Mari R. Candelore

Peroxisome Proliferator-Activated Receptors γ and α Mediate in Vivo Regulation of Uncoupling Protein (UCP-1, UCP-2, UCP-3) Gene Expression

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Human β3-adrenergic receptor agonists containing 1,2,3-triazole-substituted benzenesulfonamides

Compounds containing a 1,2,3-triazole-substituted benzenesulfonamide were prepared and found to be potent and selective human beta3-adrenergic receptor agonists. The most interesting compound, trifluoromethylbenzyl analogue 12e (beta3 EC50 = 3.1 nM with >1500-fold selectivity over binding to both beta1- and beta2 receptors), stimulates lipolysis in the rhesus monkey (ED50 = 0.36 mg/kg) and is 25% orally bioavailable in the dog.

The carboxyl terminus of the hamster β-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

The structural basis for agonist-mediated sequestration and desensitization of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster beta AR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of beta AR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, beta AR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for cAMP-dependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the beta AR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to Gs and sequestration.

Discovery of a novel glucagon receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-β-alanine (MK-0893) for the treatment of type II diabetes.

A potent, selective glucagon receptor antagonist 9m, N-[(4-{(1S)-1-[3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-β-alanine, was discovered by optimization of a previously identified lead. Compound 9m is a reversible and competitive antagonist with high binding affinity (IC(50) of 6.6 nM) and functional cAMP activity (IC(50) of 15.7 nM). It is selective for glucagon receptor relative to other family B GPCRs, showing IC(50) values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2. Compound 9m blunted glucagon-induced glucose elevation in hGCGR mice and rhesus monkeys. It also lowered ambient glucose levels in both acute and chronic mouse models: in hGCGR ob/ob mice it reduced glucose (AUC 0-6 h) by 32% and 39% at 3 and 10 mpk single doses, respectively. In hGCGR mice on a high fat diet, compound 9m at 3, and 10 mpk po in feed lowered blood glucose levels by 89% and 94% at day 10, respectively, relative to the difference between the vehicle control and lean hGCGR mice. On the basis of its favorable biological and DMPK properties, compound 9m (MK-0893) was selected for further preclinical and clinical evaluations.

Potent, selective human β3 adrenergic receptor agonists containing a substituted indoline-5-sulfonamide pharmacophore

A series of compounds possessing an N-substituted indoline-5-sulfonamide pharmacophore was prepared and evaluated for their human beta3 adrenergic receptor agonist activity. The SAR of a wide range of urea and heterocyclic substituents is discussed. 4-Octyl thiazole compound 8c was the most potent and selective compound in the series, with 2800-fold selectivity over beta1 binding and 1400-fold selectivity over beta2 binding.

Phytosphingosine 1-phosphate: a high affinity ligand for the S1P(4)/Edg-6 receptor.

It has been reported recently that the phosphorylated form of the immunomodulator FTY720 activates sphingosine 1-phosphate G protein-coupled receptors. Therefore, understanding the biology of this new class of receptors will be important in clarifying the immunological function of bioactive lysosphingolipid ligands. The S1P(4) receptor has generated interest due to its lymphoid tissue distribution. While the S1P(4) receptor binds the prototypical ligand, S1P, a survey of other lysosphingolipids demonstrated that 4D-hydroxysphinganine 1-phosphate, more commonly known as phytosphingosine 1-phosphate (PhS1P), binds to S1P(4) with higher affinity. Using radiolabeled S1P (S133P), the affinity of PhS1P for the S1P(4) receptor is 1.6nM, while that of S1P is nearly 50-fold lower (119+/-20nM). Radiolabeled PhS1P proved to be superior to S133P in routine binding assays due to improved signal-to-noise ratio. The present study demonstrates the utility of a novel radiolabeled probe, PhS133P, for in vitro studies of the S1P(4) receptor pharmacology.

L-770,644 : A potent and selective human β3 adrenergic receptor agonist with improved oral bioavailability

L-770,644 (9c) is a potent and selective agonist of the human beta3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.

Human β3 andrenergic receptor agonists containing imidazolidinone and imidazolone benzenesulfonamides

The cyclopentylpropylimidazolidinone L-766,892 is a potent beta3 AR agonist (EC50 5.7 nM, 64% activation) with 420- and 130-fold selectivity over binding to the beta1 and beta2 ARs, respectively. In anesthetized rhesus monkeys, L-766,892 elicited dose-dependent hyperglycerolemia (ED50 0.1 mg/kg) with minimal effects on heart rate.

3-pyridylethanolamines: Potent and selective human β3 adrenergic receptor agonists

The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.

Anti-diabetic efficacy and impact on amino acid metabolism of GRA1, a novel small-molecule glucagon receptor antagonist.

Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.