PT  - JOURNAL ARTICLE
AU  - Hannah M Stoveken
AU  - Laura L Bahr
AU  - M W Anders
AU  - Andrew P Wojtovich
AU  - Alan V Smrcka
AU  - Gregory G Tall
TI  - Dihydromunduletone is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.
AID  - 10.1124/mol.116.104828
DP  - 2016 Jan 01
TA  - Molecular Pharmacology
PG  - mol.116.104828
4099  - http://molpharm.aspetjournals.org/content/early/2016/06/23/mol.116.104828.short
4100  - http://molpharm.aspetjournals.org/content/early/2016/06/23/mol.116.104828.full
AB  - Adhesion GPCRs (aGPCRs) have emerging roles in development and tissue maintenance and are the most prevalent GPCR sub-class mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCRs have large extracellular domains (ECDs) containing a conserved sub-domain that mediates constitutive self-cleavage at a site near the start of the 7-transmembrane domain (7TM). The two resultant receptor protomers, ECD or N-terminal fragment (NTF), and the 7TM or C-terminal fragment remain non-covalently bound at the plasma membrane in an inactive receptor state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix (ECM) proteins that bind the NTF and may act to dissociate it to reveal the tethered agonist. Given the perceived difficulty in working with ECM proteins to create pharmacological aGPCR probes, we developed a SRE-luciferase-based high-throughput screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000 compound library comprising known drugs and natural products was screened for inhibitors of GPR56-dependent SRE activation that did not inhibit constitutively active Gα13-dependent SRE activation. Three compounds were identified and Dihydromunduletone (DHM), a rotenoid derivative, was selected for further validation using cell-free aGPCR/heterotrimeric G protein GTPγS binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5 which have similar tethered agonists, but did not inhibit the aGPCR GPR110/ADGRF1, Class A M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered- or synthetic-peptide agonist-stimulated GPR56, but did not inhibit basal GPR56 activity, demonstrating that it antagonizes the peptide agonist. These data demonstrate identification of DHM as a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic.