray-Rust, 2004), giving PKC the capacity to play
a role in both neurotransmitter and neurotrophin stimulated
neuronal responses. Studies in knockout mouse models have
shown that cPKC gamma (Abeliovich et al., 1993a, 1993b; Bowers
et al., 2000) and nPKC epsilon (Lesscher et al., 2008, 2009;
Wallace et al., 2009) in particular have roles in memory and
behavior.
In terms of PKC inhibitors there are two separate challenges: the
first is to specifically inhibit the PKCs without affecting other kinase
pathways, such as PKA or MAPK signaling; while the second, more
recent development is to attempt to selectively inhibit individual
PKC isoforms. As with other kinases there are two major classes of
PKC kinase inhibitor e ATP-binding competitors and pseudosubstrate molecules. Other approaches to eliminating PKC activity,
such as pseudo-RACK peptides that interfere with PKC translocation upon activation, are available.
The natural bacterial product staurosporine was reported to
have an inhibitory effect on PKC in 1986 (Tamaoki et al., 1986),
although this compound is very non-selective (Nakano et al., 1987;
Tamaoki et al., 1986). Around the same time other microbial
metabolites e the K252 compounds e were found to have similar
activities (Kase et al., 1986; Nakanishi et al., 1986), and to have some
limited selectivity between PKC and PKA (Kase et al., 1987). This
spawned a class of molecules known as bisindolylmaleimides
(Davis et al., 1992; Toullec et al., 1991) (Table 2) that have been
widely used (Yaguchi et al., 2010; P. Yang et al., 2010), as ‘selective’
PKC inhibitors. This chemical series however has been shown to
consistently inhibit S6K1, RSK2 and MSK1 in vitro (Davies et al.,
2000). For instance one of these compounds, Ro 318220, has
a greater affinity for these three ‘non-specific’ kinases than for PKC
itself, a significant problem given that both RSK2 and MSK1 have
demonstrable neuronal roles in plasticity and memory events
K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237
Table 2
The bisindolylmaleimide series of PKC inhibitors.
No. in series
CAS number/
chemical formula
Synonyms
Selectivity profiling
I
133052-90-1
GO 6850, GF
109203X
Anastassiadis et al. (2011)
II
III
IV
C27H26N4O2
C23H20N4O2
119139-23-0
Anastassiadis et al. (2011)
V
VI
VII
VIII
IX
X
XI
LY-333531
113963-68-1
C27H26N4O2
137592-47-3
125313-65-7
138489-18-6
131848-97-0
145333-02-4
169939-94-0
RO 31-6233,
Arcyriarubin A
RO 31-6045
RO-31-7549
RO-31-8220
RO-31-8425
RO-32-0432
Enzastaurin
1233
phosphorylated serine/threonine residue with alanine. Such
compounds have been generated for PKC and their modification
with a myristoyl group allows for their uptake into cells without
extreme measures of permeabilisation being taken (Eichholtz et al.,
1993). The use of pseudo-substrate peptides is subjected to the
same caveats as small molecule inhibitors e the lowest effective
concentration required should be used as higher concentrations
may allow binding to non-specific targets, as was observed
between PKC and CaMK peptides in one LTP study (Hvalby et al.,
1994).
7. Inhibition of PKMz
Bain et al. (2007)
Karaman et al. (2008)
(Chandramohan et al., 2008; Chwang et al., 2007; Harum et al.,
2001). LY-33531 (Enzastaurin) is a member of this family of
compounds and has been reported to show some selectivity
amongst PKC isoforms (Graff et al., 2005), although profiling has
shown that this compound can still target RSKs (Karaman et al.,
2008). LY-33531 has however progressed into clinical trails for
cancer. Anilino-monoindolylmaleimides have also been described
as PKC inhibitors (Tanaka et al., 2004), however selectivity profiling
for one (‘PKC inhibitor b’, CAS 257879-35-9) demonstrated that
these compounds also inhibit RSKs (Anastassiadis et al., 2011).
In 2008, Skvara et al. reported a study using AEB071 (also known
as sotrastaurin), to inhibit the PKC-dependent immune response
underlying psoriasis (Skvara et al., 2008). The development of this
compound, by targeted modification of a bisindolylmaleimide
skeleton, was reported the following year (Wagner et al., 2009). The
authors reported that their new, improved compound selectively
inhibited PKC over a panel of 200 other kinases, though the exact
composition of this panel was not detailed. However when the
compound was identified as an activator of Wnt signaling (likely
due to a previously unreported effect on GSK isoforms) by Verkaar
et al., a second kinase screen was carried out, in which it was shown
that while AEB071 still effectively inhibited RSKs, it was less potent
against MSK1 (Verkaar et al., 2010). This is a distinct improvement
on the bisindolylmaleimides, as the potential non-specific effect on
the RSKs could be tested for by comparison with the effects of an
RSK inhibitor such as BI-D1870 (Sapkota et al., 2007). The alteration
of GSK3 activity by AEB071 and its consequences for Wnt signaling
are, however, a concern in neuronal systems, in which these
molecules are known to influence plasticity events (Ataman et al.,
2008; Chen et al., 2006; Hall et al., 2002).
