The M₄ muscarinic acetylcholine receptor (M₄ mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M₄ mAChR in complex with the heterotrimeric G_i1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M₄ mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M₄ mAChR. These findings provide a basis for further understanding xanomeline’s complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.

M ₄毒蕈碱乙酰胆碱受体 (M ₄ mAChR) 已成为具有高度治疗意义的药物靶点，因为它在涉及精神病、认知和成瘾调节的大脑区域中表达。 mAChR 激动剂 xanomeline 在治疗精神分裂症患者的 II 期临床试验中显着改善了阳性和阴性症状量表 (PANSS) 评分。在这里，我们报告了 xanomeline 与异源三聚体 G _i1转导蛋白复合物与人 M ₄ mAChR 结合的活性状态冷冻电镜结构。出乎意料的是，发现两个 xanomeline 分子同时与单体 M ₄ mAChR 结合，其中一个分子结合在正构（乙酰胆碱结合）位点，第二个分子结合在细胞外前庭变构位点。分子动力学模拟支持了结构发现，药理学验证证实 xanomeline 作为人类 M ₄ mAChR 的双重正构和变构配体。这些发现为进一步了解 xanomeline 的复杂药理学奠定了基础，并强调了临床相关配体结合和调节 GPCR 的多种方式。