Beta2-adrenergic receptor mutagenesis for studies of biased agonism

用于偏激动研究的 β2-肾上腺素受体诱变

• 批准号: 8645023
• 负责人: Laura Michele Wingler
• 金额: $ 5.33万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8645023
• 关键词: Adrenergic Receptor; Adverse effects; Affinity; Animal Disease Models; Arrestins; Binding; Biochemical; Biological; Biological Assay; Cardiovascular Diseases; Cardiovascular Physiology; Cell surface; Cellular Assay; Collection; Cyclic AMP; Data; Drug Targeting; Event; Exhibits; Family; Foundations; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Health; Heterotrimeric GTP-Binding Proteins; Human; In Vitro; Investigation; Laboratories; Libraries; Ligands; Lung diseases; Measures; Mediating; Molecular; Molecular Conformation; Mutagenesis; Pathway interactions; Pharmaceutical Preparations; Physiological; Physiology; Play; Proteins; Reagent; Receptor Activation; Regulation; Role; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Signaling Protein; Specificity; Tachyphylaxis; Testing; Therapeutic; Therapeutic Uses; Validation; Work; arrestin3; base; cardiovascular disorder therapy; desensitization; design; design and construction; directed evolution; high throughput screening; improved; in vivo; mutant; next generation; novel therapeutics; protein activation; prototype; receptor; receptor expression; receptor internalization; repository; research study; scaffold; screening; second messenger; skills; therapeutic target; tool

DESCRIPTION (provided by applicant): Seven-transmembrane receptors (7TMRs), also known as G protein-coupled receptors (GPCRs), serve cardinal roles in the regulation of cardiovascular function and are the targets of many of the most prevalent therapies for cardiovascular disease (e.g., the ß2-adrenergic receptor (ß2AR) targeted by ß-blockers). Activation of these receptors has long been known to initiate signaling cascades through the action of G proteins. More recent discoveries have demonstrated that 7TMRs also initiate non-G protein-dependent signaling events mediated by ß-arrestins (ßarrs), which, in addition to their originally characterized role in 7TMR desensitization, serve as scaffolds for a variety of signalin proteins. It has further become apparent that certain ligands can preferentially activate subsets of 7TMRs' multiple downstream effects, a phenomenon known as "biased agonism" that could be exploited to develop "biased drugs" with fewer off-target effects. However, characterization of the molecular determinants of biased signaling and investigation of this phenomenon's therapeutic potential have been hindered by the dearth of robust reagents for probing bias. Accordingly, the objective of this proposal is to identify a series of "biased mutants" of the ß2AR for use in diverse studies. The ß2AR has been characterized in vivo and in vitro far more extensively than any other 7TMR, making it a logical target for studies of biased signaling, but no strongly biased ß2AR ligands are known. Since receptor mutagenesis is a validated alternative to ligands for biasing 7TMR signaling, we aim first to systematically screen libraries of ß2AR mutants for those exhibiting biased signaling. We will construct focused, semi-rationally designed libraries of mutants and blindly screen constructs for G protein activation, ßarr recruitment, and expression in a multiplexed, medium-throughput assay format. Second, we will subject candidate mutants from this screen to a panel of assays to confirm their bias more rigorously and evaluate their potential for use in downstream applications. Successful completion of these objectives will provide a valuable repository of tools for the study of biased signaling, which could ultimately guide the design of next- generation medications targeting the ß2AR and other 7TMRs that possess novel therapeutic profiles and improved specificities compared to existing drugs.

描述（由申请人提供）：七跨膜受体（7 TMR），也称为G蛋白偶联受体（GPCR），在心血管功能的调节中起主要作用，并且是许多最流行的心血管疾病治疗的靶点（例如，β 2-肾上腺素能受体（β 2AR）被β受体阻滞剂靶向）。这些受体的激活早已被认为通过G蛋白的作用启动信号级联。最近的发现表明，7 TMR还启动由β-arrestins（β arrs）介导的非G蛋白依赖性信号传导事件，β-arrestins除了它们在7 TMR脱敏中最初表征的作用之外，还充当各种信号蛋白的支架。进一步变得明显的是，某些配体可以优先激活7 TMR的多个下游效应的子集，这种现象被称为“偏向激动”，可以利用其开发具有更少脱靶效应的“偏向药物”。然而，由于缺乏强有力的探测偏倚的试剂，对偏倚信号传导的分子决定簇的表征和对这种现象的治疗潜力的研究受到阻碍。因此，本建议的目的是鉴定一系列β 2 AR的“偏向突变体 用于各种研究。β 2 AR在体内和体外的特征比任何其他7 TMR都要广泛得多，使其成为偏向性信号传导研究的逻辑靶点，但没有强偏向性β 2 AR配体是已知的。由于受体诱变是一种经验证的替代配体的偏置7 TMR信号，我们的目标首先是系统地筛选那些表现出偏置信号的β 2AR突变体库。我们将构建集中的、半理性设计的突变体文库，并以多重、中等通量测定形式盲筛选G蛋白活化、β-内酰胺酶募集和表达的构建体。其次，我们将从这个屏幕上的候选突变体进行一组检测，以更严格地确认他们的偏见，并评估其在下游应用中的潜力。这些目标的成功完成将为偏性信号传导的研究提供有价值的工具库，其可以最终指导靶向β 2 AR和其他7 TMR的下一代药物的设计，与现有药物相比，这些药物具有新的治疗特性和改进的特异性。