Allosteric Enhancement of Adenosine Receptors

描述(由申请人提供)：这项竞争性更新建议继续我们对A1腺苷受体(A1AR)的变构增强剂(AE)的研究。这些化合物结合到受体上的变构部位，以增加腺苷与A1AR上的正构部位的亲和力。我们已经确定了新的和改进的氨基噻吩类AEs，我们已经发现了一类新的更有效和更有效的氨基噻唑AEs。我们还发现一些二硫化物和H_2O_2对A1AR和A2AAR具有类似AE的效应。为了更多地了解AE作用的分子机制，我们研究了受体突变体和嵌合体。我们开发了一种新的系统，根据这些化合物防止激动剂放射性配体125I-ABA与GTP-GammaS反应中的A1AR快速解离的能力，对增强子活性进行“评分”。我们建议进行新的研究，以实现三个具体目标。第一个目标是合成两类新的增强子结构，并基于与受体的放射性配基结合和G蛋白的鸟嘌呤核苷酸交换来表征它们。这一目标是通过对现有结构的构象分子场分析(CoMFA)和新合成技术的使用来指导的。目的2是有效地评估完整细胞中的增强子功能，并评估选定的化合物在调节脑片中腺苷A1受体功能和刺激血管生成方面的活性。目的3将通过分析硫醇烷基化试剂、表位标记受体、定点突变和蛋白质测序来研究二硫键在AE和H_2O_2作用中的作用和二硫键的形成。我们还将研究H_2O_2可能是A2AAR信号的生理调节因子。我们假设A1AR胞外环中的半胱氨酸参与了二硫键交联反应。AES是重要的候选治疗药物，可以减轻慢性疼痛或促进特定部位的血管生成。此外，我们对硫醇/二硫化物在增强剂和H_2O_2作用中的作用的研究可能揭示关于GPCRG蛋白偶联和运输的一般机制的重要新信息。

DESCRIPTION (provided by applicant): This competitive renewal proposes to continue our investigation of allosteric enhancers (AEs) of the A1 adenosine receptor (A1AR). These compounds bind to an allosteric site on the receptor to increase the affinity of adenosine to the orthosteric site on the A1AR. We have identified new and improved aminothiophene AEs and we have discovered a new chemical class of more potent and effective aminothiazole AEs. We also have discovered that some disulfides and H2O2 have AE-like effects on the A1AR and the A2AAR. To learn more about the molecular mechanisms of AE action we investigated receptor mutants and chimera. We have developed a novel system to "score" enhancer activity based on the ability of these compounds to prevent rapid dissociation of an agonist radioligand, 125I-ABA, from the A1AR in response to GTPgammaS. We propose new studies to achieve three specific aims. Aim 1 is to synthesize new enhancer structures in two chemical classes and to characterize them based on radioligand binding to receptors and guanine nucleotide exchange from G proteins. This aim is guided by conformational molecular field analysis (CoMFA) of existing structures and the use of new synthetic techniques. Aim 2 is to efficiently evaluate enhancer function in intact cells and to evaluate selected compounds for activity in modulating adenosine A1 receptor function in brain slices and stimulation of angiogenesis. Aim 3 will investigate the role and disulfide bond formation in AE and H2O2 action through analysis of thiol alkylating agents, epitope-tagged receptors, site-directed mutagenesis and protein sequencing. We will also investigate the possibility the H2O2 is a physiological regulator of A2AAR signaling. We hypothesize that cysteines in the extracellular loops of the A1AR participate in disulfide cross linking reactions. AEs are significant therapeutic candidates that may reduce chronic pain or promote site-specific angiogenesis. Moreover, our investigation of the role of thiols/disulfides in enhancer and H2O2 action may reveal important new information about general mechanisms of GPCR G protein coupling and trafficking.