A selective cysteinyl leukotriene receptor 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice.

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

High throughput screening for orphan and liganded GPCRs.

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.

Potential regulatory roles for G protein-coupled receptor kinases and beta-arrestins in gonadotropin-releasing hormone receptor signaling.

GnRH stimulates gonadotropin secretion, which desensitizes unless the releasing hormone is secreted or administered in a pulsatile fashion. The mechanism of desensitization is unknown, but as the GnRH receptor is G protein coupled, it might involve G protein-coupled receptor kinases (GRKs). Such kinases phosphorylate the intracellular regions of seven-transmembrane receptors, permitting beta-arrestin to bind, which prevents the receptor from activating G proteins. Here, we tested the effect of GRKs and beta-arrestins on GnRH-induced inositol trisphosphate (IP3) production in COS cells transfected with the GnRH receptor complementary DNA. GRK2, -3, and -6 overexpression inhibited IP3 production by 50-75% during the 30 sec of GnRH treatment. Coexpression of GRK2 and beta-arrestin-2 suppressed GnRH-induced IP3 production more than that of either alone. Immunocytochemical staining of rat anterior pituitary revealed that all cells expressed GRK2, -3, and -6; all cells also expressed the beta-arrestins. Western blots on cytosolic extracts of rat pituitaries revealed the presence of GRK2/3 and beta-arrestin-1 and -2. The expression of GRKs and beta-arrestins by gonadotropes and their inhibition of GnRH-stimulated IP3 production in COS-1 cells expressing the GnRH receptor suggest a potential regulatory role for the GRK/beta arrestin paradigm in GnRH receptor signaling.