DESIGN OF INHIBITORS OF EPINEPHRINE BIOSYNTHESIS

肾上腺素生物合成抑制剂的设计

• 批准号: 6079046
• 负责人: GARY L GRUNEWALD
• 金额: $ 3.09万
• 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/6079046
• 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：苯乙醇胺 N-甲基转移酶（PNMT，E.C.2.1.1.28） 是催化生物合成最终步骤的酶 肾上腺素（Epi）。 Epi 约占中枢神经系统的 5% （中枢神经系统）儿茶酚胺含量，它与许多 大脑中的神经调节过程。 已被证明（我们的实验室 等）PNMT 抑制剂可以降低血压 自发性高血压大鼠 然而，目前所有的抑制剂 可用的α2-肾上腺素能受体具有高亲和力，这可以 对观察到的药理作用有显着贡献。 一个数字 探针以确定 PNMT 的活性位点结合要求 a2-肾上腺素受体已在我们实验室合成并用于 开发计算机图形模型（比较分子场分析； CoMFA) 的两个站点。 这些模型已用于设计新的配体 根据初步研究，合成具有以下潜力： 对 PNMT 活性位点表现出所需水平的选择性 a2-肾上腺素受体。 这些配体的评估结果将 用于完善我们的计算机模型并提高其辅助能力 设计一种有效且选择性的 PNMT 抑制剂，具有足够的 具有亲脂性，可穿过血脑屏障。 人脑 PNMT (hPNMT) 最近已被克隆和表达（与 M. McLeish 合作） 以及在三种 PNMT 存在下的三种不同的 hPNMT 晶体 具有不同结合特性的抑制剂已经被培育出来（在 与 J. Martin 合作）；晶体结构正在等待解决。 计算机建模、蛋白质晶体学（基于结构的 配体设计）、定点突变和同源建模 hPNMT 的活性位点将使我们能够开发出更具选择性和更有效的药物 抑制剂。 PNMT 的高通量筛选（基于与 AdoHcy 水解酶（与 R. Borchardt 合作）将被开发并 用于筛选结构多样的化合物的独特库 斯米斯曼和默特斯在堪萨斯大学收集的样品。 线索将是 在适当的情况下使用并行合成技术进​​行优化。 毛细管电泳微透析实验（合作 与 S. Lunte）将允许测量对中枢神经系统 Epi 水平的影响 清醒大鼠体内新的 PNMT 抑制剂。 全部结果 子项目将协同作用，从而形成一个高度综合的项目 PNMT 的有效选择性抑制剂，可用作 探索 Epi 在 CNS 中发挥的作用的药理学工具，以及 特别是，有可能确定药物治疗的新机制 高血压。