Rational Drug Design Based on Chemical Mechanisms

描述（由申请人提供）：在申请支持期间， 在本申请中，我们计划进一步研究两种酶的作用， 叶酸聚-γ-谷氨酸合成酶（FPGS，EC 6.3.2.17）和γ-谷氨酰 水解酶（GH，EC 3.4.19.9），参与生物合成和水解 叶酰聚-γ-谷氨酸盐使叶酸盐缀合物。“一系列磷酸肽 和含氟谷氨酸的肽被提议用于研究， FPGS和GH的机制探针和/或抑制剂。的动力学 酶催化的γ-谷氨酰连接（FPGS）和水解（GH） 天然底物和我们的机制为基础的类似物将在 详细在与分子生物学家的一系列合作中， 晶体学家和药理学家，我们将使用这些新合成的 分子来研究细胞内聚-γ-谷氨酸形成在 叶酸一碳生化和抗叶酸化疗。具体目标 如下：第 1.完成次膦酸的立体选择性合成 Glu-γ-Glu的含酸类似物。其外消旋形式是一种有效 当掺入叶酸或抗叶酸类似物中时的FPGS抑制剂。完成 用于细胞培养研究的这些抑制剂的前药形式的合成。 2. 完成同位素标记的DDATHF基底物的合成 在动力学实验中。确定详细的动力学机制， 由FPGS催化的反应，特别是与可能的持续合成能力有关的反应 在连接多个谷氨酸。 3.与X射线合作 晶体学家使用含有磷酸氨基酸的叶酸盐和抗叶酸盐， 研究FPGS催化的结构基础，特别是在 多重连接的假设进行机制。 4.确定 GH催化反应的动力学机理。特别感兴趣的是 探索反应中间体的可能稳定化， 含有氟氨基酸的γ-谷氨酰肽。完成合成 在C-末端含有谷氨酸半醛的γ-谷氨酰肽， 潜在的GH抑制剂。引发含环氧化物的合成 作为半胱氨酸乳清蛋白酶抑制剂的肽模拟物。 5.完成 （2S）3.3-二氟谷氨酸立体选择性合成及其应用 掺入适当的γ-谷氨酰肽中用于机械 GH和FPGS催化的研究。

DESCRIPTION (provided by applicant): During the period of support requested in this application, we plan to investigate further the action of two enzymes, folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) and gamma-glutamyl hydrolase (GH, EC 3.4.19.9), involved in the biosynthesis and hydrolysis of folylpoly-gamma.glutamates the folate "conjugates." A series of phosphapeptides and fluoroglutamate-containing peptides are proposed for investigation as mechanistic probes and/or inhibitors of FPGS and GH. The kinetics of enyzyme-catalyzed gamma-glutamyl ligation (FPGS) and hydrolysis (GH) of the natural substrates and our mechanism-based analogs will be investigated in detail. In a series of collaborations with molecular biologists, crystallographers, and pharmacologists, we will use these newly synthesized molecules to study the role of intracellular poly-gamma-glutamate formation in folate one-carbon biochemistry and antifolate chemotherapy. The specific aims are as follows. 1. Complete the stereoselective synthesis of a phosphinic acid-containing analog of Glu-gamma-Glu. the racemic form of which is a potent FPGS inhibitor when incorporated in folate or antifolate analogs. Complete the synthesis of prodrug forms of these inhibitors for cell culture studies. 2. Complete the synthesis of isotopically labeled DDATHF-based substrates for use in kinetics experiments. Determine the detailed kinetic mechanism of the reaction catalyzed by FPGS especially as it pertains to possible processivity in the ligation of multiple glutamates. 3. In collaboration with x-ray crystallographers use folates and antifolates containing phosphoamino acids to investigate the structural basis of FPGS catalysis, especially in terms of the hypothesized processive mechanism of multiple ligations. 4. Determine the kinetic mechanism of the reaction catalyzed by GH. Of special interest is probing for possible stabilization of reaction intermediates with several gamma-glutamyl peptides containing fluoroamino acids. Complete the synthesis of gammaglutamyl peptides containing glutamate semialdehyde at the C-terminus as potential GH inhibitors. Initiate the synthesis of epoxide-containing peptidomimetics as cysteine orotease inhibitors. 5. Complete the stereoselective synthesis of (2S) 3.3-difluoroglutamic acid and its incorporation into appropriate gammaglutamyl peptides for use in mechanistic studies of GH and FPGS catalysis.