Pharmacology of GPR55 in Yeast and Identification of GSK494581A as a Mixed-Activity Glycine Transporter Subtype 1 Inhibitor and GPR55 Agonist

Abstract

GPR55 is a G protein-coupled receptor activated by L-␣-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB 1 and CB 2 receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4Ј-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure-activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4Ј-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl) aniline), which is approximately 60-fold selective for GPR55 (pEC 50 ϭ 6.8) over GlyT1 (pIC 50 ϭ 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.

A Selective Antagonist Reveals a Potential Role of G Protein–Coupled Receptor 55 in Platelet and Endothelial Cell Function

“…Derivation of yeast strains and yeast reporter gene assays was described previously (Olesnicky et al, 1999;Brown et al, 2003Brown et al, , 2011. In brief, yeast strain YIG151 contains FLAG-GPR55 and Gpa1/G a13 chimeric G-protein a-subunit, both integrated chromosomally.…”

“…In addition, several synthetic CB 1 receptor inverse agonists/ antagonists, such as 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM251), 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281), and rimonabant (SR141716A; 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), have been shown to activate GPR55 (Oka et al, 2007;Ryberg et al, 2007;Henstridge et al, 2009Henstridge et al, , 2010Kapur et al, 2009;Yin et al, 2009;Brown et al, 2011). Although several cannabinoid ligands can activate GPR55, the receptor lacks the classic "cannabinoid binding pocket" (Kotsikorou et al, 2011).…”

“…The lipid ligand LPI; the CB 1 receptor inverse agonists/ antagonists SR141716A, AM251, and AM281; and selective GPR55 agonists were described to activate transcription factors in HEK-293 cells transiently or stably expressing human GPR55 (Ryberg et al, 2007;Henstridge et al, 2009Henstridge et al, , 2010Brown et al, 2011;Kargl et al, 2012a). The phytocannabinoid cannabidiol (CBD) was reported to antagonize O-1602-mediated GPR55 activation in HEK-GPR55 cells and human osteoclasts (Ryberg et al, 2007;Whyte et al, 2009).…”

The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55

“…So far, the most potent and endogenous GPR55 ligand is L-␣-lysophosphatidylinositol (LPI) (22,25,26,33). In addition, three groups recently described several potent and selective GPR55 agonists and antagonists (24,34,35). These new compounds are reported to be inactive at CB1 and CB2 receptors and are promising tools to elucidate the pharmacology and (patho-) physiological functions of GPR55.…”

“…In addition, numerous synthetic CB1R inverse agonists/ antagonists have been described (30,31), among which SR141716A (rimonabant) recently gained attention as a promising treatment for obesity and smoking cessation (32). Interestingly, the CB1R inverse agonists/antagonists, SR141716A, AM251, and AM281, act as agonists on GPR55 (9,22,24,33). So far, the most potent and endogenous GPR55 ligand is L-␣-lysophosphatidylinositol (LPI) (22,25,26,33).…”