BIOCHEMICAL STUDIES AND CLONING OF DELTA SUBTYPES

Delta 亚型的生化研究和克隆

• 批准号: 6104059
• 负责人: HENRY I YAMAMURA
• 金额: $ 11.27万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104059
• 关键词: analgesics; clone cells; drug screening /evaluation; human genetic material tag; inhibitor /antagonist; laboratory mouse; laboratory rat; molecular cloning; opioid receptor; polymerase chain reaction; protein isoforms; radiotracer; receptor binding; stimulant /agonist

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.

该提案是一个计划项目的一部分，该项目旨在生产和 表征作用于三角洲阿片类受体的非肽化合物。 该计划项目的一个基本假设是作用于三角洲的药物 阿片类药物受体将是有效的镇痛药，但缺乏许多 现在使用的阿片类药物的不良副作用。 此外， 假设有多种三角洲阿片类受体亚型 并且可以额外提高治疗特异性 通过药物选择特定的三角洲受体亚型的药物实现。 二 最近的发展，发现非肽化合物BW373U86 和小鼠三角洲阿片受体的克隆，为 这些假设的检验和这里提出的工作。 BW373U86是唯一已知的选择性三角体受体激动剂，不是 肽。 BW373U86的外围混合物被解析到其两个（+）中 （ - ）肯纳·赖斯（Kenner Rice）博士的异构体，他建议将每种形式标记为 与放射性结合进行研究的Tritium。 我们建议这样做 通过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。 平行线 既定配体的放射性标记形式的研究[4'-cl-- PHE4] DPDPE和NALTRINDOLE除了结合抑制研究 旨在表征由BW373U86标记的站点的表征 定义其在中枢神经系统受体上的特性。 此信息将有助于 BW373U86的新类似物的设计，并有助于解释一些不寻常的 这种新颖化合物的药理特性。 其中一些 特性，包括小鼠VAS中的三角洲受体的高效力 延期但低镇痛效力表明BW373U86可能是选择性的 用于三角洲受体亚型。 我们还打算探讨三角洲阿片类受体的假设 亚型通过克隆小鼠和人类cDNA编码所提出的亚型。 由于至少一种小鼠三角洲阿片类药物受体的亚型 克隆，我们将使用聚合酶链反应方法与产品 寡核苷酸探针选择用于三角洲阿片类药物的受体。 这些 探针将用于最初筛选鼠标，然后使用人类 脑cDNA文库，用于存在多种三角洲阿片类药物受体。 Delta受体亚型的CDNA的鉴定很重要 由于没有选择性的放射性配体，这会阻碍 开发这些亚型的选择性药物。 此外，唯一的 可以研究任何人类三角洲阿片受体的方法是通过 使用表达这些受体的重组细胞的使用。 三角洲的cdnas 受体将在哺乳动物细胞中表达以产生细胞系 已经定义了三角体受体亚型。 表达细胞系 不同的人类三角洲阿片类药物受体亚型将用于 开发特定的放射性结合和功能测定 在其他地方提出的新化合物的筛选和表征 通过此计划项目建议。