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Two small-molecule disruptors of the glucokinase-glucokinase-regulatory-protein complex, AMG-1694 and AMG-3969, are identified that decrease blood glucose levels in various models of hyperglycaemic rodents. Novel glucose-lowering agents Glucokinase (GK) is involved in the regulation of glucose homeostasis and is an important target for drug discovery against diabetes, but some current GK activators are associated with an increased risk for hypoglycaemia in the clinic. This paper presents an alternative therapeutic point of intervention, focused on the liver-specific GK regulatory protein (GKRP) that binds and inhibits GK in the nucleus. David Lloyd et al. report on a small molecule, AMG-1694, that specifically disrupts the GK-GKRP complex and reduces blood glucose in hyperglycaemic rodent models. Importantly AMG-1694 has no effect in normoglycaemic animals. Disruption of the GK-GKRP complex offers a liver-specific approach of upregulating GK activity in type II diabetes while reducing the risk of hypoglycaemia. Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus.sup.1. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate.sup.2,3 in pancreatic [beta]-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes.sup.4. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production.sup.3,5, and is subject to the endogenous inhibitor GK regulatory protein (GKRP).sup.6,7,8. During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus.sup.1,9,10. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators.sup.11. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus., Author(s): David J. Lloyd [sup.1] , David J. St Jean Jr [sup.2] , Robert J. M. Kurzeja [sup.2] , Robert C. Wahl [sup.2] , Klaus Michelsen [sup.2] , Rod Cupples [...]

Murine models of ovalbumin (OA)-induced pulmonary inflammation were utilized to role of vascular cell adhesion molecule-1 (VCAM-1) and alpha4-integrins expression in the leukocyte trafficking. Analysis of the airways of mice with OA-induced inflammation indicated the presence of OA-induce elevation of VCAM-1 expression in the murine pulmonary vascular tissue. Furthermore, inhibition of VCAM-1 and alpha4-integrin expression reduced the accumulation of eosinophils and lymphocytes.

Lymphocytes can kill target cells including hepatocytes during various inflammatory diseases by Fas receptor–mediated apoptosis. Caspase-8 is activated at the receptor level, thereby initiating the processing of downstream effector caspases. The aim of this study was to investigate the time course of caspase-8 activation and to evaluate the efficacy of the caspase-8 inhibitor IETD-CHO in a model of Fas-induced apoptosis in vivo. C3Heb/FeJ mice were treated with the anti-Fas antibody Jo-2 (0.6 mg/kg). Western blot analysis demonstrated increased cytochrome c in the cytosol (20 min), which was followed by the progressive activation of caspase-3, -9 (40–120 min), and caspase-8 (120 min). At 90 and 120 min, extensive hemorrhage was observed, indicating damage to sinusoidal lining cells. In addition, high plasma ALT levels (997 ± 316 U/L) and histological evaluation indicated severe parenchymal cell injury. Parenchymal and nonparenchymal cells showed a similar increase in caspase-3 activity and DNA fragmentation. Treatment with IETD-CHO (10 mg/kg) attenuated the increase in caspase-3 activity and DNA fragmentation by 80–90% and completely prevented hemorrhage and parenchymal cell damage. IETD-CHO also prevented the early release of mitochondrial cytochrome c and the processing of caspase-3, -8, and -9. Thus, our data support the hypothesis that Fas-mediated apoptosis is dependent on caspase-8 activation in hepatocytes and nonparenchymal cells. However, the bulk of procaspase-8 is processed late, suggesting that only a small amount of procaspase-8 may actually be activated at the Fas receptor. This initial signal may be amplified by further activation of caspase-8 by effector caspases, i.e., after mitochondrial activation. Caspase-8 is a promising therapeutic target for inhibition of Fas-mediated apoptosis. [ABSTRACT FROM PUBLISHER]

The tumor suppressor gene RASSF1A is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologs of the Drosophila Hippo kinase, RASSF1A is a potential regulator of the Hippo tumor suppressor pathway. RASSF1A shares these properties with the scaffold protein SAV1. The role of this pathway in human cancer has remained enigmatic inasmuch as Hippo pathway components are rarely mutated in tumors. Here we show that Rassf1a homozygous knockout mice develop liver tumors. However, heterozygous deletion of Sav1 or codeletion of Rassf1a and Sav1 produced liver tumors with much higher efficiency than single deletion of Rassf1a. Analysis of RASSF1A-binding partners by mass spectrometry identified the Hippo kinases MST1, MST2, and the oncogenic IkB kinase TBK1 as the most enriched RASSF1Ainteracting proteins. The transcriptome of Rassf1a-/- livers was more deregulated than that of Sav1+/- livers, and the transcriptome of Rassf1a-/-, Sav1+/- livers was similar to that of Rassf1a-/- mice. We found that the levels of TBK1 protein were substantially upregulated in livers lacking Rassf1a. Furthermore, transcripts of several β-tubulin isoforms were increased in the Rassf1a-deficient livers presumably reflecting a role of RASSF1A as a microtubule-stabilizing protein. In human liver cancer, RASSF1A frequently undergoes methylation at the promoter but this was not observed for MST1, MST2, or SAV1. Our results suggest a multifactorial role of RASSF1A in suppression of liver carcinogenesis. [ABSTRACT FROM AUTHOR]