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％，并且与许多神经调节过程有关。我们（和其他人）表明，苯甲胺N-甲基转移酶的抑制剂（PNMT，E。C. 2.1.1.28; EPI生物合成中的末端酶）可以降低自发性高血压大鼠的血压。但是，所有检查的血压效应抑制剂对α2-肾上腺素受体的亲和力也很高，这可能对观察到的药理作用产生重大贡献。现在，我们拥有非常有选择性的铅抑制剂（非常低的α2亲和力），并且具有足够的亲脂性（使用BBMEC模型）来渗透血脑屏障。我们已经开发了微透析技术，以使我们能够测量关键大脑区域（例如下丘脑）PNMT抑制剂的关键大脑区域（例如下丘脑）中CNS儿茶酚胺水平（DA，NE和EPI）的变化，我们已经确认，我们可以确认，该方法可以与大脑构成的脑料相一致的文献pNMT抑制剂提供该方法，从而为数据获得的研究提供了脑料研究的结果。我们已经克隆并表达了人脑PNMT（HPNMT）。 HPNMT的X射线晶体结构与S-腺苷-L-型 - 黑ossteine和SK＆F 29661（PNMT的竞争性抑制剂）复合在一起，已在2.4埃埃斯特罗姆斯下确定。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铅抑制剂，以增强其PNMT活性并减少其α2亲和力。我们已经根据现有的铅抑制剂和新的晶体结构提出了几个模板骨架，用于基于结构的设计工作。我们还建议开发一个高吞吐量的PNMT屏幕，以搜索化合物的库，以识别其他潜在客户，尤其是已故教授E. 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的合成方法。我们已经开发了PNMT活性位点和α2肾上腺素受体的比较分子场分析（COMFA）模型。后一种模型将被改进，因为获得新的结果是为了实现新抑制剂的高选择性。抑制剂设计的进一步有助于位置的诱变实验，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（1）选择性的有效抑制剂（α2肾上腺素受体和其他神经递质受体的最小亲和力），（2）足够进入CNS的亲脂性，以及（3）在i.p后降低CNS EPI水平。将确定对这些抑制剂对血压和听力率的影响的研究。这些抑制剂将是第一个可用来帮助确定CNS中EPI功能的药理工具。该项目已经成熟，并准备出现重要的新结果。