DESIGN OF INHIBITORS OF EPINEPHRINE BIOSYNTHESIS

肾上腺素生物合成抑制剂的设计

• 批准号: 2840155
• 负责人: GARY L GRUNEWALD
• 金额: $ 2.77万
• 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 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2840155
• 关键词: X ray crystallography; active sites; alpha adrenergic receptor; blood brain barrier; catechol methyltransferase; chemical models; computer simulation; conformation; drug design /synthesis /production; drug screening /evaluation; enzyme inhibitors; enzyme mechanism; enzyme structure; epinephrine; isozymes; laboratory rat; methyltransferase; microdialysis; receptor binding; structural biology

DESCRIPTION: Phenylethanolamine N-methyltransferase (PNMT, E. C. 2.1.1.28) is the enzyme that catalyzes the terminal step in the biosynthesis of epinephrine (Epi). Epi comprises about 5 percent of central nervous system (CNS) catecholamine content and it has been implicated in a number of neuroregulator processes in the brain. It was demonstrated (our laboratory and others) that inhibitors of PNMT can lower blood pressure in spontaneously hypertensive rats However, all of the inhibitors presently available have high affinity for a2-adrenergic receptors, which could contribute significantly to the observed pharmacological effects. A number of probes to determine the active site binding requirements for PNMT and for the a2-adrenoceptor have been synthesize in our laboratory and used to develop computer graphics models (comparative molecular field analysis; CoMFA) of the two sites. These models have been used to design new ligands for synthesis that, based on preliminary studies, have the potential of exhibiting the desired level of selectivity for the PNMT active site over the a2-adrenoceptor. The results from the evaluation of these ligands will be used to refine our computer model and improve its ability to aid in the design of a potent and selective inhibitor of PNMT with sufficient lipophilicity to cross the blood brain barrier. Human brain PNMT (hPNMT) has recently been cloned and expressed (in collaboration with M. McLeish) and thre different crystals of hPNMT in the presence of three PNMT inhibitors with different binding characteristics have been grown (in collaboration with J. Martin); the crystal structures are awaiting solution. A combination of computer modeling, protein crystallography (structure-based ligand design), site-directed mutagenesis and homology modeling of the active site of hPNMT will allow us to develop more selective and potent inhibitors. A high throughput screen for PNMT (based on coupling with AdoHcy hydrolase, in collaboration with R. Borchardt) will be developed and used to screen unique libraries of structurally diverse compounds in the Smissman and Mertes sample collections at the U. of Kansas. Leads will be optimized with parallel synthesis techniques where appropriate. Microdialysis experiments with capillary electrophoresis (in collaboration with S. Lunte) will allow measurement of the effects on CNS Epi levels of new PNMT inhibitors in vivo in conscious rats. Results from all sub-projects will act synergistically to lead to the synthesis of a highly potent and selective inhibitor of PNMT, which would be useful as a pharmacological tool to probe the role(s) played by Epi i the CNS, and, in particular, potentially identify a new mechanism for drug treatment of hypertension.

描述：苯甲醇胺N-甲基转移酶（PNMT，E。C。2.1.1.28） 是催化终末步骤的酶 肾上腺素（EPI）。 EPI约占中枢神经系统的5％ （CNS）儿茶酚胺含量，并与许多 大脑中的神经调节器过程。 它被证明了（我们的实验室 其他）PNMT抑制剂可以降低血压 但是，自发性高血压大鼠目前所有抑制剂 可用的对A2-肾上腺素能受体具有高亲和力，可以 对观察到的药理学作用做出了重大贡献。 一个数字 探针以确定PNMT的主动位点结合要求和 A2-肾上腺素受体已在我们的实验室中合成，曾经习惯 开发计算机图形模型（比较分子场分析； 两个站点的comfa）。 这些模型已用于设计新的配体 对于综合，基于初步研究，具有 在PNMT主动部位表现出所需的选择性水平 A2-肾上腺素受体。 评估这些配体的结果将 被用来完善我们的计算机模型并提高其协助的能力 PNMT的有效和选择性抑制剂的设计 跨血脑屏障的亲脂性。 人脑PNMT（HPNMT） 最近被克隆并表达（与M. McLeish合作） 在存在三个PNMT的情况下，HPNMT的三个不同的晶体 具有不同结合特征的抑制剂已增长（在 与J. Martin合作）；晶体结构正在等待解决方案。 计算机建模，蛋白质晶体学的组合（基于结构的 配体设计），定向的诱变和同源性建模 HPNMT的活跃站点将使我们能够发展出更具选择性和有效性 抑制剂。 PNMT的高吞吐量屏幕（基于与 将开发与R. Borchardt合作的Adohcy水解酶） 用于筛选在结构上不同化合物的独特库 Smissman和Mertes在堪萨斯州的样本收集。 线索将是 在适当的情况下，用并行合成技术优化。 毛细管电泳的微透析实验（合作 使用S. lunte）将允许测量对CNS EPI水平的影响 新的PNMT抑制剂体内有意识的大鼠。 所有人的结果 亚项目将协同起作用，导致高度合成 PNMT的有效和选择性抑制剂，可作为一个 探测Epi I The CNS扮演的作用的药理工具，并在 特别，有可能确定一种用于药物治疗的新机制 高血压。