RATIONAL DRUG DESIGN BASED ON CHEMICAL MECHANISMS

描述：在本申请中要求的支持期间，我们 计划进一步研究两种酶的作用机制， 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 3.4.22.17），参与生物合成和水解 叶酸聚-γ-谷氨酸盐;即，叶酸“共轭物”。 最近 合成的底物和抑制剂，以及选择的 待合成的含磷肽和含氟氨基酸的肽 在此支持期间，将用作机械探针和/或 FGPS和GH的抑制剂。 与约翰·麦奎尔博士和约翰· Galivan，我们将使用这些新合成的分子来研究 叶酸单碳中细胞内聚γ-谷氨酸形成 生物化学和抗叶酸化疗。 具体目标如下： 如下：1. 使用重组人FPGS，评价 在预稳态条件下的反应，以区分 进行性和非进行性动力机制。 2. 确定 “位置依赖性”增强或终止的机制基础 使用含3，3-二氟谷氨酸的 寡肽。 3. 用纯化的GH分离大鼠肝癌细胞，测定 GH催化水解的金属含量和机理 寡-γ-谷氨酰肽。 使用新合成的 含氟代谷氨酸盐的二肽，确定四面体 中间体在催化过程中积累。 4. 完成合成 两个类似四面体中间体的磷酸肽类似物， 在FPGS和GH催化的反应过程中形成。 评估这些新 作为FPGS和/或GH抑制剂的化合物，并且如果发现有效于 无细胞测定，研究这些化合物作为调节剂的作用， 在完整细胞中的聚谷氨酸生物合成和/或聚谷氨酸水解 哺乳动物细胞

DESCRIPTION: During the period of support requested in this application, we plan to investigate further the mechanism of action of two enzymes, folypoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) and gamma-glutamyl hydrolase (GH, EC 3.4.22.17), involved in the biosynthesis and hydrolysis of folypoly-gamma-glutamates; i.e., the folate "conjugates". Recently synthesized substrates and inhibitors, together with selected phosphapeptides and fluoroamino acid containing peptides to be synthesized during this period of support, will be used as mechanistic probes and/or inhibitors of FGPS and GH. In collaboration with Drs. John McGuire and John Galivan, we will use these newly synthesized molecules to study the role of intracellular poly-gamma-glutamate formation in folate one-carbon biochemistry and in antifolate chemotherapy. The specific aims are as follows: 1. Using recombinant human FPGS, evaluate the kinetics of the reaction under pre-steady-state conditions in order to distinguish between a processive and non-processive kinetic mechanism. 2. Determine the mechanistic basis for the "position-dependent" enhancement or termination of FPGS-catalyzed ligation using 3,3-difluoroglutamate-containing oligopeptides. 3. Using purified GH isolated rat hepatoma cells, determine the metal content and mechanism of GH-catalyzed hydrolysis of olgi-gamma-glutamyl peptides. Using newly synthesized fluoroglutamate-containing dipeptides, determine if tetrahedral intermediates accumulate during catalysis. 4. Complete the synthesis of phosphapeptide analogs of two similar tetrahedral intermediates postulated to form during the FPGS- and GH-catalyzed reaction. Evaluate these new compounds as FPGS and/or GH inhibitors and, if found to be effective in cell-free assays, investigate the action of these compounds as modulators of polyglutamate biosynthesis and/or polyglutamate hydrolysis in intact mammalian cells.