Alastair D. Reith

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

BACKGROUND Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

The Parkinson Progression Marker Initiative (PPMI)

The Parkinson Progression Marker Initiative (PPMI) is a comprehensive observational, international, multi-center study designed to identify PD progression biomarkers both to improve understanding of disease etiology and course and to provide crucial tools to enhance the likelihood of success of PD modifying therapeutic trials. The PPMI cohort will comprise 400 recently diagnosed PD and 200 healthy subjects followed longitudinally for clinical, imaging and biospecimen biomarker assessment using standardized data acquisition protocols at twenty-one clinical sites. All study data will be integrated in the PPMI study database and will be rapidly and publically available through the PPMI web site- www.ppmi-info.org. Biological samples including longitudinal collection of blood, cerebrospinal fluid (CSF) and urine will be available to scientists by application to an independent PPMI biospecimen review committee also through the PPMI web site. PPMI will rely on a partnership of government, PD foundations, industry and academics working cooperatively. This approach is crucial to enhance the potential for success of this ambitious strategy to develop PD progression biomarkers that will accelerate research in disease modifying therapeutics.

Selective small‐molecule inhibitors of glycogen synthase kinase‐3 activity protect primary neurones from death

The phosphatidylinositol 3‐kinase (PI 3‐kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase‐3 (GSK‐3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK‐3 in response to insulin and growth factors, which inhibits GSK‐3 activity and leads to the modulation of multiple GSK‐3 regulated cellular processes. We show that the novel potent and selective small‐molecule inhibitors of GSK‐3; SB‐415286 and SB‐216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3‐kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK‐3 activity and modulation of GSK‐3 substrates tau and β‐catenin. Thus, in addition to the previously assigned roles of GSK‐3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK‐3 activity in primary neurones is sufficient to prevent death, implicating GSK‐3 as a physiologically relevant principal regulatory target of the PI 3‐kinase/PKB neuronal survival pathway.

Inhibition of LRRK2 kinase activity leads to dephosphorylation ofSer910/Ser935, disruption of 14-3-3 binding and altered cytoplasmiclocalization

LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinsons 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 Parkinsons 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 Parkinsons 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 Parkinsons disease.

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

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinsons 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. DOI: http://dx.doi.org/10.7554/eLife.12813.001

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

The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinsons 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 Parkinsons 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.

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

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for some forms of Parkinsons 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.

Expression and Characterization of GSK-3 Mutants and Their Effect on β-Catenin Phosphorylation in Intact Cells

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 β-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of β-catenin by GSK-3 does not require primingin 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-3β. 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 Ser33, Ser37, and Thr41 in β-catenin requires priming through prior phosphorylation at Ser45 in intact cells. We have shown that the Arg96 mutant does not induce β-catenin degradation but instead stabilizes β-catenin, indicating that it is unable to phosphorylate β-catenin in intact cells. Furthermore, if Ser45 in β-catenin is mutated to Ala, β-catenin is markedly stabilized, and phosphorylation of Ser33, Ser37, and Thr41 in β-catenin by wild type GSK-3β 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 β-catenin in intact cells although it has reduced activity. Mutation of Tyr216 to Phe markedly reduces the ability of GSK-3β to phosphorylate and down-regulate β-catenin. In conclusion, we have found that the Arg96 mutant has a dominant-negative effect on GSK-3β-dependent phosphorylation of β-catenin and that targeting of β-catenin for degradation requires prior priming through phosphorylation of Ser45.

GSK-3 inhibition by adenoviral FRAT1 overexpression is neuroprotective and induces Tau dephosphorylation and β-catenin stabilisation without elevation of glycogen synthase activity

Glycogen synthase kinase 3 (GSK‐3) has previously been shown to play an important role in the regulation of apoptosis. However, the nature of GSK‐3 effector pathways that are relevant to neuroprotection remains poorly defined. Here, we have compared neuroprotection resulting from modulation of GSK‐3 activity in PC12 cells using either selective small molecule ATP‐competitive GSK‐3 inhibitors (SB‐216763 and SB‐415286), or adenovirus overexpressing requently earranged in dvanced ‐cell lymphomas 1 (FRAT1), a protein proposed as a negative regulator of GSK‐3 activity towards Axin and β‐catenin. Our data demonstrate that cellular overexpression of FRAT1 is sufficient to confer neuroprotection and correlates with inhibition of GSK‐3 activity towards Tau and β‐catenin, but not modulation of glycogen synthase (GS) activity. By comparison, treatment with SB‐216763 and SB‐415286 proved more potent in terms of neuroprotection, and correlated with inhibition of GSK‐3 activity towards GS in addition to Tau and β‐catenin.

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

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.