RATIONAL DRUG DESIGN BASED ON CHEMICAL MECHANISMS

基于化学机制的合理药物设计

• 批准号: 6124477
• 负责人: JAMES K COWARD
• 金额: $ 21.68万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124477
• 关键词: HTC cell; antineoplastics; cell free system; chemical kinetics; chemical structure; dipeptides; drug design /synthesis /production; enzyme activity; enzyme inhibitors; enzyme substrate complex; fluoroaminoacid; folate antagonist; glutamate dehydrogenase; glutaminase; hydrolase; ligands; neoplasm /cancer pharmacology; oligopeptides; outcomes research; peptide analog; phosphopeptides; recombinant proteins; tetrahydrofolylpolyglutamate synthase; zinc

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.

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理， Folypoly-Gamma--谷氨酸合成酶（FPGS，EC 6.3.2.17）和γ-谷氨基 水解酶（GH，EC 3.4.22.17），参与生物合成和水解的水解 Forypoly-Gamma-Glutamates；即，叶酸“缀合物”。 最近 合成的底物和抑制剂以及选定的 磷脂肽和含有含有合成肽的氟氨基酸酸 在这段支持期间，将用作机械探针和/或 FGP和GH的抑制剂。 与Drs合作。约翰·麦奎尔（John McGuire）和约翰 加利文，我们将使用这些新合成的分子来研究 叶酸一碳中细胞内聚粉 - 谷氨酸形成 生物化学和抗叶酸化学疗法。 具体目的是 如下：1。使用重组人FPG，评估动力学 在稳态条件下的反应，以区分 过程和非过程动力学机制。 2。确定 “依赖位置”增强或终止的机械基础 使用3,3-二氟谷氨酸的FPG催化结扎 寡肽。 3。使用纯化的GH分离的大鼠肝癌细胞，确定 GH催化的水解的金属含量和机制 OLGI-GAMMA-谷氨酰肽。 使用新合成的 含氟谷氨酸二肽，确定四面体 中间体在催化过程中积累。 4。完成合成 假定两个类似四面体中间体的磷酸肽类似物 在FPGS和GH催化反应期间形成。 评估这些新 化合物作为FPG和/或GH抑制剂，如果发现有效 无细胞测定，研究这些化合物作为调节剂的作用 完整的聚谷氨酸生物合成和/或聚谷氨酸水解 哺乳动物细胞。