Design of Inhibitors of Epinephrine Biosynthesis

肾上腺素(Epi)约占中枢神经系统(CNS)儿茶酚胺的5%，参与多种神经调节过程。我们(和其他人)已经证明苯乙醇胺N-甲基转移酶的抑制剂(PNMT，E.C.2.1.1.28；Epi生物合成的末端酶)可以降低自发性高血压大鼠的血压。然而，所有研究血压效应的抑制剂对α2-肾上腺素能受体也有很高的亲和力，这可能对观察到的药理作用有很大贡献。我们现在有非常有选择性的铅抑制剂(非常低的α2亲和力)，并且有足够的亲脂性(使用BBMEC模型)来穿透血脑屏障。我们已经开发了微透析技术，使我们能够测量在腹膜注射PNMT抑制剂后，中枢关键脑区(如下丘脑)中枢儿茶酚胺(DA、NE和Epi)的变化。我们已经证实，这种方法可以提供与脑匀浆研究数据完全一致的文献中的PNMT抑制剂的结果。我们克隆并表达了人脑PNMT(HPNMT)。最近测定了人PNMT与S-腺苷-L-同型半胱氨酸和SK&F 29661(PNMT的竞争性抑制剂)形成的络合物的X射线晶体结构为2.4埃。我们建议充分利用这些高度集成的结果，并使用基于结构的药物设计来优化我们的先导抑制剂，以增强它们的PNMT活性，降低它们的α2亲和力。我们基于现有的铅抑制剂和新的晶体结构，提出了几种模板骨架用于基于结构的设计工作。我们还建议开发一种高通量的PNMT屏幕来搜索化合物的库以确定其他铅，特别是堪萨斯大学已故教授E.E.Smissman和M.P.Mertes的样本库。牵头优化研究将在适当的情况下采用并行综合方法。我们建立了PNMT活性部位和α2肾上腺素受体的比较分子场分析(CoMFA)模型。后一种模型将随着新结果的获得而得到改进，以帮助实现新抑制剂的高选择性。对抑制剂设计的进一步帮助将是旨在确定PNMT抑制剂结合的重要氨基酸残基的定点突变实验。一旦我们确定了有效的抑制剂，它们是(1)选择性的(在α2肾上腺素受体和其他神经递质受体上的最小亲和力)，(2)足够亲脂的进入中枢神经系统，以及(3)显示出在I.P.后降低中枢神经系统Epi水平。给药后，将确定这些抑制剂对血压和心率的影响。这些抑制剂将是第一批可用来帮助确定中枢神经系统中EPI功能的药理学工具(S)。这个项目已经成熟，准备推出重要的新成果。

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.