ALLOSTERIC ENHANCEMENT OF A1 ADENOSINE RECEPTORS

2-氨基-3-苄基噻吩如PD 81723（PD）已被证明是 A1腺苷受体的选择性变构增强剂。在高 这些化合物作为竞争性拮抗剂。我们 初步数据表明，PD增强了重组体的偶联， 人A1受体对G蛋白，但PD对其他受体没有增强作用。 人腺苷受体。A1增强剂具有巨大的治疗潜力 因为它们可能产生最小副作用或脱敏作用， 选择性地在缺血组织中内源性腺苷被 制作。在六个目标中，有四个是针对确定分子 PD引起人A1腺苷变构增强的机制 受体。我们已经稳定表达了所有四种人腺苷受体， 亚型，并通过用 六组氨酸和FLAG表位，以使H/F-受体， 被净化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 已经制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，以及均质重组G proteins.目的1是评价的增强和拮抗活性， 对所有四种重组人腺苷受体的PD。目标二是 测定PD对纯化的H/F-A1之间相互作用的影响 受体和纯化的G蛋白的确定亚基组成， 重构测定。目的3是检查PD对A1/A2 A的影响 我们已经构建了一种显示A1药理学和Gs 偶联（A1和A2 A受体分别偶联至Gi和Gs）。目标4 构建A1/A3受体嵌合体和A1突变体， 增强子活性所需的酸。另外两个目标是 在共同研究者Olsson博士的参与下， 药物化学专家目的5是合成和筛选新化合物 它们是比基于PD的更有效的增强剂， 结合，生化和功能（Langendorff灌注豚鼠 心脏）测定。有可能合成具有很少或几乎不具有任何活性的增强子。 无拮抗剂活性。目的6是合成增强子放射性配体， 用于表征增强子结合的光亲和标记 域将对亲和标记的纯化受体进行测序，以鉴定 标记的氨基酸。除了有助于可能的 开发有前途的治疗药物，这些研究将是有用的 增加了我们对调节G受体的因素的理解 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.