PRO-LEU-GLY-NH2 AMD DOPAMINE RECEPTOR MODULATION STUDY

PRO-LEU-GLY-NH2 AMD 多巴胺受体调节研究

• 批准号: 2263754
• 负责人: RODNEY L JOHNSON
• 金额: $ 19.61万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2263754
• 关键词: G protein; Parkinson's disease; X ray crystallography; chemical structure function; disease /disorder model; dopamine agonists; dopamine receptor; gene expression; haloperidol; hormone receptor; laboratory mouse; laboratory rat; lactams; neuropeptide receptor; nuclear magnetic resonance spectroscopy; nucleic acid probes; peptide chemical synthesis; peptide hormone; peptide hormone analog; piperazines; radiotracer; receptor binding; tardive dyskinesia

The long term objective of this research is to understand the structural basis by which Pro-Leu-Gly-NH2 (PLG) and its peptidomimetic analogues modulate CNS dopamine receptors and to elucidate the molecular mechanism underlying this modulation. In pursuing this objective, we plan to synthesize analogues of highly potent PLG peptidomimetics to test the hypothesis that the carbonyl groups in these molecules are important for their intrinsic activity. Analogues will also be made which are substituted with hydrophobic groups capable of mimicking the leucyl side chain found in PLG. Synthesis of analogues of the diketopiperazine lactam peptidomimetic will be made to test the hypothesis that it is the type II beta-turn conformation that is the important conformational feature of dopamine receptor modulating activity while the N-terminal C5 conformation is the structural feature responsible for the potency of the PLG peptidomimetics. Different sized ring analogues of the highly constrained spiro bicyclic lactam PLG peptidomimetic will be made so as to alter the phi2, psi2, and phi3 torsion angles and thereby determine the effect this has on dopamine receptor modulating activity. Finally, two general types of potential radioligands for the putative PLG receptor will be obtained and evaluated. One type will be a tritiated diketopiperazine lactam peptidomimetic,while in a second approach the 4- hydroxybenzyl group will be incorporated into the structures of the highly potent PLG analogues for the purpose of radiolabeling these compounds with 125I. The PLG peptidomimetics synthesized will be evaluated for their ability to modulate (3H]spiroperidol/N- propylnorapomorphine competitive binding to D2 receptors in the presence or absence of 5 prime-guanylylimidodiphosphate, a norhydrolyzable analog of GTP. They will also be evaluated for their ability to enhance the binding of dopamine receptor agonists to either the D1, D2, D3 D4 or D5 receptors using SH-SY5Y human neuroblastoma cell lines transfected with either the D1,D2, D3, D4 or D5 receptor genes. Selected PLG peptidomimetics will be tested for their activity in in vivo animal models of Parkinson's disease (6- hydroxydopamine-lesioned rat model and the MPTP mouse model) and dyskinesia disorders (haloperidol-induced D2 receptor supersensitivity model and the L-DOPA-induced dyskinesia model). Finally, studies will be conducted to further determine the functional interaction of PLG and its bioactive analogues with G-proteins in the striatum through the use of specific antisense phosphothioate oligonucleotides by examining: (1) the ability of Gs and Gi antisense oligonucleotides to modulate rotational behavior induced by dopamine agonists in 6-hydroxydopamine lesioned rats in the presence and absence of PLG analogues; (2) the G protein levels and mRNA expression for Gi alter treating rats with D2 receptor agonists in the absence and presence of PLG peptidomimetics, and (3) the effect of PLG peptidomimetics on GTPase activity of purified G-proteins uncoupled from the receptor. These studies will enhance our understanding of dopamine receptor modulation and provide groundwork for the development of new therapeutic agents for the treatment of such neurological disorders as Parkinson's disease and tardive dyskinesia.

这项研究的长期目标是了解结构 Pro-Leu-Gly-NH 2（PLG）及其拟肽类似物 调节中枢神经系统多巴胺受体，并阐明其分子机制 在这种调制下。为达致这个目标，我们计划 合成高效PLG肽模拟物的类似物，以测试 假设这些分子中的羰基对于 他们的内在活动。也将作出类似的， 被能够模拟亮氨酰基侧的疏水基团取代 在PLG中发现的链。二酮哌嗪内酰胺类似物的合成 拟肽将测试的假设，它是类型 II β-转角构象，这是重要的构象特征 多巴胺受体调节活性，而N-末端C5 构象是负责药物效力的结构特征。 PLG肽模拟物。不同大小的环类似物的高度 将制备受约束的螺双环内酰胺PLG肽模拟物， 以改变φ 2、φ 12和φ 3扭转角，从而确定 这对多巴胺受体调节活性的影响。最后， 假定PLG受体的两种一般类型的潜在放射性配体 将得到并评估。一种是氚化的 二酮哌嗪内酰胺肽模拟物，而在第二种方法中， 羟基苄基将被引入到化合物的结构中。 用于放射性标记这些化合物的高效PLG类似物 含125 I的化合物。将合成的PLG肽模拟物 评价它们调节（3 H]螺哌利多/N- 丙去甲阿扑吗啡与D2受体的竞争性结合 或不存在去甲水解类似物5-伯-鸟苷酰亚氨基二磷酸 的GTP。他们还将评估他们提高 多巴胺受体激动剂与D1、D2、D3、D4或D5的结合 使用转染了受体的SH-SY 5 Y人神经母细胞瘤细胞系， D1、D2、D3、D4或D5受体基因。选择的PLG 将测试肽模拟物在体内动物中的活性 帕金森病模型（6-羟基多巴胺损伤的大鼠模型和 MPTP小鼠模型）和运动障碍病症（氟哌啶醇诱导的D2 受体超敏模型和L-DOPA诱导的运动障碍模型）。 最后，将进行研究，以进一步确定功能 PLG及其生物活性类似物与G-蛋白的相互作用 纹状体通过使用特定的反义 硫代磷酸寡核苷酸通过检查：（1）Gs和 Gi反义寡核苷酸调节由G1诱导的旋转行为 多巴胺激动剂在6-羟基多巴胺损伤大鼠中的存在和 不存在PLG类似物;（2）G蛋白水平和mRNA表达 对于Gi，在不存在的情况下用D2受体激动剂处理大鼠， PLG肽模拟物的存在，和（3）PLG的作用 肽模拟物对纯化的G-蛋白的GT3活性的影响 受体。这些研究将增进我们对多巴胺的了解 受体调节，并为开发新的 用于治疗神经障碍的治疗剂， 帕金森病和迟发性运动障碍。