BIOCHEMICAL STUDIES AND CLONING OF DELTA SUBTYPES

该提案是一个旨在生产和 表征作用于δ阿片受体的非肽化合物。 该项目的一个基本假设是， 阿片受体将是有效的镇痛剂，但将缺乏许多 现在使用的鸦片类药物的不良副作用。 此外，委员会认为， 假设存在多种δ阿片受体亚型， 并且治疗特异性的额外增加可以 通过对特定δ受体亚型有选择性的药物来实现。 两 最近的发展，非肽化合物BW 373U86的发现 以及小鼠δ阿片受体的克隆，为 这些假设的测试和这里提出的工作。 BW373U86是唯一已知的选择性δ受体激动剂， 肽。 将BW373U86的外消旋混合物拆分成其两个（+） 和（-）异构体由肯纳赖斯博士谁建议有每一个形式标记 用氚进行放射性配体结合研究。 我们建议这样做 组织匀浆结合和受体表征 使用小鼠和大鼠神经组织的放射自显影研究。 平行 用已建立的配体[4'-Cl-]的放射性标记形式进行的研究 Phe4] DPDPE和纳曲吲哚以及结合抑制研究 设计用于表征BW373U86标记的部位， 定义其在CNS受体上的特性。 这些信息将有助于 BW373U86的新类似物的设计，并帮助解释一些不寻常的 这种新型化合物的药理学性质。 其中一些 特性，包括对小鼠血管中δ受体的高效力 但镇痛效力较低，表明BW 373 U86可能具有选择性 δ受体亚型的基因。 我们还打算探讨δ阿片受体假说 通过克隆编码所提出的亚型的小鼠和人cDNA， 由于小鼠δ阿片受体的至少一种亚型已经被发现， 克隆，我们将使用聚合酶链反应方法， 对δ阿片受体具有选择性的寡核苷酸探针。 这些 探针将用于最初筛选小鼠，随后筛选人 脑cDNA文库中存在多种δ阿片受体。 δ受体亚型的cDNA鉴定是重要的 由于选择性放射性配体不可用，这阻碍了 开发针对这些亚型的选择性药物。 此外，唯一 研究任何人类δ阿片受体的方法是通过 使用表达这些受体的重组细胞。 delta的cDNA 受体将在哺乳动物细胞中表达以产生细胞系 具有确定的δ受体亚型。 表达的细胞系 不同的人δ阿片样物质受体亚型将用于 开发特异性放射性配体结合和功能测定， 其他地方提出的新化合物的筛选和表征 这个项目的提案。

This proposal is part of a program project that seeks to produce and characterize non-peptidic compounds acting at delta opioid receptors. A basic hypothesis of this program project is that drugs acting on delta opioid receptors will be potent analgesics, but will lack many of the undesirable side effects of the opiate drugs now in use. Furthermore, it is hypothesized that there are multiple delta opioid receptor subtypes and that an additional increase in therapeutic specificity can be achieved by drugs selective for particular delta receptor subtypes. Two recent developments, the discovery of the non-peptidic compound BW373U86 and the cloning of mouse delta opioid receptors, provide a foundation for the testing of these hypotheses and the work proposed here. BW373U86 is the only known selective delta receptor agonist that is not a peptide. The racemic mixture of BW373U86 was resolved into its two (+) and (-) isomers by Dr. Kenner Rice who proposes to have each form labeled with tritium for studies by radioligand binding. We propose to do this characterization by tissue homogenate binding and receptor autoradiography studies using mouse and rat neural tissue. Parallel studies with radiolabeled forms of the established ligands [4'-Cl- Phe4]DPDPE and naltrindole in addition to binding inhibition studies designed to characterize the site(s) labeled by BW373U86 will be used to define its properties at CNS receptors. This information will assist the design of new analogs of BW373U86 and help to explain some of the unusual pharmacological properties of this novel compound. Some of these properties, including high potency at delta receptors in the mouse vas deferens but low analgesic potency suggest that BW373U86 may be selective for delta receptor subtypes. We also intend to explore the hypothesis of delta opioid receptor subtypes by cloning mouse and human cDNAs encoding the proposed subtypes. Since at least one subtype of the mouse delta opioid receptor has been cloned, we will use polymerase chain reaction methods to product oligonucleotide probes selective for delta opioid receptors. These probes will be used to initially screen mouse and subsequently human brain cDNA libraries for the presence of multiple delta opioid receptors. The identification of cDNAs for delta receptor subtypes is important since selective radioligands are not available which impedes the development of selective drugs for these subtypes. Furthermore, the only way by which any human delta opioid receptors can be studied is through the use of recombinant cells expressing these receptors. cDNAs for delta receptors will be expressed in mammalian cells to produce cell lines having defined delta receptor subtypes. The cell lines expressing different human delta opioid receptor subtypes will be used for the development of specific radioligand binding and functional assays for the screening and characterization of the new compounds proposed elsewhere by this program project proposal.