RATIONAL DRUG DESIGN BASED ON CHEMICAL MECHANISMS

描述：在本申请所要求的支持期间，我们 计划进一步研究两种酶的作用机制， 叶多聚-谷氨酸合成酶和γ-谷氨酰基 水解酶(GH，EC 3.4.22.17)，参与生物合成和水解酶 叶酸-多-伽马-谷氨酸；即叶酸“结合物”。最近 合成底物和抑制剂，以及选定的 待合成的磷多肽和含氟氨基酸多肽 在此支撑期内，将用作机械探头和/或 FGPS和GH的抑制剂。与John McGuire博士和John McGuire博士合作 Galivan，我们将利用这些新合成的分子研究 叶酸一碳的胞内聚γ-谷氨酸的形成 在生物化学和抗叶酸化疗中。具体目标如下 研究结果如下：1.利用重组人FPGS，评价其代谢动力学。 在预稳定状态条件下的反应，以区分 进行性和非进行性的动力机制。2.确定 “位置依赖”增强或终止的机制基础 含3，3-二氟谷氨酸的FPGS催化的连接反应 寡肽。3.用纯化的生长激素分离的大鼠肝癌细胞，测定 生长激素催化水解物的金属含量及机理 奥吉-伽马-谷氨酰多肽。使用新合成的 含氟谷氨酸二肽，测定四面体 中间体在催化过程中积累。4.完成……的合成 假设两个相似的四面体中间体的磷肽类似物 在FPGS和GH催化的反应中形成。评估这些新功能 化合物作为FPGS和/或GH抑制剂，如果被发现在 无细胞分析，研究这些化合物作为调节剂的作用 完整的聚谷氨酸的生物合成和/或聚谷氨酸的水解 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.