Allosteric Enhancement of Adenosine Receptors

这项竞争性更新建议继续我们对A1的变构增强剂(AE)的研究 腺苷受体(A^R)。这些化合物结合到受体上的变构部位，以增加 腺苷与A^R上正构位点的亲和力我们已经确定了新的和改进的 和我们发现了一种新的化学类更有效和更有效的 氨基噻唑类药物。我们还发现，一些二硫化物和H_2O_2具有类似AE的作用。 为了更多地了解AE作用的分子机制，我们研究了受体 变种人和嵌合体。我们已经开发了一种新的系统来对增强子活性进行“评分”，该系统基于 这些化合物可以防止激动剂放射性配基125I-ABA从A^R快速解离。 对GTPYS的回应。我们建议进行新的研究，以实现三个具体目标。目标1是合成新的 两种化学类别的增强子结构及其基于与放射性配基结合的特征 受体与G蛋白的鸟嘌呤核苷酸交换。这一目标是由构象分子指导的 现有结构的场分析(CoMFA)和新合成技术的使用。目标2是高效地 评估完整细胞中的增强子功能，并评估所选化合物的调节活性 腺苷A1受体在脑片中的作用和刺激血管生成。目标3将调查这一角色 通过对硫醇烷基化试剂、表位和表位的分析，得出在AE和H_2O_2作用下形成二硫键的结论。 标记受体、定点突变和蛋白质测序。我们还将调查这一可能性 H_2O_2是A^R信号的生理调节因子。我们假设细胞外的半胱氨酸 AiAR的环参与二硫键的交联反应。AES是重要的治疗候选药物 这可能会减轻慢性疼痛或促进特定部位的血管生成。此外，我们对这一角色的调查 增强剂中的硫醇/二硫化物和H_2O_2的作用可能揭示有关一般情况的重要新信息 GPCRG蛋白偶联和转运的机制。

This competitive renewal proposes to continue our investigation of allosteric enhancers (AEs) of the A1 adenosine receptor (A^R). These compounds bind to an allosteric site on the receptor to increase the affinity of adenosine to the orthosteric site on the A^R. 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 AiAR and the A^R. 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 A^R in response to GTPyS. We propose newstudies 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 A^R signaling. We hypothesize that cysteines in the extracellular loops of the AiAR 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.