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The discovery and optimization of novel N-(3-(1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yloxy)phenyl)benzenesulfonamide GPR119 agonists is described. Modification of the pyridylphthalimide motif of the molecule with R(1)=-Me and R(2)=-(i)Pr substituents, incorporated with a 6-fluoro substitution on the central phenyl ring offered a potent and metabolically stable tool compound 22.

The present report describes our efforts to convert an existing LXR agonist into an LXR antagonist using a structure-based approach. A series of benzenesulfonamides was synthesized based on structural modification of a known LXR agonist and was determined to be potent dual liver X receptor (LXR α/β) ligands. Herein we report the identification of compound 54 as the first reported LXR antagonist that is suitable for pharmacological in vivo evaluation in rodents.

Structural modification of a series of dual LXRα/β agonists led to the identification of a new class of LXRβ partial agonists. An X-ray co-crystal structure shows that a representative member of this series, pyrrole 5, binds to LXRβ with a reversed orientation compared to 1.

Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes.

The objective of this study was to demonstrate the efficacy of a novel peroxisome proliferator-activated receptor (PPAR) agonist and known PPARalpha and PPARdelta agonists to increase HDL-cholesterol (HDL-C) in the St. Kitts vervet, a nonhuman primate model of atherosclerosis. Four groups (n = 6) were studied and each group was assigned one of the following "treatments": a) vehicle only (vehicle); b) the PPARdelta selective agonist GW501516 (GW); c) the PPARalpha/delta agonist T913659 (T659); and d) the PPARalpha agonist TriCor (fenofibrate). No statistically significant changes were seen in body weight, total plasma cholesterol, plasma triglycerides, VLDL-C, LDL-C, or apolipoprotein B (apoB) concentrations. Each of the PPARalpha and PPARdelta agonists investigated in this study increased plasma HDL-C, apoA-I, and apoA-II concentrations and increased HDL particle size in St. Kitts vervets. The maximum percentage increase in HDL-C from baseline for each group was as follows: vehicle, 5%; GW, 43%; T659, 43%; and fenofibrate, 20%. Treatment with GW and T659 resulted in an increase in medium-sized HDL particles, whereas fenofibrate showed increases in large HDL particles. These data provide additional evidence that PPARalpha and PPARdelta agonists (both mixed and selective) have beneficial effects on HDL-C in these experimental primates.

Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G i -signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G i -coupled receptor (Ro1), we provide evidence that increased G i signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G i signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G i signaling in causing cardiac pathology.

To control G protein signaling in vivo, we have modified G protein-coupled receptors to respond exclusively to synthetic small molecule agonists and not to their natural agonist(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). A prototype RASSL (Ro1) based on the Gi-coupled K opioid receptor was expressed in transgenic mice under the control of the tetracycline transactivator (tet) system. Activation of Ro1 expressed in the heart decreased heart rate by up to 80%, an expected effect of increased Gi signaling. Maximal heart rate changes occurred in less than 1 min, demonstrating the speed of this inducible signaling system. This Ro1-mediated slowing of heart rate was also subject to desensitization, which lasted more than 24 h. Both the initial effect on heart rate and the desensitization occurred, even though Ro1 is derived from a human opioid receptor not normally involved in heart rate control. In addition, the tet system was used to induce Ro1 expression in hepatocytes and salivary gland, where Gi signaling is known to control physiologic events such as proliferation and secretion. These studies demonstrate that a RASSL can be inducibly expressed in several mouse tissues and used in vivo to activate G protein signaling in a controllable fashion.

We are developing a system to control G protein signaling in vivo to regulate a broad range of physiologic responses. Our system utilizes G protein-coupled peptide receptors engineered to respond exclusively to synthetic small molecule ligands and not to their natural ligand(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). We have made two prototype RASSLs that are based on the human κ opioid receptor. Small molecule drugs that activate the κ receptor are nonaddictive and safe to administer in vivo . Binding and signaling assays reveal 200–2000-fold reductions in the ability of our RASSLs to bind or be activated by dynorphin, an endogenous peptide ligand of the κ opioid receptor. In a high-throughput signaling assay, these prototype RASSLs expressed in Chinese hamster ovary K1 cells showed little or no response to a panel of 21 opioid peptides but still signaled normally in response to small molecule drugs such as spiradoline. Activation of a RASSL by spiradoline also caused proliferation of rat-1a tissue culture cells. These data provide evidence that G protein-coupled receptors can be made into RASSLs. The potential in vivo applications for RASSLs include the positive enrichment of transfected cells and the development of new animal models of disease.

Glucokinase (GK) is a hexokinase isozyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate. Glucokinase activators are being investigated as potential diabetes therapies because of their effects on hepatic glucose output and/or insulin secretion. Here, we have examined the efficacy and mechanisms of action of a novel glucokinase activator, GKA23. In vitro, GKA23 increased the affinity of rat and mouse glucokinase for glucose, and increased glucose uptake in primary rat hepatocytes. In vivo, GKA23 treatment improved glucose homeostasis in rats by enhancing beta cell insulin secretion and suppressing hepatic glucose production. Sub-chronic GKA23 treatment of mice fed a high-fat diet resulted in improved glucose homeostasis and lipid profile.

The discovery and optimization of a series of potent PPARdelta full agonists with partial agonistic activity against PPARgamma is described.

037 A Critical Period of Vulnerability to Hyperactive Gq Signaling Revealed by Inducible and Heart-Specific Expression of Gq* in Transgenic Mice Nathalie Cotte, 1 Charles H. Redfern, 1'2 Peter Mikus, 1 Nathan Salomonis, 1 Michael Y. Degtyarev] Peter S. Coward, 1 Andrew Kwa, 1 Tetsuo Shioi, 4 Nila Shah, 5 Tim McKinsey, 3 Eric Olson, s Seigo Izumo, a Janet Wartington, s Anthony J. Baker, 2 Brace R. Conklin~'2; 1Gladstone Institute of Cardiovascular Disease, Gladstone Institute of Neurological Disease, 2Department of Medicine, University of California, San Francisco, CA, 3Hamon Center for Basic Research in Cancer, University of Texas Southwestern Medical Center, Dallas, TX, 4Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, SAffymetrix Inc, Santa Clara, CA