Dihydromunduletone (DHM) is an adhesion G protein-coupled receptor (aGPCR) antagonist that acts as a chemical probe for the inhibition of GPR56 and GPR114/ADGRG5, which have similar tethered agonists.

Assays are initiated by the addition of [35S]GTPγS, and the rates of aGPCR-stimulated G protein activation (Gα binding to [35S]GTPγS) are measured with or without the influence of added compounds. Dihydromunduletone (DHM) inhibits the kinetics of GPR56 7TM-stimulated G13 GTPγS binding to varying degrees. Among the inhibitory compounds tested, Dihydromunduletone is the most effective, reducing the rate at which GPR56 7TM activates G13 by over 75% (from 0.18 to 0.04 minute−1)[1].At a concentration of Dihydromunduletone (DHM) that maximally inhibits GPR56 (50 μM), the rate of GPR114 7TM-stimulated Gs activity is also dramatically inhibited. However, when Dihydromunduletone (50 μM) is applied to GPR110 7TM, it fails to inhibit GPR110 stimulation of Gq GTPγS binding[1].
Cells transfected with GPR56 A386M 7TM are incubated with increasing concentrations of Dihydromunduletone. After adding the P7 peptide agonist, SRE-luciferase activity is measured. Dihydromunduletone inhibits P7 peptide-induced luciferase activity in a concentration-dependent manner. Additionally, cells treated with a fixed concentration of 3 µM Dihydromunduletone and then stimulated with an increasing concentration of P7 peptide agonist show that Dihydromunduletone treatment blunts P7 peptide activation at each concentration. In conclusion, Dihydromunduletone antagonizes synthetic-peptide agonist and tethered-peptide agonist-mediated aGPCR activation in isolated membranes and HEK293T cell-based assays, but it does not inhibit basal receptor signaling[1].