BIOCHEMICAL STUDIES AND CLONING OF DELTA SUBTYPES

这项提案是一个计划项目的一部分，该项目寻求生产和 鉴定作用于阿片受体的非肽类化合物。 该计划项目的一个基本假设是作用于三角洲的药物 阿片受体将是有效的止痛剂，但将缺乏许多 目前正在使用的鸦片类药物的不良副作用。此外， 推测存在多种增量阿片受体亚型。 而且治疗特异性的额外增加可以是 通过对特定的Delta受体亚型有选择性的药物来实现。二 非肽类化合物BW373U86的新发现 并克隆了小鼠β阿片受体，为进一步研究奠定基础 对这些假说的检验和这里提出的工作。 BW373U86是唯一已知的选择性Delta受体激动剂 一种多肽。BW373U86的外消旋混合物被拆分成两个(+) 和(-)异构体，由肯纳·赖斯博士提出，他建议将每种形式标记为 用氚进行放射性配基结合研究。我们建议这样做 组织匀浆结合和受体的特性 用小鼠和大鼠神经组织进行放射自显影研究。平行 已建立的配体[4‘-Cl-]放射性标记形式的研究 DPDPE和纳曲吲哚的结合抑制研究 设计用于表征由BW373U86标记的站点(S)将用于 定义其在中枢神经系统受体上的特性。这些信息将有助于 设计了BW373U86的新类比，并帮助解释了一些不寻常的现象 该新化合物的药理性质。其中一些 特性，包括在小鼠输精管中的Delta受体的高活性 输精管但镇痛效力低提示BW373U86可能是选择性的 用于Delta受体亚型。 我们还打算探索增量阿片受体的假说。 通过克隆小鼠和人类编码建议的亚型的cDNA来实现亚型的克隆。 因为至少有一个亚型的小鼠增量阿片受体 克隆后，我们将使用聚合酶链式反应的方法来生产 对阿片受体有选择性的寡核苷酸探针。这些 探测器最初将用于筛查老鼠，随后将用于筛查人类 脑内存在多个阿片受体的cDNA文库。 识别Delta受体亚型的cDNA是很重要的 由于没有选择性的放射性配基，这阻碍了 针对这些亚型的选择性药物的开发。此外，唯一的 研究任何人类三角洲阿片受体的方法都是通过 使用表达这些受体的重组细胞。三角洲的cDNA 受体将在哺乳动物细胞中表达，以产生细胞系 已经定义了Delta受体亚型。表达基因的细胞系 不同的人类三角洲阿片受体亚型将用于 特异性放射性配基结合及功能检测方法的建立 别处提出的新化合物的筛选和表征 由该方案项目建议书。

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.