Kinetics of Ligand Binding in Dopamine D1 Receptor Biased Signaling

脑神经通路中多巴胺（DA）神经传递不足是脑缺血的病理生理基础。 许多致残性神经和精神疾病，包括物质使用障碍、注意力缺陷 精神分裂症中的多动症和认知障碍。DA D1受体（D1 R）是一种G蛋白- 偶联受体（GPCR）被认为在运动控制、奖赏、注意和工作中起核心作用 记忆虽然D1 R的激活可以为治疗不同的大脑提供有价值的治疗策略， 疾病，建立的儿茶酚配体的不期望的性质已经阻止了治疗疾病的发展， 超过40年我们的研究小组最近解决了这个儿茶酚问题，并报告了第一个非儿茶酚D1 R 具有前所未有的药物样特性的选择性激动剂。包括D1 R在内的许多GPCR可以发出信号， 不仅通过G蛋白，而且通过与其他信号蛋白的G蛋白独立的相互作用 包括最显著的β-抑制蛋白。出乎意料的是，几种新的非儿茶酚D1 R激动剂显示出 通过G蛋白的偏向信号传导活性，而不参与β-抑制蛋白。这种G蛋白偏向信号传导， 新的D1 R激动剂可以导致相对于无偏D1 R的上级激活和/或降低的副作用特征 激动剂，提供了创新的机会，微调D1 R活性的神经治疗。从历史上看，GPCR 配体已经基于其效力、功效和特异性进行了优化;然而，另一个关键参数 影响受体信号传导的是配体与受体结合的持续时间（即，结合动力学）。我们 假设配体结合的持续时间是决定选择性免疫应答的信号偏差的关键机制。 针对G蛋白的D1 R激动剂与β-抑制蛋白信号传导。该研究项目的目标是验证和 定量这些新型D1 R激动剂的偏向激动剂活性，并评估更快的配体-受体结合是否 动力学是驱动偏置信号传导的机制。目标1将采用多种方法，包括功能性 D1 R信号传导测定、竞争和动力学结合测定，以确定D1 R激动剂的信号传导偏倚的等级 以及无偏和有偏激动剂的动力学结合参数（Koff，Kon）。然后我们将这些 激动剂与β-抑制蛋白介导的脱敏信号传导结果的动力学结合参数， 内化目的2将确定更快的结合动力学是否驱动体内非儿茶酚D1 R激动剂功效。我们 将评估来自小鼠的无偏倚和偏倚非儿茶酚D1 R激动剂的动力学结合参数（Koff，Kon） 纹状体膜，并比较D1 R激动剂对可卡因诱导的运动行为的功能功效 使用野生型和β-arrestin 2敲除小鼠。这个研究项目将扩大我们高度有限的欣赏 配体的性质和机制D1 R偏信号。如果我们发现激动剂的作用时间 结合是偏D1 R信号传导的基础机制，我们将提供一个定义和可测量的配体 性质，以帮助设计偏向激动剂。该项目还将验证相关性和潜力 G蛋白偏向激动对D1 R功能的有用性。

Inadequate dopamine (DA) neurotransmission in brain neural pathways is a pathophysiological underpinning of many disabling neurologic and psychiatric illnesses including substance use disorders, attention deficit hyperactivity disorder and cognitive impairments in schizophrenia. The DA D1 receptor (D1R) is a G protein- coupled receptor (GPCR) identified as playing a central role in motor control, reward, attention and working memory. While activation of the D1R could provide a valuable therapeutic strategy for treating diverse brain disorders, undesirable properties of established catechol ligands have prevented therapeutic development for over 40 years. Our research group recently solved this catechol problem and reported the first non-catechol D1R selective agonists that have unprecedented drug-like properties. Many GPCRs, including the D1R, can signal not only through G proteins, but also via G protein-independent interactions with other signaling proteins including, most prominently, β-arrestins. Unexpectedly, several of the novel non-catechol D1R agonists show biased signaling activity via G proteins, without engagement of β-arrestins. This G protein biased signaling by novel D1R agonists may result in superior activation and/or reduced side effect profiles relative to unbiased D1R agonists, providing the innovative opportunity to fine tune D1R activity for neurotherapeutics. Historically, GPCR ligands have been optimized based on their potency, efficacy and specificity; however, another crucial parameter that impacts receptor signaling is the duration of ligand binding to the receptor (i.e., binding kinetics). We hypothesize that the duration of ligand binding is a key mechanism that determines signaling bias of selective D1R agonists towards G proteins versus β-arrestin signaling. The goal of this research project is to validate and quantify the biased agonist activity of these novel D1R agonists and evaluate if faster ligand-receptor binding kinetics is a mechanism driving biased signaling. Aim 1 will use a combination of approaches including functional D1R signaling assays, competition and kinetic binding assays to define a scale of signaling bias for D1R agonists and the kinetic binding parameters (Koff, Kon) for unbiased and biased agonists. We will then correlate these kinetic binding parameters of agonists to β-arrestin-mediated signaling outcomes of desensitization and internalization. Aim 2 will determine if faster binding kinetics drive non-catechol D1R agonist efficacy in vivo. We will assess kinetic binding parameters (Koff, Kon) for unbiased and biased non-catechol D1R agonists from mouse striatal membranes and compare the functional efficacy of D1R agonists on cocaine-induced locomotor behavior using wildtype and β-arrestin2 knockout mice. This research project will expand our highly limited appreciation of ligand properties and mechanisms governing D1R biased signaling. If we discover that the duration of agonist binding is a mechanism underlying biased D1R signaling, we will provide a defining and measurable ligand property to aid in the design of biased agonists. This project will also validate the relevance and potential usefulness of G protein biased agonism for D1R function.