Publications by authors named "Alastair D Reith"

19 Publications

  • Page 1 of 1

5-Substituted-N-pyridazinylbenzamides as potent and selective LRRK2 inhibitors: Improved brain unbound fraction enables efficacy.

Bioorg Med Chem Lett 2019 01 28;29(2):212-215. Epub 2018 Nov 28.

Neurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, PR China. Electronic address:

We describe the discovery and optimization of 5-substituted-N-pyridazinylbenzamide derivatives as potent and selective LRRK2 inhibitors. Extensive SAR studies led to the identification of compounds 18 and 23, which demonstrated good in vitro pharmacokinetic profile and excellent selectivity over 140 other kinases. Both compounds demonstrated high unbound fractions in both blood and brain. Compound 18 proved to be brain penetrant, and the high unbound fraction of compound 18 in brain enabled its in vivo efficacy in CNS, wherein a significant inhibition of LRRK2 Ser935 phosphorylation was observed in rat brain following intravenous infusion at 5 mg/kg/h.
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http://dx.doi.org/10.1016/j.bmcl.2018.11.054DOI Listing
January 2019

LRRK2 is a negative regulator of phagosome maturation in macrophages.

EMBO J 2018 06 22;37(12). Epub 2018 May 22.

Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced phagosome maturation and mycobacterial control independently of autophagy. , LRRK2 deficiency in mice resulted in a significant decrease in burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls replication by regulating phagosome maturation.
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http://dx.doi.org/10.15252/embj.201798694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003659PMC
June 2018

Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent, selective and orally bioavailable LRRK2 inhibitors.

Bioorg Med Chem Lett 2018 05 19;28(9):1615-1620. Epub 2018 Mar 19.

Neurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, PR China. Electronic address:

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson's disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100 mg/kg) and mice (45 mg/kg) following oral administration.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.045DOI Listing
May 2018

Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and orally bioavailable LRRK2 inhibitors.

Bioorg Med Chem Lett 2017 09 21;27(17):4034-4038. Epub 2017 Jul 21.

Neurodegeneration DPU, Neurosciences Therapeutic Area Unit, GSK Pharmaceuticals R&D, 898 Halei Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, PR China. Electronic address:

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson's disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.
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http://dx.doi.org/10.1016/j.bmcl.2017.07.052DOI Listing
September 2017

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.

Biochem J 2016 09 29;473(17):2671-85. Epub 2016 Jul 29.

MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser(935) and Ser(1292) biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser(910) and Ser(935) and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.
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http://dx.doi.org/10.1042/BCJ20160557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003698PMC
September 2016

Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.

Elife 2016 Jan 29;5. Epub 2016 Jan 29.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.
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http://dx.doi.org/10.7554/eLife.12813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769169PMC
January 2016

A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease Using RapidFire Mass Spectrometry.

J Biomol Screen 2016 Feb 24;21(2):145-55. Epub 2015 Sep 24.

Neurodegeneration DPU, Neurosciences Therapy Area Unit, GlaxoSmithKline, Pharmaceuticals R&D, Hertfordshire, UK, and Pudong, China.

LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson's disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.
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http://dx.doi.org/10.1177/1087057115606707DOI Listing
February 2016

MSK1 and MSK2 inhibit lipopolysaccharide-induced prostaglandin production via an interleukin-10 feedback loop.

Mol Cell Biol 2013 Apr 4;33(7):1456-67. Epub 2013 Feb 4.

MRC Protein Phosphorylation Unit, Sir James Black Complex, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

Prostaglandin production is catalyzed by cyclooxygenase 2 (cox-2). We demonstrate here that MSK1 and MSK2 (MSK1/2) can exert control on the induction of cox-2 mRNA by Toll-like receptor (TLR) agonists. In the initial phase of cox-2 induction, MSK1/2 knockout macrophages confirmed a role for MSK in the positive regulation of transcription. However, at later time points both lipopolysaccharide (LPS)-induced prostaglandin and cox-2 protein levels were increased in MSK1/2 knockout. Further analysis found that while MSKs promoted cox-2 mRNA transcription, following longer LPS stimulation MSKs also promoted degradation of cox-2 mRNA. This was found to be the result of an interleukin 10 (IL-10) feedback mechanism, with endogenously produced IL-10 promoting cox-2 degradation. The ability of IL-10 to do this was dependent on the mRNA binding protein TTP through a p38/MK2-mediated mechanism. As MSKs regulate IL-10 production in response to LPS, MSK1/2 knockout results in reduced IL-10 secretion and therefore reduced feedback from IL-10 on cox-2 mRNA stability. Following LPS stimulation, this increased mRNA stability correlated to an elevated induction of both of cox-2 protein and prostaglandin secretion in MSK1/2 knockout macrophages relative to that in wild-type cells. This was not restricted to isolated macrophages, as a similar effect of MSK1/2 knockout was seen on plasma prostaglandin E2 (PGE2) levels following intraperitoneal injection of LPS.
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http://dx.doi.org/10.1128/MCB.01690-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624268PMC
April 2013

GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.

