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.

中文翻译：

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靶向蛋白质组学与亲和质谱分析相结合揭示了拮抗剂E7充当孤儿受体GPR52的细胞内共价配体

GPR52是一种A类孤儿G蛋白偶联受体（GPCR），被认为是治疗亨廷顿氏病和多种精神病的有希望的治疗靶标。尽管最近解决的GPR52结构揭示了所有报道的激动剂可能共有的结合机制，但小分子拮抗剂E7不能放入该激动剂结合袋中，其与受体的相互作用方式仍然未知。在这里，我们采用了靶向蛋白质组学和亲和质谱方法来揭示E7的独特结合模式，该模式可以作为GPR52的共价和变构配体。在这项研究中鉴定出的三个与Cys形成共价结合物的Cys残基中，细胞内C156 ^4.40对E7的拮抗活性做出了最重要的贡献。发现用于拮抗剂共价连接的这种新的细胞内位点将促进GPR52-选择性负变构调节剂的设计，其可以用作治疗亨廷顿氏病的潜在疗法。