High Throughput Screening for Activators and Inhibitors of GPR56 (ADGRG1)
Authors: Vizurraga Alexander Luis , Stoveken Hannah , McEwen Dyke , Tall Gregory 
Published: June 2021

GPR56 (ADGRG1) is a member of a subset of G protein coupled receptors called adhesion GPCRs (AGPCRs), comprising 33 different receptors. AGPCRs uniquely possess an extracellular GPCR autoproteolysis‐incuding (GAIN) domain proximal to the N‐terminus of the first transmembrane span (TM1). This domain alone is sufficient to catalyze an autoproteolysis event, splitting the receptor into an extracellular N‐terminal fragment (NTF), and a membrane‐intercalated C‐terminal fragment (CTF), bound together non‐covalently. Dissociation of the NTF via possible shear force(s) exposes a short extracellular sequence of the CTF termed the tethered‐peptide‐agonist. Exposure of the tethered‐peptide‐agonist allows it to bind to its orthosteric binding pocket, thereby activating G‐protein signaling. GPR56 is a model AGPCR that couples to G12/13 family G‐proteins. Its dysfunction causes the neurodevelopmental disorder bilateral frontoparietal polymicrogyria (BFPP). Pilot small molecule high throughput screens were conducted to find agonists and antagonists of GPR56. HEK293T cells were transfected with either an engineered receptor with a truncated tethered‐peptide‐agonist (GPR56 A386M) or a fully‐active receptor with an intact tethered‐peptide‐agonist (GPR56 7TM) to screen for agonists and antagonists, respectively. The serum response element luciferase (SRE‐Luc) gene reporter was co‐transfected to provide an activity readout of G‐protein signaling in response to various small molecules. Hits from the screen were vetted in counterscreens; inhibitors were counterscreened with cells expressing constitutively active Ga13‐Q226L, and agonists were counterscreened with cells transfected with the reporter alone. This screen identified a first‐in‐class GPR56 partial agonist, 3‐a‐acetoxydihydrodeoxygedunin (3aDOG) and an antagonist, dihydromunduletone (DHM). Our current work is expanding to a large‐scale screens of over 180,000 total compounds at the Center for Chemical Genomics (CCG). Large‐scale 293T transfections (~1 billion cells) were extensively optimized for signal‐to‐noise ratios by calculating Z′ assay quality scores. Cells with optimal Z′‐scores were screened in conjunction with the CCG. Screens began with the Maybridge library of 24k compounds and the Natural Product Extracts library of 35k extracts. From these screens, a small pool of high‐activity activators and inhibitors were vetted in counterscreening. Hits that survived counterscreen testing were verified by directed dual luciferase assays and an in vitro GPCR reconstitution assay. We are currently working on screening the ChemDiv set of over 120,000 compounds. Knowledge of these receptors is still sparse; the discovery of high affinity agonists or antagonists will be useful probes to explore AGPCR activities in tissue and organ systems. These compounds may also serve as leads for development of AGPCR‐directed therapeutic compounds.

Support or Funding Information

GM120110, NS103946, T32‐GM007315 (NIH CMB Training Grant)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.