School of Life Sciences, The Medical School, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, Nottingham, UK.
Methods Mol Biol. 2021;2268:249-274. doi: 10.1007/978-1-0716-1221-7_17.
An understanding of the kinetic contributions to G protein-coupled receptor pharmacology and signaling is increasingly important in compound profiling. Nonequilibrium conditions are commonly present in vivo, for example, as the drug competes with dynamic changes in hormone or neurotransmitter concentration for the receptor. Under such conditions individual binding kinetic properties of the ligands can influence duration of action, local ligand concentration, and functional properties such as the degree of insurmountable inhibition. Mapping the kinetic patterns of GPCR signaling events elicited by agonists, rather than a peak response at a single timepoint, is often key to predicting their functional impact. This is also a path to a better understanding of the origins of ligand bias, and whether such ligands demonstrate their effects through selection of distinct GPCR conformations, or via their kinetic properties. Recent developments in complementation approaches, based on a small bright shrimp luciferase Nanoluc, provide a new route to kinetic analysis of GPCR signaling in living cells that is amenable to the throughput required for compound profiling. In the NanoBiT luciferase complementation system, GPCRs and effector proteins are tagged with Nanoluc fragments optimized for their low interacting affinity and stability. The interactions brought about by GPCR recruitment of the effector are reproduced by a rapid and reversible increase in NanoBiT luminescence, in the presence of its substrate furimazine. Here we discuss the methods for optimizing and validating the GPCR NanoBiT assays, and protocols for their application to study endpoint and kinetic aspects of agonist and antagonist pharmacology. We also describe how timecourse families of agonist concentration response curves, derived from a single NanoBiT assay experiment, can be used to evaluate the kinetic components in operational model derived parameters of ligand bias.
理解动力学对 G 蛋白偶联受体药理学和信号转导的贡献在化合物分析中变得越来越重要。例如，在体内通常存在非平衡条件，因为药物与激素或神经递质浓度的动态变化竞争受体。在这种情况下，配体的个体结合动力学特性会影响作用持续时间、局部配体浓度以及功能特性，如不可逾越抑制的程度。绘制激动剂引起的 GPCR 信号事件的动力学模式，而不是在单个时间点的峰值反应，通常是预测其功能影响的关键。这也是更好地理解配体偏倚的起源的途径，以及这些配体是否通过选择不同的 GPCR 构象或通过其动力学特性来发挥其作用。基于小的亮虾荧光素 Nanoluc 的互补方法的最新进展，为在活细胞中进行 GPCR 信号动力学分析提供了一条新途径，该方法适用于化合物分析所需的通量。在 NanoBiT 荧光素互补系统中，GPCR 和效应蛋白被标记有优化的 Nanoluc 片段，以实现其低相互作用亲和力和稳定性。在其底物 furimazine 的存在下，通过快速和可逆地增加 NanoBiT 发光，重现由 GPCR 招募效应蛋白引起的相互作用。在这里，我们讨论了优化和验证 GPCR NanoBiT 测定的方法，以及将其应用于研究激动剂和拮抗剂药理学的终点和动力学方面的方案。我们还描述了如何从单个 NanoBiT 测定实验中获得的激动剂浓度反应曲线的时间曲线家族，用于评估配体偏倚的操作模型衍生参数中的动力学成分。
