Chemical Probe Identification Platform for Orphan GPCRs Using Focused Compound Screening: GPR39 as a Case Example.

Orphan G protein-coupled receptors (oGPCRs) are a class of integral membrane proteins for which endogenous ligands or transmitters have not yet been discovered. Transgenic animal technologies have uncovered potential roles for many of these oGPCRs, providing new targets for the treatment of various diseases. Understanding signaling pathways of oGPCRs and validating these receptors as potential drug targets requires the identification of chemical probe compounds to be used in place of endogenous ligands to interrogate these receptors. A novel chemical probe identification platform was created in which GPCR-focused libraries were screened against sets of oGPCR targets, with a goal of discovering fit-for-purpose chemical probes for the more druggable members of the set. Application of the platform to a set of oGPCRs resulted in the discovery of the first reported small molecule agonists for GPR39, a receptor implicated in the regulation of insulin secretion and preservation of beta cells in the pancreas. Compound 1 stimulated intracellular calcium mobilization in recombinant and native cells in a GPR39-specific manner but did not potentiate glucose-stimulated insulin secretion in human islet preparations.

Discovery of new piperidine amide triazolobenzodiazepinones as intestinal-selective CCK1 receptor agonists.

New cholecystokinin-1 receptor (CCK1R) agonist 'triggers' were identified using iterative library synthesis. Structural activity relationship studies led to the discovery of compound 10e, a potent CCK1R agonist that demonstrated robust weight loss in a diet-induced obese rat model with very low systemic exposure. Pharmacokinetic data suggest that efficacy is primarily driven through activation of CCK1R's located within the intestinal wall.

Design of a Potent CB1 Receptor Antagonist Series: Potential Scaffold for Peripherally-Targeted Agents.

Antagonism of cannabinoid-1 (CB1) receptor signaling has been demonstrated to inhibit feeding behaviors in humans, but CB1-mediated central nervous system (CNS) side effects have halted the marketing and further development of the lead drugs against this target. However, peripherally restricted CB1 receptor antagonists may hold potential for providing the desired efficacy with reduced CNS side effect profiles. In this report we detail the discovery and structure-activity-relationship analysis of a novel bicyclic scaffold (3) that exhibits potent CB1 receptor antagonism and oral activity in preclinical feeding models. Optimization of physical properties has led to the identification of analogues which are predicted to have reduced CNS exposure and could serve as a starting point for the design of peripherally targeted CB1 receptor antagonists.

Quantitative in vitro and in vivo pharmacological profile of CE-178253, a potent and selective cannabinoid type 1 (CB1) receptor antagonist.

BACKGROUND: Cannabinoid 1 (CB1) receptor antagonists exhibit pharmacological properties favorable for the treatment of obesity and other related metabolic disorders. CE-178253 (1-[7-(2-Chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a]-[1,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid hydrochloride) is a recently discovered selective centrally-acting CB1 receptor antagonist. Despite a large body of knowledge on cannabinoid receptor antagonists little data exist on the quantitative pharmacology of this therapeutic class of drugs. The purpose of the current studies was to evaluate the quantitative pharmacology and concentration/effect relationships of CE-178253 based on unbound plasma concentration and in vitro pharmacology data in different in vivo preclinical models of FI and energy expenditure. RESULTS: In vitro, CE-178253 exhibits sub-nanomolar potency at human CB1 receptors in both binding (Ki = 0.33 nM) and functional assays (Ki = 0.07 nM). CE-178253 has low affinity (Ki > 10,000 nM) for human CB2 receptors. In vivo, CE-178253 exhibits concentration-dependent anorectic activity in both fast-induced re-feeding and spontaneous nocturnal feeding FI models. As measured by indirect calorimetry, CE-178253 acutely stimulates energy expenditure by greater than 30% in rats and shifts substrate oxidation from carbohydrate to fat as indicated by a decrease the respiratory quotient from 0.85 to 0.75. Determination of the concentration-effect relationships and ex vivo receptor occupancy in efficacy models of energy intake and expenditure suggest that a greater than a 2-fold coverage of the Ki (50-75% receptor occupancy) is required for maximum efficacy. Finally, in two preclinical models of obesity, CE-178253 dose-dependently promotes weight loss in diet-induced obese rats and mice. CONCLUSIONS: We have combined quantitative pharmacology and ex vivo CB1 receptor occupancy data to assess concentration/effect relationships in food intake, energy expenditure and weight loss studies. Quantitative pharmacology studies provide a strong a foundation for establishing and improving confidence in mechanism as well as aiding in the progression of compounds from preclinical pharmacology to clinical development.

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB(1) receptor antagonist for the management of obesity.

Cannabinoid CB(1) receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB(1) receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB(1) receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB(1) receptors in both binding (K(i)=0.7 nM) and functional assays (K(i)=0.2 nM). The compound has low affinity (K(i)=7600 nM) for human CB(2) receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB(1) receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system.

New bicyclic cannabinoid receptor-1 (CB1-R) antagonists.

A series of conformationally constrained bicyclic derivatives derived from SR141716 was prepared and evaluated as hCB(1)-R antagonists and inverse agonists. Optimization of the structure-activity relationships around the 2,6-dihydro-pyrazolo[4,3-d]pyrimidin-7-one derivative 2a led to the identification of two compounds with oral activity in rodent feeding models (2h and 4a). Replacement of the PP group in 2h with other bicyclic groups resulted in a loss of binding affinity.

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes.

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.