Kinetics of Ligand Binding in Dopamine D1 Receptor Biased Signaling

脑神经通路中多巴胺(DA)神经传递不足是脑血管疾病的病理生理基础 许多致残的神经和精神疾病，包括物质使用障碍、注意力缺陷 精神分裂症患者的多动障碍和认知障碍。DA D1受体(D1R)是一种G蛋白- 偶联受体被认为在运动控制、奖赏、注意力和工作中起着中心作用 记忆。而D1R的激活可以为治疗不同的大脑提供一种有价值的治疗策略 疾病，已建立的儿茶酚配体的不良性质阻碍了治疗的发展 40多年了。我们的课题组最近解决了这个儿茶酚问题，并报道了第一个非儿茶酚D1R 具有前所未有的类药物特性的选择性激动剂。包括D1R在内的许多GPCR可以发出信号 不仅通过G蛋白，还通过G蛋白与其他信号蛋白的非依赖性相互作用 最突出的是，包括反逮捕(β-Drastins)。出乎意料的是，几种新型非儿茶酚D1R激动剂显示 偏向的信号活性通过G蛋白，而不参与β-拦阻蛋白。这种G蛋白通过以下途径偏向信号 与无偏倚的D1R相比，新的D1R激动剂可以产生更好的激活和/或减少的副作用 激动剂，为神经疗法提供微调D1R活性的创新机会。从历史上看，gpcr 根据配基的效力、效力和特异性对其进行了优化；然而，另一个关键参数 影响受体信号传递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信号偏向的关键机制 D1R激动剂对G蛋白和β-arrestin信号的作用。本研究项目的目标是验证和 量化这些新的D1R激动剂的偏向激动剂活性，并评估更快的配体-受体结合 动力学是一种驱动偏向信号的机制。目标1将使用多种方法的组合，包括功能 D1R信号分析、竞争和动力学结合分析以确定D1R激动剂的信号偏向范围 以及无偏置和偏置激动剂的动力学结合参数(Koff，Kon)。然后我们将把这些关联起来 激动剂对β-arrestin介导的脱敏和信号转导结果的动力学结合参数 内部化。目的2将确定更快的结合动力学是否在体内驱动非儿茶酚D1R激动剂的疗效。我们 将评估无偏向和偏向非儿茶酚D1R激动剂的动力学结合参数(Koff，Kon) 纹状体膜和比较D1R激动剂对可卡因诱导的运动行为的作用效果 在2只基因敲除小鼠中使用野生型和β抑制。这项研究项目将扩大我们有限的鉴赏力 配基性质和支配D1R偏向信号的机制。如果我们发现激动剂的持续时间 结合是偏向D1R信号的一种机制，我们将提供一个定义的和可测量的配体 属性以帮助设计有偏向的激动剂。该项目还将验证其相关性和潜力 G蛋白偏向激动剂对D1R功能的有效性。

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.