Discovery of Small Molecule Adhesion GPCR Modulators

Abstract

GPR56 (ADGRG1) is a member of a subset of G protein coupled receptors called adhesion GPCRs (AGPCRs). These GPCRs are unique in their possession of an autoproteolytic extracellular domain that cleaves the receptors as a prerequisite to activation. GPR56 couples to G12/13 family G proteins and we recently discovered that it functions as a platelet collagen receptor that is important for hemostasis. We hypothesize that GPR56 is an early-acting collagen sensor for platelets – enabling them to first recognize blood vessel injury and to initiate the platelet program that leads to repair. Despite being biologically relevant in a number of cellular processes and diseases, there is a distinct lack of available small molecule tool compounds for AGPCRs, including GPR56. We conducted a large-scale high-throughput screen to find agonists and antagonists of GPR56. Over 150,000 small molecules and extracts were screened at UM's Center for Chemical Genomics (CCG) using a cell-based serum response element (SRE) luciferase gene reporter assay in HEK293T cells. Primary screening yielded 47 initial candidate agonists that activated GPR56 in secondary cell-based reporter and biochemical GPCR reconstitution assays. GPR56 reconstitution assays with pure G proteins further revealed that 19 of these compounds caused at least a 5-fold increase over control in G protein GTPγS binding. A notable structural cluster was identified among these molecules with some members exhibiting a 10-fold increase in potency (~30 μM to ~3 μM EC50) and equivalent efficacy compared to the full agonist positive control, a synthetic peptide mimetic of the GPR56 tethered-peptide-agonist. Follow-up GPR56 activation assays investigating the structure-activity relationship (SAR) of these pharmacophores are being pursued in conjunction with orthogonal platelet activity assays. Knowledge of AGPCR mechanisms and pathologies is rapidly emerging. As such, the discovery of high affinity agonists and antagonists will be useful probes to explore their activities in endogenous contexts. These compounds may also serve as leads for the development of GPR56-targeted pro- or anticoagulants.