Another early PKC inhibitor group was the calphostins, also
produced by microorganisms (Iida et al., 1989). These molecules are
structurally unrelated to the bisindolylmaleimides and were found
to inhibit c- and n-PKC’s through their interaction with the C1
domain facilitating irreversible oxidative damage to the protein
(Gopalakrishna et al., 1992). Conclusions drawn from the use of
calphostins in neurons were put into a questionable light by the
report that calphostin C, the most potent PKC inhibitor of the group,
has a similar effect on L-type calcium channels, blocking their
activity (Hartzell and Rinderknecht, 1996). This could alter the
excitability of neuronal systems, making electrophysiological
recordings difficult to interpret, and suggests that calphostin is
a poor choice of inhibitor in neuronal PKC research.
The second potential target for kinase inhibitors is the substratebinding site, as opposed to the ATP-binding site. The easiest way to
target such a site is with a pseudo-substrate peptide, mimicking the
specific sequence targeted by the kinase but substituting the
PKMz is a brain specific isoform of the atypical PKCz, driven from
an alternative promoter that bypasses the regulatory N-terminal
section of the protein (Hernandez et al., 2003; Marshall et al.,
2000). This results in a constitutively active kinase molecule
which is highly expressed in a number of regions of the brain,
including the hippocampus (Oster et al., 2004). Elevated levels of
PKMz have been associated with the maintenance of long term
potentiation (Osten et al., 1996; Powell et al., 1994; Sacktor et al.,
1993), with a complementary down regulation of PKMz observed
in long term depression (Hrabetova and Sacktor, 1996, 2001).
Further work to establish the roles of PKMz in learning and memory
have however relied heavily on the use of molecules reported to be
z-isoform specific PKC inhibitors (Cai et al., 2011; Drier et al., 2002;
Hrabetova and Sacktor, 1996; Moncada and Viola, 2008; Serrano
et al., 2008; Shema et al., 2009).
One of the plant derived molecules identified in the early years
of PKC inhibition research, chelerythrine (Herbert et al., 1990), has
since been reported to have some isoform selectivity among the
PKC family, in that it is effective against PKMz at far lower
concentrations than for the other PKC isoforms tested (a, b and 3)
and full length PKCz (Ling et al., 2002; Thompson and Fields, 1996).
While no large scale kinase screens are available for the compound,
it has been shown in independent studies to inhibit CaMKII at
similar concentrations to full length PKC (Ling et al., 2002), and
MRCK (Myotonic dystrophy kinase-related Cdc42-binding kinase)
at similar concentrations to PKMz (Tan et al., 2011). Additionally,
a number of other proteins whose functions or interactions are
disrupted by chelerythrine have been identified, including several
that have relevance to neuronal cells. These include the glycine
transporter GlyT1 (Jursky and Baliova, 2011), the apoptosis regulator Bcl-X (Bernardo et al., 2008), MKP-1, a MAPK phosphatase
(Vogt et al., 2005), the cannaboid receptor CB1 (Bisset et al., 2011),
and the dopaminergic enzyme Aromatic Amino-acid Decarboxylase (Drsata et al., 1996). The presence of the molecule in a cell also
introduces a source of oxidative stress (Matkar et al., 2008; Yu et al.,
2000). Together, these observations mean the use of chelerythrine
alone to study PKMz is not ideal. Attempts to design a more targeted, small molecule inhibitor for PKC/PKMz are ongoing. The 2(6-phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles developed by
Pfizer show some PKC isoform selectivity, but the best of them still
inhibited more than 20 out of 37 unrelated kinases screened,
including PKA, PKBg, and CaMKII (Trujillo et al., 2009). An independent study by Yuan et al. identified the 3-hydroxy-2-(3hydroxyphenyl)-4H-1-benzopyran-4-ones, a group of molecules
whose most selective isoform targeted PKCz without significantly
inhibiting PKCb, PKC3, PKBa and PKA, however more detailed
screening information is currently not available (Yuan et al., 2009).
The second commonly used PKMz inhibitor is a pseudosubstrate peptide sequence derived from the regulator domain of
PKCz, known as zeta inhibitor peptide (ZIP) (Laudanna et al., 1998).
This relies on sequence differences in the regulatory domains of the
PKC isoforms to provide specificity (Hofmann, 1997). In the absence
1234
K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237
of any comprehensive screening data the use of ZIP alone may not
provide conclusive evidence for PKMz function, particularly as
when used at high concentrations it can target CaMKII (Ling et al.,
2002). Given the very different chemical natures of chelerythrine
and ZIP, however, a consistent result from both compounds would
give much better evidence for a role of PKMz (Sacktor and Fenton,
2011).
8. Summary
The deployment of selective kinase inhibitors by the pharmaceutical industry and academic labs has seen considerable progress
in the last 10e15 years. As a result many compounds are now
available that can be used for the study of specific kinases in
cells. However, as for any method, care must be taken with these
studies, and it will always be prudent to back up inhibitor studies
with genetic or siRNA based methods, and vice versa. Very few
compounds are completely specific for one kinase and therefore
the possibility of off target effects must be considered. In this
respect, the recent development of kinase screening panels
provides an excellent starting point for the prediction of nonspecific cellular effects of kinases inhibitors. The careful use of
small molecule kinase inhibitors has allowed, and will continue to
facilitate, significant progress in our understanding of intracellular
signaling.
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