Targeted Proteomics Combined with Affinity Mass Spectrometry Analysis Reveals Antagonist E7 Acts As an Intracellular Covalent Ligand of Orphan Receptor GPR52

The GPR52, a class A orphan G protein-coupled receptor (GPCR), is regarded as a promising therapeutic target for the treatment of Huntington's disease and multiple psychiatric disorders. Although the recently solved structure of GPR52 has revealed a binding mechanism likely shared by all reported agonists, the small molecule antagonist E7 cannot fit into this agonist-binding pocket, and its interaction mode with the receptor remains unknown. Here, we employed targeted proteomics and affinity mass spectrometry approaches to uncover a unique binding mode of E7 which acts as a covalent and allosteric ligand of GPR52. Among three Cys residues identified in this study to form covalent conjugates with E7, the intracellular C156^4.40 makes the most significant contribution to the antagonism activity of E7. Discovery of this novel intracellular site for covalent attachment of an antagonist would facilitate the design of GPR52-selective negative allosteric modulators which could serve as potential therapeutics for treating Huntington's disease.