Molecular properties of B-adrenergic receptors in Asthma

哮喘中 B-肾上腺素能受体的分子特性

• 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
• 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' 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

 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，用于哮喘的急性（挽救）和慢性（维持）治疗。由于许多哮喘患者未能得到控制，因此有必要了解在长期治疗过程中观察到的急性β受体激动剂疗效的丧失以及耐受性或快速耐受的发展。这些问题部分是由于缺乏对 HASM 中 β2AR 信号传导基本方面的了解。该提案有三个目标来缩小这一差距，其广泛、长期的目标是开发最佳的 β-激动剂治疗哮喘以降低发病率。在目标 1 中，我们将筛选 4000 万个化合物库，以发现能够稳定有利于哮喘的特定 β2AR 构象的 β-激动剂。这种“偏向”将倾向于 Gs/cAMP 偶联（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、然后测量 ß-arrestin 募集、然后测量人体气道中的生理功能的顺序筛选方法来完成。来自这次筛选的化合物将成为哮喘临床前试验的候选化合物。此外，它们的结构将告诉我们工程激动剂从受体发挥特定功能的分子基础。在目标 2 中，将确定 β2AR 与另一种支气管扩张的气道受体（苦味受体 (TAS2R)）之间的相互作用。用于治疗哮喘的 TAS2R 激动剂正在开发中，预计 TAS2R 和 β2AR 激动剂将同时给药。然而，这两种受体似乎在细胞表面以异二聚体的形式交织在一起，其中一种受体的激活会改变另一种受体的功能。将使用双分子荧光互补、免疫共沉淀、生物素化和共聚焦显微镜研究异二聚体及其功能。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，miRNA 翻译抑制 β2AR 基因的机制将定义如何在 HASM 上建立 β2AR 蛋白的静息水平。该水平也是对 β-激动剂的急性反应的决定因素，并且 miRNA 本身似乎受到激动剂的调节。这些事件将通过模型细胞和 HASM 中的过表达、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-激动剂途径中新疗法的机制，以改善哮喘的治疗。