Covalent Allosteric Probe for the Metabotropic Glutamate Receptor 2: Design, Synthesis, and Pharmacological Characterization

[Image: see text] Covalent labeling of G protein-coupled receptors (GPCRs) by small molecules is a powerful approach to understand binding modes, mechanism of action, pharmacology, and even facilitate structure elucidation. We report the first covalent positive allosteric modulator (PAM) for a class C GPCR, the mGlu(2) receptor. Three putatively covalent mGlu(2) PAMs were designed and synthesized. Pharmacological characterization identified 2 to bind the receptor covalently. Computational modeling combined with receptor mutagenesis revealed T791(7.29×30) as the likely position of covalent interaction. We show how this covalent ligand can be used to characterize the PAM binding mode and that it is a valuable tool compound in studying receptor function and binding kinetics. Our findings advance the understanding of the mGlu(2) PAM interaction and suggest that 2 is a valuable probe for further structural and chemical biology approaches.