Astraea Therapeutics, Mountain View, California (J.J.L., W.E.P., N.T.Z.); and Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany (A.M., P.D., S.S.).
Astraea Therapeutics, Mountain View, California (J.J.L., W.E.P., N.T.Z.); and Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany (A.M., P.D., S.S.)
Mol Pharmacol. 2021 Jul;100(1):7-18. doi: 10.1124/molpharm.120.000076. Epub 2021 May 6.
Agonists at the nociceptin opioid peptide receptor (NOP) are under investigation as therapeutics for nonaddicting analgesia, opioid use disorder, Parkinson's disease, and other indications. NOP full and partial agonists have both been of interest, particularly since NOP partial agonists show a reduced propensity for behavioral disruption than NOP full agonists. Here, we investigated the in vitro pharmacological properties of chemically diverse NOP receptor agonists in assays measuring functional activation of the NOP receptor such as guanosine 5'-O-[gamma-thio]triphosphate (GTPS) binding, cAMP inhibition, G protein-coupled inwardly rectifying potassium (GIRK) channel activation, phosphorylation, β-arrestin recruitment and receptor internalization. When normalized to the efficacy of the natural agonist nociceptin/orphanin FQ (N/OFQ), we found that different functional assays that measure intrinsic activity produce inconsistent levels of agonist efficacy, particularly for ligands that were partial agonists. Agonist efficacy obtained in the GTPS assay tended to be lower than that in the cAMP and GIRK assays. These structurally diverse NOP agonists also showed differential receptor phosphorylation profiles at the phosphosites we examined and induced varying levels of receptor internalization. Interestingly, although the rank order for β-arrestin recruitment by these NOP agonists was consistent with their ability to induce receptor internalization, their phosphorylation signatures at the time point we investigated were not indicative of the levels of β-arrestin recruitment or internalization induced by these agonists. It is possible that other phosphorylation sites, yet to be identified, drive the recruitment of NOP receptor ensembles and subsequent receptor trafficking by some nonpeptide NOP agonists. These findings potentially help understand NOP agonist pharmacology in the context of ligand-activated receptor trafficking. SIGNIFICANCE STATEMENT: Chemically diverse agonist ligands at the nociceptin opioid receptor G protein-coupled receptor showed differential efficacy for activating downstream events after receptor binding, in a suite of functional assays measuring guanosine 5'-O-[gamma-thio]triphosphate binding, cAMP inhibition, G protein-coupled inwardly rectifying protein channel activation, β-arrestin recruitment, receptor internalization and receptor phosphorylation. These analyses provide a context for understanding nociceptin opioid peptide receptor (NOP) agonist pharmacology driven by ligand-induced differential NOP receptor signaling.
阿片类药物受体（NOP）的激动剂正在被研究作为非成瘾性镇痛、阿片类药物使用障碍、帕金森病和其他适应症的治疗方法。NOP 完全激动剂和部分激动剂都引起了人们的兴趣，特别是因为 NOP 部分激动剂显示出比 NOP 完全激动剂更低的行为中断倾向。在这里，我们研究了化学多样性 NOP 受体激动剂在测量 NOP 受体功能激活的测定中的体外药理学特性，例如鸟苷 5'-O-[γ-硫]三磷酸（GTPS）结合、cAMP 抑制、G 蛋白偶联内向整流钾（GIRK）通道激活、磷酸化、β-抑制蛋白募集和受体内化。当与天然激动剂孤啡肽/孤啡肽 FQ（N/OFQ）的效力归一化时，我们发现，测量内在活性的不同功能测定产生了不一致的激动剂效力水平，特别是对于那些部分激动剂的配体。在 GTPS 测定中获得的激动剂效力往往低于 cAMP 和 GIRK 测定。这些结构不同的 NOP 激动剂在我们检查的磷酸化位点上也表现出不同的受体磷酸化谱，并诱导不同水平的受体内化。有趣的是，尽管这些 NOP 激动剂募集β-抑制蛋白的顺序与它们诱导受体内化的能力一致，但在我们研究的时间点上，它们的磷酸化特征并不能说明这些激动剂诱导的β-抑制蛋白募集或内化水平。有可能是其他尚未确定的磷酸化位点驱动了一些非肽类 NOP 激动剂的 NOP 受体集合的募集和随后的受体转运。这些发现有助于在配体激活的受体转运的背景下理解 NOP 激动剂药理学。意义声明：阿片类药物受体（NOP）的化学多样性激动剂配体在一系列测量 GTPS 结合、cAMP 抑制、G 蛋白偶联内向整流蛋白通道激活、β-抑制蛋白募集、受体内化和受体磷酸化的功能测定中，对激活受体结合后下游事件的效力显示出差异。这些分析为理解由配体诱导的 NOP 受体信号转导驱动的 NOP 肽受体（NOP）激动剂药理学提供了一个背景。
