Shaker Kv1 (1.1-1.7, 1.10) Kv2 (2.1, 2.2) Kir1 (1.1-1.3) TWIK (TWIK1-2) Kv3 (3.1-3.4) Kir2 (2.1-2.4) TREK Kv4 (4.1-4.3) Kir3 (3.1-3.4) TASK Kv5 (5.1) Kir4 (4.1) TRAAK Kv6 (6.1, 6.2) Kir6 (6.1-6.2) Kv8 (8.1) Kir7 (7.1) Kv9 (9.1-9.3) ether-a-go-go EAG hERG LQT-related KCNQ1 (KvLQT1) KCNQ 2-5 Ca 2+ -activated Slo (R) SK (SK1-3) IK (IK1) auxiliary subunits Kv (β1-β3) SUR1, SUR2 K Ca (β1-β4) Mink miRP1, miRP2

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

OBJECTIVEThe clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor.RESEARCH DESIGN AND METHODSScreening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo.RESULTSNovel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment.CONCLUSIONSThese studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

1 A hallmark for unstable bladder contractions is hyperexcitability and changes in the nature of spontaneous phasic activity of the bladder smooth muscle. In this study, we have characterized the spontaneous activity of the urinary bladder smooth muscle from the pig, a widely used model for studying human bladder function. 2 Our studies demonstrate that phasic activity of the pig detrusor is myogenic and is in¯uenced by the presence of urothelium. Denuded strips exhibit robust spontaneous activity measured as mean area under the contraction curve (AUC=188.9+15.63 mNs) compared to intact strips (AUC=7.3+1.94 mNs). 3 Spontaneous phasic activity, particularly the amplitude, is dependent on both calcium entry through voltage-dependent calcium channels and release from ryanodine receptors as shown by inhibition of spontaneous activity by nifedipine and ryanodine respectively. 4 Inhibition of BK Ca channels by iberiotoxin (100 nM) resulted in an increase in contraction amplitude (89.1+20.4%) and frequency (92.5+31.0%). The SK Ca channel blocker apamin (100 nM) also increased contraction amplitude (69.1+24.3%) and frequency (53.5+13.6%) demonstrating that these mechanisms are critical to the regulation of phasic spontaneous activity. 5 Inhibition of K ATP channels by glyburide (10 mM) did not signi®cantly alter myogenic contractions (AUC=18.5+12.3%). However, K ATP channel openers (KCOs) showed an exquisite sensitivity for suppression of spontaneous myogenic activity. KCOs were generally 15 fold more potent in suppressing spontaneous activity compared to contractions evoked by electrical ®eld-stimulation. These studies suggest that potassium channel modulation, particularly K ATP channels, may o er a unique mechanism for controlling spontaneous myogenic activity especially those associated with the hyperexcitability occurring in unstable bladders.

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Background: Insulin-degrading enzyme (IDE) is the best characterized catabolic enzyme implicated in insulin proteolysis. Results: Newly discovered dual exosite IDE inhibitors do not significantly affect insulin action or clearance. Conclusion: IDE catabolism does not appear to be the primary mechanism of insulin clearance in vivo. Significance: These IDE inhibitors will enable broader investigation of IDE function.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.