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

这项研究的长期目标是理解结构 Pro-Leu-Gly-NH2(PLG)及其模拟肽类似物的基础 中枢神经系统多巴胺受体的调控及其分子机制 在这种调制的基础上。为达致这个目标，我们计划 合成高效的PLG模拟多肽的类似物以测试 假设这些分子中的羰基对 它们的内在活动。还将生产类似的产品，这些产品 被能够模拟亮氨酸侧基的疏水基团取代 在PLG中发现链条。二酮基哌嗪内酰胺类似物的合成 将进行多肽模拟来检验它是这种类型的假设 IIβ转角构象，这是重要的构象特征 多巴胺受体的调节活性，而N端C5 构象是结构特征，负责的效力 PLG多肽模拟剂。不同大小的环类似物的高度 约束螺环双环内酰胺PLG多肽仿制品 以改变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， 推测的PLG受体的两种可能的放射性配体 将被获取和评估。其中一种类型将是一种氚 二酮哌嗪内酰胺类多肽，而在第二种方法中，4- 羟基苄基将被引入到结构中 用于放射性标记目的的高度有效的PLG类似物 含125I的化合物。合成的PLG多肽类药物将被 评价它们调节(~3H)螺哌利多/N-的能力 丙基去甲吗啡与D2受体的竞争结合 或不含5-基鸟氨酰二磷酸，这是一种可去水解的类似物 GTP的一部分。他们还将接受评估，以确定他们是否有能力提高 多巴胺受体激动剂与D1、D2、D3、D4或D5的结合 人神经母细胞瘤细胞系SH-SY5Y转导受体的研究 D1、D2、D3、D4或D5受体基因。选定的PLG 多肽类药物将在活体动物身上进行活性测试 帕金森病模型(6-羟基多巴胺损毁大鼠模型和 MPTP小鼠模型)和运动障碍障碍(氟哌啶醇诱导的D2 受体超敏模型和L-多巴诱发的运动障碍模型)。 最后，将进行研究，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 通过使用特定反义的纹状体 硫代寡核苷酸通过检测：(1)Gs和 GI反义寡核苷酸对小鼠旋转行为的调控作用 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 在没有D2受体激动剂的情况下用D2受体激动剂治疗大鼠 PLG多肽模拟物的存在，以及(3)PLG的作用 肽模拟法对分离纯化的G蛋白GTP酶活性的影响 感受器。这些研究将加深我们对多巴胺的理解。 受体的调节，为开发新的 用于治疗神经疾病的治疗剂，如 帕金森氏症和迟发性运动障碍。

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.