Bioorg Med Chem Lett 2012 Sep 7;22(17):5625-9. Epub 2012 Jul 7.

External Alliances & Development, R&D China, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK.

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinson's disease. Here we report the discovery and characterization of 2-arylmethyloxy-5-subtitutent-N-arylbenzamides with potent LRRK2 activities exemplified by GSK2578215A which exhibits biochemical IC(50)s of around 10 nM against both wild-type LRRK2 and the G2019S mutant. GSK2578215A exhibits exceptionally high selectivity for LRRK2 across the kinome, substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.3-1.0 μM in cells and in mouse spleen and kidney, but not in brain, following intraperitoneal injection of 100mg/kg.
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http://dx.doi.org/10.1016/j.bmcl.2012.06.104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208292PMC
September 2012

Characterization of the cellular action of the MSK inhibitor SB-747651A.

Biochem J 2012 Jan;441(1):347-57

MRC Protein Phosphorylation Unit, Sir James Black Complex, University of Dundee, Dundee DD1 5EH, Scotland, U.K.

MSK1 (mitogen- and stress-activated kinase 1) and MSK2 are nuclear protein kinases that regulate transcription downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38α MAPKs (mitogen-activated protein kinases) via the phosphorylation of CREB (cAMP-response-element-binding protein) and histone H3. Previous studies on the function of MSKs have used two inhibitors, H89 and Ro 31-8220, both of which have multiple off-target effects. In the present study, we report the characterization of the in vitro and cellular properties of an improved MSK1 inhibitor, SB-747651A. In vitro, SB-747651A inhibits MSK1 with an IC50 value of 11 nM. Screening of an in vitro panel of 117 protein kinases revealed that, at 1 μM, SB-747651A inhibited four other kinases, PRK2 (double-stranded-RNA-dependent protein kinase 2), RSK1 (ribosomal S6 kinase 1), p70S6K (S6K is S6 kinase) (p70RSK) and ROCK-II (Rho-associated protein kinase 2), with a similar potency to MSK1. In cells, SB-747651A fully inhibited MSK activity at 5-10 μM. SB-747651A was found to inhibit the production of the anti-inflammatory cytokine IL-10 (interleukin-10) in wild-type, but not MSK1/2-knockout, macrophages following LPS (lipopolysaccharide) stimulation. Both SB-747651A and MSK1/2 knockout resulted in elevated pro-inflammatory cytokine production by macrophages in response to LPS. Comparison of the effects of SB-747651A, both in vitro and in cells, demonstrated that SB-747651A exhibited improved selectivity over H89 and Ro 31-8220 and therefore represents a useful tool to study MSK function in cells.
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http://dx.doi.org/10.1042/BJ20110970DOI Listing
January 2012

Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.

Biochem J 2010 Sep;430(3):405-13

University of Dundee, Scotland, UK.

LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients. Since a common mutation that replaces Gly2019 with a serine residue enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms via its phosphorylation of Ser910 and Ser935. In the present study we show that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These findings indicate that dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess the relative activity of LRRK2 inhibitors in vivo. These results will aid the elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.
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http://dx.doi.org/10.1042/BJ20100784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3631100PMC
September 2010

Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease.

Biochem J 2009 Oct 23;424(1):47-60. Epub 2009 Oct 23.

MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.

The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson's disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.
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http://dx.doi.org/10.1042/BJ20091035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759966PMC
October 2009

The identification of potent, selective and CNS penetrant furan-based inhibitors of B-Raf kinase.

Bioorg Med Chem Lett 2008 Aug 24;18(15):4373-6. Epub 2008 Jun 24.

Department of Medicinal Chemistry, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

Modification of the potent imidazole-based B-Raf inhibitor SB-590885 resulted in the identification of a series of furan-based derivatives with enhanced CNS penetration. One such compound, SB-699393 (17), was examined in vivo to challenge the hypothesis that selective B-Raf inhibitors may be of value in the treatment of stroke.
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http://dx.doi.org/10.1016/j.bmcl.2008.06.070DOI Listing
August 2008

FRAT1, a substrate-specific regulator of glycogen synthase kinase-3 activity, is a cellular substrate of protein kinase A.

J Biol Chem 2006 Nov 18;281(46):35021-9. Epub 2006 Sep 18.

Discovery Research Biology, Neurology Centre of Excellence in Drug Discovery, Computational, Analytical, and Structural Sciences, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AD, United Kingdom.

