Molecular properties of B-adrenergic receptors in Asthma

哮喘中 B-肾上腺素能受体的分子特性

基本信息

批准号：
9130410
负责人：
Stephen B Liggett
金额：
$ 37.38万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9130410
关键词：
3&apos Untranslated Regions ADRB2 gene Acute Adrenergic Agents Adrenergic Agonists Adrenergic Receptor Affect Agonist Anti-Inflammatory Agents Anti-inflammatory Arrestins Asthma Binding Biotinylation Bronchial Spasm Bronchodilation Bronchodilator Agents Cell model Cell surface Chronic Clinical Co-Immunoprecipitations Complex Confocal Microscopy Coupling Cyclic AMP Development Disease Down-Regulation Educational process of instructing Engineering Event Family Fluorescence G protein coupled receptor kinase G-Protein-Coupled Receptors Gene Expression Goals Grant Health Human In Vitro Kinetics Knowledge Lead Libraries Maintenance Therapy Measurement Measures Mediating MicroRNAs Molecular Molecular Conformation Morbidity - disease rate Muscle Nodal Outcome Pathway interactions Phosphorylation Physiological Property Receptor Gene Receptor Signaling Regimen Regulation Relaxation Reporter Genes Rest Role Second Messenger Systems Signal Transduction Structure System Tachyphylaxis Taste Buds Testing Texture Translational Repression adrenergic analog asthmatic base improved knock-down locked nucleic acid member novel novel therapeutic intervention novel therapeutics overexpression preclinical study protein expression receptor receptor expression receptor function research study respiratory smooth muscle response scaffold screening second messenger therapeutic target

项目摘要

DESCRIPTION (provided by applicant): β-agonists acting at the β2-adrenergic receptor (β2AR) on human airway smooth muscle (HASM) relax the muscle and thus dilate the airways, and are used for acute (rescue) and chronic (maintenance) therapy for asthma. With many asthmatics not achieving control, there is a need to understand the loss of efficacy observed with acute ß-agonists and the development of tolerance, or tachyphylaxis, during chronic therapy. These issues are due in part to a lack of understanding of fundamental aspects of β2AR signaling in HASM. This proposal has three aims to close this gap, with the broad, long-term objective of developing optimal ß-agonist treatment for asthma to reduce morbidity. In Aim 1, we will screen a 40 million compound library to discover ß-agonists that stabilize a specific β2AR conformation that is favorable for asthma. This "biasing" would be towards Gs/cAMP coupling (improves bronchodilation) and away from ß-arrestin recruitment (thus minimizing tolerance). This will be accomplished through a sequential screening approach that measures cAMP, then ß-arrestin recruitment, and then physiologic function in human airways. The compounds that come from this screening will be candidates for preclinical trials in asthma. Moreover, their structures will teach us the molecular basis for engineering agonists to exert specific functions from receptors. In Aim 2, the interactions between β2AR and another airway receptor that bronchodilates, the bitter taste receptor (TAS2R), will be ascertained. Agonists for TAS2Rs are in development for treating asthma, and it is envisioned that TAS2R and β2AR agonists will be administered concomitantly. Yet, the two receptors appear to be intertwined at the cell surface in heterodimers, where activation of one receptor alters function of the other. Heterodimers and their function will be studied using bimolecular fluorescent complementation, co-immunoprecipitation, biotinylation, and confocal microscopy. Function will be ascertained by measuring the second messengers from each monomeric component (Ca2+ for TAS2R; cAMP for β2AR). In Aim 3, the mechanism of translational repression of the β2AR gene by miRNAs will define how the resting level of β2AR protein is established on HASM. This level is also a determinant of the acute response to ß-agonist, and miRNAs themselves appear to be modulated by agonists. These events will be studied with selected members of the let7, miR15, and miR30 miRNA families by overexpression, knock-downs, and gene and protein expression measurements in model cells and HASM. Collectively, these studies will define mechanisms that point to new therapeutics within the ß-agonist pathway for improved therapy of asthma.

描述（申请人提供）：β-激动剂作用于人气道平滑肌（HASM）上的β2-肾上腺素能受体（β2AR），放松肌肉，从而扩张气道，用于急性（抢救）和慢性（维持）由于许多哮喘患者未能得到控制，因此有必要了解急性β受体激动剂在慢性治疗期间观察到的疗效丧失以及耐受性或快速耐受的发展。这些问题部分是由于对 HASM 中 β2AR 信号转导的基本方面缺乏了解，该提案的三个目标是缩小这一差距，其广泛的长期目标是开发最佳的 β-激动剂治疗哮喘。在目标 1 中，我们将筛选 4000 万个化合物库，以发现稳定有利于哮喘的特定 β2AR 构象的 β-激动剂。（改善支气管扩张）并远离 ß-arrestin 募集（从而最大限度地减少耐受性），这将通过测量 cAMP、然后 ß-arrestin 募集、然后测量人类气道中的化合物的顺序筛选方法来实现。此外，它们的结构将告诉我们工程激动剂通过受体发挥特定功能的分子基础。β2AR 与另一种受体之间的相互作用。支气管扩张的气道受体，即苦味受体 (TAS2R)，将被确定用于治疗哮喘的 TAS2R 激动剂，预计 TAS2R 和 β2AR 激动剂将同时给药，然而，这两种受体似乎是相互交织的。在异二聚体的细胞表面，其中一种受体的激活改变了另一种异二聚体的功能，并且将使用它们来研究它们的功能。双分子荧光互补、免疫共沉淀、生物素化和共聚焦显微镜通过测量每个单体成分的第二信使（TAS2R 的 Ca2+；β2AR 的 cAMP）来确定功能。在目标 3 中，β2AR 基因的翻译抑制机制。由 miRNA 确定的 β2AR 蛋白的静息水平是如何在 HASM 上建立的，该水平也是急性的决定因素。这些事件将通过在模型细胞中过表达、敲低以及基因和蛋白质表达测量来研究let7、miR15和miR30 miRNA家族的选定成员。总的来说，这些研究将确定β-激动剂途径中改善哮喘治疗的新疗法的机制。