ALLOSTERIC ENHANCEMENT OF A1 ADENOSINE RECEPTORS

2-氨基-3-苯基噻吩类化合物，如PD81723(Pd)已被证明是 腺苷A1受体的选择性变构增强剂。在高处 这些化合物在浓度上充当竞争性拮抗剂。我们的 初步数据表明，Pd增强了重组人 人对G蛋白的A1受体，但PD对其他蛋白没有促进作用 人的腺苷受体。A1增强剂具有巨大的治疗潜力 因为它们可能产生最小的副作用或脱敏和作用 选择性地在缺血组织中内源性腺苷 制作。在六个目标中，有四个是针对确定分子 帕金森病引起人A1腺苷变构增强的机制 感受器。我们已经稳定表达了所有四种人类腺苷受体 亚型，并通过延长氨基末端来修饰这些受体 六氢组氨酸和FLAG表位使H/F受体易于 是纯净的。PD增强激动剂与纯化的H/F-A1受体的结合， 证明它直接与受体上的变构位点结合。我们 已经准备了腺苷受体嵌合体和突变体，几个独特的 放射性配体和光亲和标记以及均相重组G 蛋白质。目的1是评价马兜铃酸的增强和拮抗活性 PD在四种重组人腺苷受体上均有表达。目标2是 测定钯对纯化H/F-A1相互作用的影响 确定亚基组成的受体和纯化的G蛋白 重建分析。目的3是研究PD对A1/A2a的影响 我们构建的显示A1药理和Gs的杂交受体 偶联(A1和A2a受体分别与Gi和Gs偶联)。目标4 构建A1/A3受体嵌合体和A1突变体以鉴定氨基酸 促进剂活性所需的酸类。另外两个目标将是 在联合调查员奥尔森博士的参与下， 专业的药物化学家。目标5是合成和筛选新化合物 它们是比钯为基础的更有效和更有效的增强剂 结合、生化和功能(朗宁多夫灌流豚鼠) 心脏)化验。有可能用很少或很少的原料合成增强剂 没有对抗活动。目标6是合成增强剂放射性配体和 用于表征增强子结合的光亲和标记 域。亲和标记的纯化受体将被测序以识别 标记的氨基酸。除了为可能的 开发有前景的治疗剂，这些研究将是有用的 以增加我们对调节受体-G的因素的了解 蛋白质偶联。

2-Amino-3-benzylthiophenes such as PD81723 (PD) have been shown to be selective allosteric enhancers of A1 adenosine receptors. At high concentrations these compounds act as competitive antagonists. Our preliminary data indicate that PD enhances the coupling of recombinant human A1 receptors to G proteins, but PD has no enhancing effect on other human adenosine receptors. A1 enhancers have great therapeutic potential since they may produce minimal side effects or desensitization and act selectively in ischemic tissues at sites where endogenous adenosine is produced. Of six aims, four are directed at determining the molecular mechanisms by which PD causes allosteric enhancement of human A1 adenosine receptors. We have stably expressed all four human adenosine receptor subtypes, and modified these receptors by extending the amino termini with hexahistidine and the FLAG epitope to make H/F- receptors that can readily be purified. PD enhances agonist binding to purified H/F-A1 receptors, proving that it binds directly to an allosteric site on the receptor. We have prepared adenosine receptor chimera and mutants, several unique radioligands and photoaffinity labels, and homogeneous recombinant G proteins. Aim 1 is to evaluate the enhancing and antagonist activities of PD on all four recombinant human adenosine receptors. Aim 2 is to determine the effects of PD on the interaction between purified H/F-A1 receptors and purified G proteins of defined subunit composition in reconstitution assays. Aim 3 is to examine the effects of PD on an A1/A2A hybrid receptor we have constructed that displays A1 pharmacology and Gs coupling (A1 and A2A receptors coupled to Gi and Gs, respectively). Aim 4 is to construct A1/A3 receptor chimera and A1 mutants to identify amino acids required for enhancer activity. Two additional aims will be facilitated by the participation of the co-investigator, Dr. Olsson, an expert medicinal chemist. Aim 5 is to synthesize and screen new compounds that are more potent and possibly more efficacious enhancers than PD based on binding, biochemical and functional (Langendorff perfused guinea pig heart) assays. It may be possible to synthesize enhancers with little or no antagonist activity. Aim 6 is to synthesize enhancer radioligands and photoaffinity labels for use in characterizing the enhancer binding domain. Affinity labeled purified receptors will be sequenced to identify labeled amino acids. In addition to contributing to the possible development of promising therapeutic agents, these studies will be useful for increasing our understanding of factors that regulate receptor-G protein coupling.