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Neuropharmacology 63 (2012) 1227e1237 Contents lists available at SciVerse ScienceDirect 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., 1228 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 K.J. Martin, J.S.C. Arthur / Neuropharmacology 63 (2012) 1227e1237 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. 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