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，旨在产生和 表征作用于 δ 阿片受体的非肽化合物。 该计划项目的一个基本假设是作用于 delta 的药物 阿片受体将是有效的镇痛药，但缺乏许多 目前使用的阿片类药物的不良副作用。 此外， 假设存在多种 δ 阿片受体亚型 并且可以进一步增加治疗特异性 通过对特定δ受体亚型具有选择性的药物来实现。 二 最新进展，非肽化合物BW373U86的发现 以及小鼠δ阿片受体的克隆，为 对这些假设的检验和此处提出的工作。 BW373U86 是唯一已知的选择性 δ 受体激动剂，但不 一种肽。 BW373U86 的外消旋混合物被分解成两个 (+) 和 (-) 异构体，由 Kenner Rice 博士提出，他建议对每种形式进行标记 与氚一起进行放射性配体结合研究。 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进行放射自显影研究。 平行线 用已建立的配体的放射性标记形式进行研究[4'-Cl- Phe4]DPDPE 和纳曲吲哚以及结合抑制研究 旨在表征 BW373U86 标记的​​位点将用于 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有助于解释一些不寻常的地方 这种新型化合物的药理学特性。 其中一些 特性，包括对小鼠血管中 δ 受体的高效力 输精管但镇痛效力较低表明 BW373U86 可能具有选择性 对于δ受体亚型。 我们还打算探索δ阿片受体的假设 通过克隆编码所提出的亚型的小鼠和人类 cDNA 来确定亚型。 由于小鼠 δ 阿片受体的至少一种亚型已被 克隆后，我们将使用聚合酶链式反应方法来生产 对δ阿片受体有选择性的寡核苷酸探针。 这些 探针将首先用于筛选小鼠，随后用于筛选人类 大脑 cDNA 文库，用于检测多个 δ 阿片受体的存在。 δ 受体亚型 cDNA 的鉴定很重要 由于选择性放射性配体不可用，这阻碍了 针对这些亚型开发选择性药物。 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 具有定义的δ受体亚型。 表达的细胞系 不同的人δ阿片受体亚型将用于 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 通过本计划项目建议书。