GPR55 is a rhodopsin-like (Class A) G protein-coupled receptor (GPCR), highly expressed in human striatum (1
), that has the potential to become an important therapeutic target (Genbank accession # NM-005683; see ). Characterizations of GPR55(−/−) knock-out mice (2
) reveal a role for GPR55 in inflammatory pain, neuropathic pain, and bone development, while other studies indicate that GPR55 activation is pro-carcinogenic(4
). The GPCR proteins most homologous to GPR55 are GPR35 (27%), P2Y (29%), GPR23 (30%), and CCR4 (23%) (1
), but GPR55 also exhibits low amino acid identity to cannabinoid CB1 (13.5%) and CB2 (14.4%) receptors. This homology to cannabinoid receptors and the potential utility of marijuana derivatives in therapy prompted a search for GPR55 ligands among known cannabinoid receptor compounds. Thus, some synthetic and natural cannabinoids were among the first chemicals recognized to bind GPR55 (7
Helix net representation of the human GPR55 sequence.
Subsequent studies of GPR55 pharmacology suggested that lysophosphatidylinositol (LPI; 1
) compounds are endogenous GPR55 agonists (8
), with 2-AGPI, a 2-arachidonoyl containing LPI species, possessing significantly higher potency and maximal efficacy of the LPI species observed to date (9
). 2-AGPI may represent the true natural ligand of GPR55. In this manuscript we reference efficacy relative to the commercially available LPI (1
). Using a β-arrestin green fluorescent protein biosensor to assess a cohort of CB1/CB2 ligands for GPR55 activity, Kapur et al
) confirmed LPI as a GPR55 agonist, while observing that the cannabinoid antagonists AM251 and SR141716A were also GPR55 agonists. These GPR55 ligands possess comparable efficacy in inducing β-arrestin trafficking, and moreover, activate the G-protein dependent signaling of PKCβII. Conversely, the potent synthetic cannabinoid agonist CP55940 acts as a GPR55 antagonist/partial agonist, inhibiting GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2 (10
More potent and selective GPR55 ligands have been unavailable predominantly because many of the known GPR55 ligands were identified from cannabinoid receptor/lipid biased compound libraries (11
). The discovery and molecular characterization of better GPR55 chemotypes is an important next step towards the design of higher quality GPR55 ligands that can serve either as research tools or as a basis for designing novel drugs. During a collaborative project between our individual laboratories and the Sanford-Burnham screening center of the Molecular Libraries Probe Production Centers Network (MLPCN), we identified a series of GPR55 agonists that belong to novel, unreported GPR55 agonist chemotypes. These were discovered by a high content, high throughput β-arrestin screen (see http://mli.nih.gov/mli/mlp-probes/
). In this manuscript we focus on three GPR55 agonists identified in this screen: CID1792197 (2
), CID1172084 (ML185, 3
), and (CID2440433; ML184, 4
) (see for compound drawings).
The structures of GPR55 agonists (1–4) and control compound (5) are illustrated here.
Only a few Class A GPCRs have been crystallized to date. These include Rhodopsin (Rho) (13
), the β2
-adrenergic receptor (β2
-adrenergic receptor (β1
), adenosine A2A receptor (20
) and most recently the CXCR4 (21
) and dopamine D3 (22
) receptors. These crystal structures reveal a common topology that includes: (1) an extracellular N terminus; (2) seven transmembrane alpha helices (TMHs) arranged to form a closed bundle; (3) loops connecting TMHs that extend intra- and extracellularly; and (4) an intracellular C terminus that begins with a short helical segment (Helix 8) oriented parallel to the membrane surface. Although no crystal structure is available for GPR55, it is possible to build a homology model of GPR55 based on available crystal structures and refine this model based on sequence dictated differences between GPR55 and the template crystal structure. This refined model can then be used to explore the GPR55 ligand binding pocket and key amino acid interactions for GPR55 ligands. We report here the development of a refined homology model of the GPR55 activated state (GPR55 R*) and its subsequent use to explore the interaction between GPR55 and the novel CID agonists (2
) identified in our screen. Our modeling data indicate that the similarity between the CID compounds and LPI enables them all to be recognized by a single GPR55 binding pocket. The chemical diversity provided by these three lead compounds, CID (2
), combined with the identification of key receptor interaction sites should provide a basis for the design of more potent and efficacious second generation GPR55 ligands that retain GPR55 selectivity.