Gregory J. Murphy

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.

PPAR-γ agonists: therapeutic role in diabetes, inflammation and cancer

Abstract The recent development of a novel class of insulin-sensitizing drugs, the thiazolidinediones (TZDs), represents a significant advance in antidiabetic therapy. One key mechanism by which these drugs exert their effects is by activation of the peroxisome proliferator activated receptor γ (PPAR-γ), a member of the nuclear receptor family. Evidence supporting this mechanism of action of the TZDs will be reviewed in this article. Recent data suggests that PPAR-γ agonists might also have therapeutic potential in the treatment of inflammatory diseases and certain cancers.

Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases

GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.

Treatment of intact hepatocytes with either the phorbol ester TPA or glucagon elicits the phosphorylation and functional inactivation of the inhibitory guanine nucleotide regulatory protein Gi

The antiserum AS7 can specifically immunoprecipitate α‐Gi from membrane extracts as well as from a mixture of purified α‐Gi and α‐Go. as ascertained using [32P]ADP‐ribosylated G‐proteins. Using this antiserum to immunoprecipitate α‐Gi from hepatocytes labelled with 32P it was evident that α‐Gi was phosphorylated under basal (resting) conditions. Challenge of hepatocytes with the tumour promoting phorbol ester TPA, however, elicited a marked enhancement of the phosphorylation state of α‐Gi. This was accompanied by the loss of inhibitory effect of Gi on adenylate cyclase, as judged by the inability of low concentrations of p[NH]ppG to inhibit forskolin‐stimulated adenylate cyclase activity. Such actions were mimicked by treatment of hepatocytes with either glucagon or TH‐glucagon, an analogue of glucagon which is incapable of activating adenylate cyclase and elevating intracellular cyclic AMP concentrations. Pre‐treatment of hepatocytes with either glucagon, TPA or insulin did not affect the ability of pertussis toxin to cause the NAD+‐dependent, [32P]ADP‐ribosylation of α‐Gi in membrane fractions isolated from such pre‐treated hepatocytes. We suggest that protein kinase C can elicit the phosphorylation and functional inactivation of α‐Gi in intact hepatocytes. As pertussis toxin only causes the ADP‐ribosylation of the holomeric form of Gi, it may be that phosphorylation leaves α‐Gi in its holomeric state.

Correlation of β3-adrenoceptor-induced activation of cyclic amp-dependent protein kinase with activation of lipolysis in rat white adipocytes

The lipolytic action of the beta 3-adrenoceptor-selective agonist 4-[2-[(2-hydroxy-2-(3-chlorophenyl)ethyl)-amino]propyl]-phenoxyacetic acid (BRL 37344) was compared to that of isoprenaline in adipocytes derived from rat white adipose tissue. Concentration-response curves for activation of lipolysis by each agonist correlated well with the dose-response curves for activation of cAMP-dependent protein kinase (A-Kinase). Addition of propranolol at a concentration (0.1 microM) sufficient to block beta 1- and beta 2-adrenoceptors did not affect the stimulation of either parameter by BRL 37344 or isoprenaline, indicating that lipolysis was predominantly dependent on beta 3-adrenoceptor stimulation. Blockade of beta 3-adrenoceptors by 3 microM propranolol antagonized both A-Kinase activation and glycerol release. Activation of lipolysis by BRL 37344 was blocked by treatment of the cells with N-[2-p-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H89) a potent and selective isoquinolinesulphonamide inhibitor of A-Kinase activity. Taken together, these results indicate that lipolysis in rat white adipocytes is primarily controlled by beta 3-adrenoceptors, and that cyclic AMP generation alone is responsible for activation of lipolysis in this tissue.

Glucagon activates two distinct signal transduction systems in hepatocytes, which leads to the desensitization of G-protein-regulated adenylate cyclase, the phosphorylation and inactivation of Gi-2 and the phosphorylation and stimulation of a specific cyclic AMP phosphodiesterase

A transient increase in the intracellular concentrations of cyclic AMP occurs as a result of the challenge of hepatocytes with glucagon. This event is determined by the initial rapid activation of adenylate cyclase, which is responsible for the production of cyclic AMP within the cell. Following on from this we observe the desensitization of adenylate cyclase; the A-kinase-mediated activation of the ‘dense-vesicle’, high affinity cyclic AMP phosphodiesterase; the phosphorylation and functional inactivation of the inhibitory G-protein Gi-2 and the establishment of a ‘selective’ insulin-resistant state. These events identify ‘interplay’ or ‘cross-talk’ occurring between distinct cellular signalling systems.

Glucagon desensitisation of adenylate cyclase in hepatocytes: an action mediated by a distinct population of glucagon receptors coupled to inositol phospholipid metabolism

Many tissues when challenged with hormones able to stimulate adenylate cyclase exhibit a rapid but brief rise in the intracellular concentrations of cyclic AMP. The transience of this response, which may endure for merely minutes, is due to desensitization.

Desensitization of Glucagon-Stimulated Adenylate Cyclase is Mediated by Stimulation of Inositol Phospholipid Metabolism

Desensitization is the process whereby responsiveness to a hormone or drug is reduced after repeated or prolonged exposure to that agonist. Although this phenomenon is widespread in biological regulation, the underlying molecular events have only recently begun to be defined. We have provided an initial characterisation of the molecular events leading to the rapid desensitization of glucagon-stimulated adenylate cyclase activity in rat hepatocytes. This, together with activation of specific cyclic AMP phosphodiesterases (1, 2), accounts for the transient rise in intracellular cyclic AMP concentrations that occurs when hepatocytes are challenged with glucagon (Fig.l).