ALLOSTERIC ENHANCEMENT OF THE CARDIAC ADENOSINE RECEPTOR

心脏腺苷受体的变构增强

• 批准号: 2226703
• 负责人: LUIZ BELARDINELLI
• 金额: $ 18.76万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226703
• 关键词: CHO cells; allosteric site; antiarrhythmic agent; anticonvulsants; chemical structure function; chemical synthesis; drug receptors; electrophysiology; guinea pigs; heart cell; heart metabolism; heart pharmacology; isolation perfusion; mathematical model; myocardial ischemia /hypoxia; purinergic receptor; site directed mutagenesis; thiophenes; tissue /cell culture; vasodilation

Recently a series of 2-amino-3 benzoylthiophenes were shown to be allosteric enhancers (AEs) of the actions of adenosine (Ado) on the A1- Ado receptor (A1-AdoR) in the rat brain and in the FRTL-5 thyroid cell line. Positive allosterism of the cardiac A1-AdoR system has not been previously investigated. This research proposal is designed to 1) characterize and establish the specificity of the enhancement caused by the prototype AE, PD 81,723 (PD), of the cardiac actions of exogenous and endogenous Ado, 2) define the selectivity of PD for the A1-AdoR vs other AdoR subtypes, and mathematically model the allosteric enhancement, 3) investigate species and receptor-subtype differences in response to PD, and determine the PD binding domain(s) using recombinant AdoRs, and 4) synthesize and screen new Aes with improved potency an selectivity for AdoR subtypes. To accomplish these broad objectives, a multidisciplinary (pharmacology, molecular biology, chemistry) approach is proposed. Studies will be carried out with guinea pig isolated hearts and freshly isolated cardiomyocytes, cultured cell lines, and cells stably transfected with native and mutated recombinant AdoR subtypes. The effect of PD on AdoR-mediated slowing of AV nodal conduction in isolated heats and on membrane currents (e.g., IKAdo) in single atrial and ventricular myocytes will be quantitated using electrophysiological techniques. The effects of PD on membrane binding of, and cardiac responses to, drugs that have effects similar to those of Ado (e.g., carbachol) will be determined to assess the specificity of PD as enhancer of the actions of Ado. To investigate potentiation by Aes of cardiac responses to endogenous Ado, hypoxia, ischemia, and inhibitors of Ado metabolism will be used to elevate the myocardial interstitial concentration of Ado in the presence and absence of PD. To determine the selectivity of PD for a given AdoR subtype, PD's effects on A1-mediated slowing of AV nodal conduction and on A2-mediated coronary vasodilation will be compared, and radioligand binding of AdoR agonists to recombinant A1,A2a,A2b and A3-AdoRs permanently expressed in CHO cells and to endogenous A1- and A2-AdoRs in DDT1MF-2 and PC-12 cell lines will be quantitated. The nature of the allosteric interaction between PD and the AdoR will be assessed by studying the effect of Aes on a) AdoR agonist, and antagonist radioligand binding parameters, b) affinity states of the A1-AdoR, and c) coupling of AdoRs to G proteins. The effects of PD on binding of AdoR agonists to cloned and expressed AdoR subtypes from various species (including human) and to chimeric receptor constructs will be used to guide site-directed mutagenesis efforts to identify the region(s) of the AdoR important for AE activity. The ultimate goal of this proposal is to demonstrate that allosteric enhancers of the actions of Ado are useful agents to increase the response to locally released Ado in the heart and thereby act as site=specific drugs. These compounds may prove to be an ideal therapeutic approach to selectively and specifically amplify the actions of endogenous adenosine.

最近一系列的 2-氨基-3 苯甲酰噻吩被证明 腺苷 (Ado) 对 A1- 作用的变构增强剂 (AE) 大鼠大脑和 FRTL-5 甲状腺细胞中的 Ado 受体 (A1-AdoR) 线。 心脏 A1-AdoR 系统的正变构尚未被证实 之前调查过。 本研究计划旨在 1) 表征并确定由以下原因引起的增强的特异性 原型 AE，PD 81,723 (PD)，外源性和外源性心脏作用 内源性 Ado，2) 定义 PD 对 A1-AdoR 相对于其他 Ado 的选择性 AdoR 亚型，并对变构增强进行数学建模，3) 研究对 PD 反应的物种和受体亚型差异， 并使用重组 AdoR 确定 PD 结合结构域，以及 4) 合成和筛选具有改进效力和选择性的新 Aes AdoR 亚型。 为了实现这些广泛的目标，需要多学科 提出了（药理学、分子生物学、化学）方法。 研究将用豚鼠离体心脏和新鲜心脏进行 分离的心肌细胞、培养的细胞系和稳定的细胞 用天然和突变的重组 AdoR 亚型转染。 这 PD 对 AdoR 介导的房室结传导减慢的影响 加热和膜电流（例如，IKAdo）在单个心房和 心室肌细胞将使用电生理学进行定量 技术。 PD对膜结合和心脏的影响 对具有与 Ado 类似作用的药物（例如， 将确定卡巴胆碱 (carbachol) 以评估 PD 作为增强剂的特异性 阿多的行动。 研究 Aes 对心脏的增强作用 对内源性 Ado、缺氧、缺血和 Ado 抑制剂的反应 新陈代谢将用于升高心肌间质 PD 存在和不存在时 Ado 的浓度。 确定 PD 对给定 AdoR 亚型的选择性，PD 对 A1 介导的影响 房室结传导减慢和 A2 介导的冠状血管舒张 将比较 AdoR 激动剂与重组体的放射性配体结合 A1、A2a、A2b 和 A3-AdoR 在 CHO 细胞中永久表达并 DDT1MF-2 和 PC-12 细胞系中的内源性 A1- 和 A2-AdoR 将被 定量。 PD和PD之间变构相互作用的本质 AdoR 将通过研究 Aes 对 a) AdoR 激动剂的影响来评估， 和拮抗剂放射性配体结合参数，b) 的亲和力状态 A1-AdoR，和 c) AdoR 与 G 蛋白的偶联。 PD对的影响 AdoR激动剂与克隆和表达的AdoR亚型的结合 各种物种（包括人类）和嵌合受体构建体 将用于指导定点诱变工作，以确定 AdoR 的区域对于 AE 活动很重要。 最终目标是 该提案旨在证明行动的变构增强剂 Ado 是有用的代理，可以提高对本地发布的 Ado 的响应 在心脏中，从而充当位点特异性药物。 这些化合物可能 被证明是一种理想的选择性和特异性治疗方法 增强内源性腺苷的作用。