Affinity mass spectrometry (MS) enables rapid screening of compound mixtures for ligands bound to a specific protein target, yet its current throughput is limited to individually assay pools of 400−2000 compounds. Typical affinity MS screens implemented in pharmaceutical industry laboratories identify putative ligands based on qualitative analysis of compound binding to the target whereas no quantitative information is acquired to discriminate high-and low-affinity ligands in the screening phase. Furthermore, these screens require purification of a stabilized form of the protein target, which poses a great challenge for membrane receptor targets. Here, we describe a new, potentially general affinity MS strategy that allows screening of 20,000 compounds in one pool for highly efficient ligand discovery toward a G protein-coupled receptor (GPCR) target. Quantitative measurement of compound binding to the receptor enables highaffinity ligand selection using both the purified receptor and receptor-embedded cell membranes. This high-throughput, label-free and quantitative affinity MS screen resulted in discovery of three new antagonists of the A 2A adenosine receptor.

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 C1564.40 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.

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