DESIGN OF INHIBITORS OF EPINEPHRINE BIOSYNTHESIS

苯乙醇胺N-甲基转移酶（PNMT，E.C. 2.1.1.28）是酶 催化肾上腺素生物合成的最后一步 这种酶的水平增加，发现在压力期间， 高血压埃皮内托与多起 大脑中的神经调节过程。我们（和其他人）已经证明了 PNMT抑制剂可以自发降低血压， 高血压大鼠然而，目前可获得的所有抑制剂都具有 对α 2肾上腺素能受体的高亲和力 与观察到的药理学作用显著相关。我们先前已经 合成了许多探针来确定活性位点结合 PNMT的要求，并使用这些信息开发了一台计算机， 活动站点的图形模型。该模型已被用于设计新的 用于合成的配体，基于初步研究，具有 表现出PNMT所需选择性水平的潜力 肾上腺素能受体上的活性部位。这些新的配体将是 合成并评价它们对PNMT的亲和力和选择性。的 结果将被纳入一个改进的计算机模型的积极 PNMT的网站，也将用于构建一个互补的模型， α肾上腺素能受体此外，我们将继续高度重视 成功的研究采用转移核Overhauser 几种关键抑制剂构象的增强测定法 以及当结合到PNMT的活性位点时的底物。的nmr 构象信息将允许改进预测 计算机模型的实用性。我们还将对氨基进行亲和标记， 酸残基的活性位点，并使用此信息，结合 与基于知识的同源性建模，构建一个可能的三个- 酶的三维模型。足够量的纯PNMT将 从牛肾上腺中获得，以允许尝试结晶 并测定其X射线晶体结构。分子 生物学实验的建议，将使我们能够获得人类 肾上腺PNMT和可能的人脑PNMT的量足以 使用更相关的人类酶评估潜在的抑制剂， 并尝试结晶和X射线晶体结构测定 所有子项目的成果将结合起来， 时尚，导致合成一个高度有效的和选择性的 PNMT抑制剂。这种抑制剂将是一种有用的药理学 一种探索肾上腺素在中枢神经系统中作用的工具 系统，特别是潜在地确定了药物 治疗高血压。

Phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) is the enzyme that catalyzes the terminal step in the biosynthesis of epinephrine. Increased levels of this enzyme are found during periods of stress and in hypertension. Epinephrine has been implicated in a number of neuroregulatory processes in the brain. We (and others) have demonstrated that inhibitors of PNMT can lower blood pressure in spontaneously hypertensive rats. However, all of the inhibitors presently available have high affinity for alpha2-adrenergic receptors that could contribute significantly to the observed pharmacological effects. We have previously synthesized a number of probes to determine the active site binding requirements for PNMT and have used this information to develop a computer graphics model of the active site. The model has 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 alpha-adrenergic receptor. These new ligands will be synthesized and evaluated for their affinity and selectivity for PNMT. The results will be incorporated into an improved computer model of the active site of PNMT, and will also be used to construct a complementary model of the alpha-adrenergic receptor. In addition, we will continue our highly successful studies employing the transferred nuclear Overhauser enhancement method to determine the conformation of a few key inhibitors and substrates when bound to the active site of PNMT. The nmr conformational information will allow improvement of the predictive utility of the computer model. We will also do affinity labeling of amino acid residues at the active site and use this information, in conjunction with knowledge-based homology modeling, to construct a possible three- dimensional model of the enzyme. A sufficient quantity of pure PNMT will be obtained from bovine adrenals to allow attempts at crystallization of the enzyme and a determination of its X-ray crystal structure. Molecular biology experiments are proposed that will allow us to obtain human adrenal PNMT and possibly human brain PNMT in quantities sufficient to evaluate potential inhibitors using the more relevant human enzyme and also to attempt crystallization and X-ray crystal structure determination of it. Results from all sub-projects will be combined in a synergistic fashion to lead to the synthesis of a highly potent and selective inhibitor of PNMT. Such an inhibitor would be a useful pharmacological tool to probe the role played by epinephrine in the central nervous system, and, in particular, potentially identify a new mechanism for drug treatment of hypertension.