FRAT1, like its Xenopus homolog glycogen synthase kinase-3 (GSK-3)-binding protein, is known to inhibit GSK-3-mediated phosphorylation of beta-catenin. It is currently unknown how FRAT-GSK-3-binding protein activity toward GSK-3 is regulated. FRAT1 has recently been shown to be a phosphoprotein in vivo; however, the responsible kinase(s) have not been determined. In this study, we identified Ser188 as a phosphorylated residue in FRAT1. The identity of the kinase that catalyzes Ser188 phosphorylation and the significance of this phosphorylation to FRAT1 function were investigated. Protein kinase A (PKA) was found to phosphorylate Ser188 in vitro as well as in intact cells. Importantly, activation of endogenous cAMP-coupled beta-adrenergic receptors with norepinephrine stimulated the phosphorylation of FRAT1 at Ser188. GSK-3 was also able to phosphorylate FRAT1 at Ser188 and other residues in vitro or when overexpressed in intact cells. In contrast, endogenous GSK-3 did not lead to significant FRAT1 phosphorylation in cells, suggesting that GSK-3 is not a major FRAT1 kinase in vivo. Phosphorylation of Ser188 by PKA inhibited the ability of FRAT1 to activate beta-catenin-dependent transcription. In conclusion, PKA phosphorylates FRAT1 in vitro as well as in intact cells and may play a role in regulating the inhibitory activity of FRAT1 toward GSK-3.
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http://dx.doi.org/10.1074/jbc.M607003200DOI Listing
November 2006

The identification of potent and selective imidazole-based inhibitors of B-Raf kinase.

Bioorg Med Chem Lett 2006 Jan 2;16(2):378-81. Epub 2005 Nov 2.

Department of Medicinal Chemistry, Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

A novel triarylimidazole derivative, SB-590885 (33), bearing a 2,3-dihydro-1H-inden-1-one oxime substituent has been identified as a potent and extremely selective inhibitor of B-Raf kinase.
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http://dx.doi.org/10.1016/j.bmcl.2005.09.072DOI Listing
January 2006

A kinase-dependent role for EphA2 receptor in promoting tumor growth and metastasis.

Oncogene 2005 Nov;24(53):7859-68

Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-2363, USA.

Receptor tyrosine kinases of the Eph family are upregulated in several different types of cancer. One family member in particular, the EphA2 receptor, has been linked to breast, prostate, lung and colon cancer, as well as melanoma. However, mechanisms by which EphA2 contributes to tumor progression are far from clear. In certain tumor cell lines, EphA2 receptor is underphosphorylated, raising the question of whether ligand-induced receptor phosphorylation and its kinase activity play a role in oncogenesis. To test directly the role of EphA2 receptor phosphorylation/kinase activity in tumor progression, we generated EphA2 receptor variants that were either lacking the cytoplasmic domain or carrying a point mutation that inhibits its kinase activity. Expression of these EphA2 mutants in breast cancer cells resulted in decreased tumor volume and increased tumor apoptosis in primary tumors. In addition, the numbers of lung metastases were significantly reduced in both experimental and spontaneous metastasis models. Reduced tumor volume and metastasis are not due to defects in tumor angiogenesis, as there is no significant difference in tumor vessel density between wild-type tumors and tumors expressing EphA2-signaling-defective mutants. In contrast, tumor cells expressing the EphA2 mutants are defective in RhoA GTPase activation and cell migration. Taken together, these results suggest that receptor phosphorylation and kinase activity of the EphA2 receptor, at least in part, contribute to tumor malignancy.
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http://dx.doi.org/10.1038/sj.onc.1208937DOI Listing
November 2005

Mitogen and stress response kinase-1 (MSK1) mediates excitotoxic induced death of hippocampal neurones.

J Neurochem 2003 Jul;86(1):25-32

Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, UK.

Activation of the mitogen-activated protein kinase (MAPK/ERK) signal transduction pathway may mediate excitotoxic neuronal cell death in vitro and during ischemic brain injury in vivo. However, little is known, of the upstream regulation or downstream consequences of ERK activation under these conditions. Magnesium removal has been described to induce hyperexcitability and degeneration in cultured hippocampal neurones. Here, we show that neurotoxicity evoked by Mg2+ removal in primary hippocampal neurones stimulates ERK, but not p38, phosphorylation. Removal of Mg2+ also resulted in induction of the MAPK/ERK substrate mitogen- and stress-response kinase 1 (MSK1) and induced phosphorylation of the MSK1 substrate, the transcription factor cAMP response element binding protein (CREB). Neuronal death and phosphorylation of components in this cascade were inhibited by the Raf inhibitor SB-386023, by the MEK inhibitor U0126, or by the MSK1 inhibitors H89 and Ro318220. Importantly, this form of cell death was inhibited in hippocampal neurones cultured from MSK1-/- mice and inhibitors of Raf or MEK had no additive neuroprotective effect. Together, these data indicate that MSK1 is a physiological kinase for CREB and that this activity is an essential component of activity-dependent neuronal cell death.
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http://dx.doi.org/10.1046/j.1471-4159.2003.01830.xDOI Listing
July 2003

SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.

Mol Pharmacol 2002 Jul;62(1):65-74

Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London, UK.

Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, adhesion, and specification of developmental fate, in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor, both serine/threonine kinases. Here, we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are very highly related to ALK5 in their kinase domains. It has no effect on the other, more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542, we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum.
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http://dx.doi.org/10.1124/mol.62.1.65DOI Listing
July 2002

Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells.

J Biol Chem 2002 Jun 19;277(26):23330-5. Epub 2002 Apr 19.

Systems Research-Kinase Biology Discovery Research, GlaxoSmithKline Pharmaceuticals, Harlow, Essex, CM19 5AD, United Kingdom.

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).
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http://dx.doi.org/10.1074/jbc.M201364200DOI Listing
June 2002