Identification of a First‐In‐Class Adhesion G protein‐Coupled Receptor Orthosteric Antagonist

Adhesion GPCRs (aGPCRs) represent untapped therapeutic targets with roles in cancer progression and tissue development. aGPCRs have large, modular extracellular domains (ECDs) containing a G PCR A utoproteolysis In ducing (GAIN) domain that mediates self‐cleavage at a conserved site ~20 residues N‐terminal to transmembrane domain‐1 (TM1) of the 7‐transmembrane domain (7TM). The resultant receptor fragments (ECD and 7TM) remain non‐covalently bound at the plasma membrane in an inactive conformation, positioned for activation. We recently showed that extracellular fragment dissociation revealed the cryptic ~20 residue TM1 stalk, allowing it to act as a tethered agonist to activate the 7TM. We propose that natural aGPCR extracellular matrix (ECM) protein ligands, such as collagens and laminins, activate G protein signaling by binding and dissociating the extracellular fragment from the 7TM, resulting in tethered agonist decryption ( Figure 1). Given this novel aGPCR activation mechanism and the perceived lack of utility of ECM ligands, this project aims to identify aGPCR GPR56/ADGRG1 small molecule modulators for research and therapeutic use. We designed a high throughput screen and counter‐screen to search for inhibitors of the constitutively active GPR56 7TM domain using a G protein 13‐dependent S erum R esponse E lement (SRE)‐luciferase reporter assay. A 2000 compound library comprised primarily of known drugs and natural compounds (SPECTRUM collection) was screened for GPR56 inhibitors that did not inhibit constitutively active Gα13 Q226L. Sixty‐six potential GPR56 inhibitors were identified, with 64 neatly eliminated by the Gα13QL counterscreen (average Z′ score screen = 0.67, counterscreen = 0.8). Dihydromunduletone (DHM), one of the identified compounds, is a rotenone derivative, a class of compounds best known for their use as insecticides and piscicides, but which have never been reported to inhibit GPCR activity. Using our established aGPCR / heterotrimeric G protein GTPγS binding reconstitution assays as a means of secondary compound validation, we directly measured compound‐dependent inhibition of receptor‐mediated G protein activation with clear results. DHM inhibited GPR56 7TM: G protein activation (IC 50 ~21 μM) ( Figure 2A–B) and related GPR114, but not GPR110, nor the M3 muscarinic or β2 adrenergic receptors. Importantly, DHM inhibited synthetic‐ or tethered‐peptide agonist induced, but not basal GPR56 activity, strongly indicating that DHM is an orthosteric antagonist that directly blocks agonist action. DHM, as one of the first‐in‐class aGPCR antagonists, may find utility as a pharmacological lead for development of therapeutic aGPCR inhibitors. Support or Funding Information This work is supported by NIH grant R01GM088242B to G.G.T and PhRMA Foundation Pre‐Doctoral Fellowship to H.M.S. 1 Schematic of Proposed aGPCR Activation MechanismaGPCRs are constitutively cleaved at a site within the GAIN domain, and the two receptor pieces remain associated non‐covalently. When ligands bind the extracellular domain or when receptors are treated with urea, the ECD is removed, unmasking the tethered agonist. The tethered agonist is liberated to bind the 7TM orthosteric site and initiate G protein signaling cascades. DHM acts by competing with the tethered agonist for binding.2 DHM is a GPR56/ ADGRG1 Orthosteric Antagonista, DHM was applied to Hi5 cell membranes expressing constitutively active GPR56 7TM and binding of [ 35 S]‐GTPγS to Gα13 (activation) was measured. DHM inhibited the rate of receptor‐mediated G protein activation. b, Increasing concentrations of DHM were applied to GPR56 7TM Hi5 membranes and the initial rates of G protein activation were measured and plotted as a function of concentration. DHM inhibited GPR56 with an IC 50 ~21μM.