Design of Inhibitors of Epinephrine Biosynthesis

肾上腺素生物合成抑制剂的设计

• 批准号: 6526684
• 负责人: GARY L GRUNEWALD
• 金额: $ 45.16万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-06-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6526684
• 关键词: X ray crystallography; alpha adrenergic receptor; antihypertensive agents; blood brain barrier; cardiovascular function; chemical models; combinatorial chemistry; conformation; drug design /synthesis /production; drug screening /evaluation; enzyme inhibitors; enzyme mechanism; enzyme structure; epinephrine; heart rate; laboratory rat; methyltransferase; microdialysis; neurotransmitter biosynthesis; receptor binding

Epinephrine (Epi) comprises about 5% of central nervous system (CNS) catecholamines and it has been implicated in a number of neuroregulatory processes. We (and others) have shown that inhibitors of phenylethanolamine N-methyltransferase (PNMT, E. C. 2.1.1.28; the terminal enzyme in Epi biosynthesis) can lower blood pressure in spontaneously hypertensive rats. However, all inhibitors examined for blood pressure effects also have high affinity for alpha2-adrenoceptors, which could contribute significantly to the observed pharmacological effects. We now have lead inhibitors that are very selective (very low alpha2 affinity) and have sufficient lipophilicity (using the BBMEC model) to penetrate the blood brain barrier. We have developed microdialysis techniques to allow us to measure the changes in CNS catecholamine levels (DA, NE and Epi) in key brain regions (e.g., hypothalamus) following intraperitoneal administration of PNMT inhibitors We have confirmed that this method can provide results on literature PNMT inhibitors in complete agreement with data obtained from brain homogenate studies. We have cloned and expressed human brain PNMT (hPNMT). The X-ray crystal structure of hPNMT complexed with S-adenosy-L-homocysteine and SK&F 29661 (a competitive inhibitor of PNMT) has recently been determined at 2.4 Angstroms. We propose to take full advantage of these highly integrated results and use structure-based drug design to optimize our lead inhibitors to enhance their PNMT activity and reduce their alpha2 affinity. We have proposed several template skeletons for the structure- based design work based on existing lead inhibitors and the new crystal structure. We also propose to develop a high throughput PNMT screen to search libraries of compounds to identify other leads, particularly the sample libraries of the late Professors E. E. Smissman and M. P. Mertes of the University of Kansas. Lead optimization studies will employ parallel synthesis methods where appropriate. We have developed comparative molecular field analysis (CoMFA) models of both the active site of PNMT and the alpha2 adrenoceptor. The latter model will be refined as new results are obtained as an aid to achieving high selectivity in the new inhibitors. A further aid in the inhibitor design will be site- directed mutagenesis experiments aimed at determining the important amino acid residues for PNMT inhibitor binding. Once we have identified potent inhibitors that are (1) selective (minimal affinity at alpha2 adrenoceptors and other neurotransmitter receptors), (2) lipophilic enough to enter the CNS, and (3) shown to lower CNS Epi levels following i.p. administration, investigation of the effects of these inhibitors on blood pressure and hear rate will be determined. These inhibitors will be the first pharmacological tools available to help determine the function(s) of Epi in the CNS. This project is ripe and ready to burst forth with important new results.

肾上腺素（Epi）约占中枢神经系统（CNS）儿茶酚胺的5%，它与许多神经调节过程有关。我们（和其他人）已经证明，苯乙醇胺n -甲基转移酶（PNMT, e.c. 2.1.1.28； Epi生物合成的终端酶）抑制剂可以降低自发性高血压大鼠的血压。然而，所有检测血压作用的抑制剂也对α 2-肾上腺素受体具有高亲和力，这可能对观察到的药理作用有重要贡献。我们现在有非常选择性的先导抑制剂（非常低的α 2亲和力），并且具有足够的亲脂性（使用BBMEC模型）来穿透血脑屏障。我们开发了微透析技术，使我们能够测量腹腔注射PNMT抑制剂后大脑关键区域（如下丘脑）CNS儿茶酚胺水平（DA， NE和Epi）的变化。我们已经证实，该方法可以提供文献PNMT抑制剂的结果，与从脑匀浆研究中获得的数据完全一致。我们克隆并表达了人脑PNMT （hPNMT）。最近在2.4埃下测定了hPNMT与s -腺苷- l -同型半胱氨酸和SK&F 29661 （PNMT的竞争抑制剂）配合的x射线晶体结构。我们建议充分利用这些高度整合的结果，并使用基于结构的药物设计来优化我们的先导抑制剂，以提高其PNMT活性并降低其alpha2亲和力。我们在现有的铅抑制剂和新的晶体结构的基础上，提出了几种基于结构的模板骨架。我们还建议开发一种高通量PNMT筛选来搜索化合物库以识别其他线索，特别是已故堪萨斯大学教授E. E. Smissman和M. P. Mertes的样本库。铅优化研究将在适当的情况下采用平行合成方法。我们建立了PNMT和α 2肾上腺素受体活性位点的比较分子场分析（CoMFA）模型。后一种模型将被改进，因为获得了新的结果，以帮助在新的抑制剂中实现高选择性。抑制剂设计的进一步帮助将是定点诱变实验，旨在确定PNMT抑制剂结合的重要氨基酸残基。一旦我们确定了有效的抑制剂(1)选择性（对α 2肾上腺素受体和其他神经递质受体的亲和力最小），(2)亲脂性足以进入中枢神经系统，(3)显示出在i.p.给药后降低中枢神经系统Epi水平，这些抑制剂对血压和心率的影响将被确定。这些抑制剂将是第一个可用的药理学工具，以帮助确定Epi在中枢神经系统中的功能。这个项目已经成熟，准备迸发出新的重要成果。