Identification of the GPR55 Antagonist Binding Site Using a Novel Set of High-Potency GPR55 Selective Ligands

Abstract

GPR55 is a Class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as L-α-lysophosphatidylinositol (LPI). While there is increasing evidence for physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ~300,000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50s in the 0.16 to 2.72 μM range (Heynen-Genel, et al., (2010) “Screening for Selective Ligands for GPR55 – Antagonists” [ML191, ML192, ML193] Bookshelf ID: NBK66153; PMID: 22091481). Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1 or CB2 up to 20 μM. Using a model of GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second generation GPR55 selective antagonists.

Structure-Activity Relationship of the GPR55 Antagonist, CID16020046

“…We address this by presenting a preliminary structure-activity relationship (SAR). We show that structural analogues of CID16020046 retain GPR55 antagonist activity, and we interpret the SAR in the context of a published GPR55 structural model [11]. Finally, we test ML193 and CID16020046 in parallel and corroborate antagonist activity of ML193.…”

“…CID16020046 was independently identified in an antagonist screen utilising U2OS (Human Osteosarcoma) cells stably expressing GFP-tagged β-arrestin and over-expressing hGPR55, measuring agonist-induced β-arrestin translocation by imaging [38]. Kotsikorou et al [11] docked CID16020046 into a model of the inactive form of hGPR55. They predicted the lactam carbonyl, which has the highest electrostatic potential in CID16020046, to hydrogen bond with residue K2.60(80), which is the only positively charged residue in the putative binding site.…”

“…Hence, an informative approach has been to study the effects of LPI on cells and tissues in combination with synthetic GPR55 antagonists identified from screening. ML193 and CID16020046 are antagonists from distinct structural classes that have found widespread use in studies of GPR55 function [11,12]. ML193 blocks LPI-evoked calcium transients in cultured neonatal cardiomyocytes [13].…”

GPR55: a new promising target for metabolism?

“…Energy homeostasis is mainly determined by the balance between energy intake (from food ingestion) and energy expenditure (from basal metabolic rate, thermogenesis and physical activity). To decipher the effects of central GPR55 signaling on food consumption, a number of pharmacological studies have been performed in animal models; however, many of them employed cannabinoid receptor/lipid biased compound libraries, which lead to lack of receptor selectivity (Kotsikorou et al 2013). For instance, rats that followed a treatment with CBD displayed reduced body weight gain and food intake; however, those effects were not detected when animals also received AM630, a selective CB2 antagonist, suggesting that the CB2 receptor might be involved in the outcomes previously observed (Ignatowska-Jankowska et al 2011).…”

Integrated Approaches for Genome-wide Interrogation of the Druggable Non-olfactory G Protein-coupled Receptor Superfamily

“…For example, PubChem lists screens for a large number of GPCRs and, from these screens, results for a handful of orphan GPCRs have been published in peer-reviewed journals (29 -31). These published findings have led to the discovery that pamoic acid is a potent agonist for GPR35 via ␤-arrestin signaling (31), as well as the discovery of novel agonists and antagonists for GPR55 (29,30).…”