Discovery and Binding Mechanism of Pyrazoloisoquinoline-Based Novel β‑Arrestin Inverse Agonists of the Kappa-Opioid Receptor

Chronic exposure to stress or unwanted stimuli has been known to activate kappa opioid receptor/dynorphin (KOR/DYN) systems, which could induce depressive states and develop into some psychiatric disorders. Here, we report the first discovery of pyrazoloisoquinoline-based novel KOR β-arrestin inverse agonists through synthesis, structure–activity relationships, optimization, and the biological evaluations of μ/κ/δ opioid receptor activities with cAMP and β-arrestin recruitment assays. The optimized compound 7q shows potent and selective β-arrestin inverse agonism at KOR with an EC50 value of 9.33 nM in contrast to lower activities at DOR and no activity at MOR. Moreover, we use molecular dynamics simulations to predict the binding mode of the inverse agonist and propose a mechanism for the inverse agonism. We find that the transmembrane helix 6 position of the activated state is different for the OR subtypes, leading to significantly different interactions between the receptor and β-arrestin.

已有研究表明，长期暴露于应激或有害刺激可激活κ阿片受体/强啡肽（kappa opioid receptor/dynorphin, KOR/DYN）系统，该系统可诱发抑郁状态并进展为部分精神类疾病。本研究首次发现基于吡唑并异喹啉骨架的新型KOR β抑制蛋白反向激动剂，通过合成、构效关系分析、化合物优化以及采用环磷酸腺苷（cyclic adenosine monophosphate, cAMP）和β抑制蛋白募集实验，对μ、κ、δ阿片受体的活性开展了生物学评价。经优化得到的化合物7q可在KOR上展现出强效且具有选择性的β抑制蛋白反向激动活性，其半数有效浓度（half maximal effective concentration, EC50）为9.33 nM；与之相比，该化合物对δ阿片受体（delta opioid receptor, DOR）活性较弱，对μ阿片受体（mu opioid receptor, MOR）则无活性。此外，本研究通过分子动力学模拟预测了该反向激动剂的结合模式，并提出了其反向激动的作用机制。本研究发现，激活状态下的跨膜螺旋6（transmembrane helix 6, TM6）位置在不同阿片受体亚型间存在差异，进而导致受体与β抑制蛋白之间的相互作用存在显著差异。