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: 2460498
• 负责人: RODNEY L JOHNSON
• 金额: $ 21.66万
• 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/2460498
• 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-NH2 (PLG) 及其拟肽类似物的基础 调节中枢神经系统多巴胺受体并阐明分子机制 这种调制的基础。为了实现这一目标，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。类似物也将被制造，它们是 被能够模拟亮氨酰侧的疏水基团取代 在 PLG 中发现的链。二酮哌嗪内酰胺类似物的合成 将制作肽模拟物来检验它是该类型的假设 II β-转角构象是重要的构象特征 多巴胺受体调节活性，而 N 端 C5 构象是负责效力的结构特征 PLG 肽模拟物。不同尺寸的高度类似环 将制备受限螺双环内酰胺 PLG 拟肽，以便 改变 phi2、psi2 和 phi3 扭转角，从而确定 这对多巴胺受体调节活性的影响。最后， 推定 PLG 受体的两种一般类型的潜在放射性配体 将获得并评估。一种类型是氚化的 二酮哌嗪内酰胺拟肽，而在第二种方法中，4- 羟基苄基将被引入到结构中 高效的 PLG 类似物，用于放射性标记这些 与 125I 形成化合物。合成的 PLG 肽模拟物将是 评估其调节 (3H]螺哌啶醇/N-的能力 存在时，丙基去甲吗啡与 D2 受体竞争性结合 或缺少 5′-鸟苷酰亚胺二磷酸酯（一种不可水解的类似物） GTP 的。他们还将接受评估，以评估他们提高 多巴胺受体激动剂与 D1、D2、D3、D4 或 D5 结合 受体使用转染的SH-SY5Y人神经母细胞瘤细胞系 D1、D2、D3、D4 或 D5 受体基因。选定的PLG 将测试肽模拟物在动物体内的活性 帕金森病模型（6-羟基多巴胺损伤大鼠模型和 MPTP 小鼠模型）和运动障碍（氟哌啶醇诱导的 D2 受体超敏模型和左旋多巴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 通过使用特定反义纹状体 硫代磷酸酯寡核苷酸通过检查：（1）Gs和的能力 Gi反义寡核苷酸调节旋转行为 多巴胺激动剂对 6-羟基多巴胺损伤大鼠的影响 缺乏PLG类似物； (2)G蛋白水平和mRNA表达 对于Gi改变，在缺乏D2受体激动剂的情况下治疗大鼠 PLG 肽模拟物的存在，以及 (3) PLG 的作用 模拟肽对纯化 G 蛋白解偶联的 GTP 酶活性的影响 受体。这些研究将增强我们对多巴胺的理解 受体调节并为新的开发奠定基础 用于治疗此类神经系统疾病的治疗剂，例如 帕金森病和迟发性运动障碍。