FOLYLPOLYGLUTAMATE SYNTHETASE IN CANCER CHEMOTHERARY

癌症化疗中的酰基聚谷氨酸合成酶

• 批准号: 3185693
• 负责人: JOHN J MCGUIRE
• 金额: $ 14.33万
• 依托单位: ROSWELL PARK CANCER INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3185693
• 关键词: aminopterin; antibody formation; antileukemic agent; cell line; chemical structure function; clone cells; cofactor; complementary DNA; cytotoxicity; drug design /synthesis /production; drug metabolism; drug resistance; enzyme inhibitors; enzyme mechanism; enzyme substrate complex; folate antagonist; laboratory rabbit; leukemia; ligase; methotrexate; neoplasm /cancer chemotherapy; neoplasm /cancer pharmacology; neoplastic cell; nonhuman therapy evaluation; nucleic acid probes; ornithine; polyglutamates; protein biosynthesis; tissue /cell culture; transfection /expression vector

The long-term goal of the proposed research is to develop improved treatment for human cancer by exploiting aspects of folyl- or antifolylpoly(gamma-glutamate) synthesis. Folylpolyglutamates are essential for cell proliferation, while polyglutamates of classical antifolates are implicated in, and often critical for, their cytotoxic action. Synthesis of polyglutamate metabolites plays a role in drug resistance and may have a role in selectivity. Complete biochemical understanding of the synthesis and function of folyl- and antifolylpolyglutamates may thus allow the development of new agents or strategies designed to exploit this critical process. This long term goal will be addressed in this grant period through the following specific aims which incorporate elements unique to this lab: 1. Design, synthesis, and characterization of human folylpolyglutamate synthetase (FPGS) inhibitors. Rational design of inhibitors is based on FPGS substrate specificity data generated in this lab using purified, well- characterized human FPGS. Novel antifolates that could potentially inhibit FPGS (based on structure) or that will add to the FPGS structure-activity data base are also studied. Syntheses are performed by recognized experts in antifol chemistry, principally Drs. M.G. Nair and J.R. Piper. All antifols will be fully characterized in this lab. Of interest are 2nd generation analogs of the first, human FPGS-specific inhibitor, 5,8-dideaza-pteroyl-ornithine (Dr. Piper), which was designed and characterized in this lab. FPGS inhibitors will be used in investigations of polyglutamate metabolism and its regulation in intact human leukemia cell lines. Basic knowledge derived from such studies may allow more effective use of current antifols or suggest new targets for therapeutic intervention. 2. Characterization of the development and properties of human leukemia cell lines that are resistant to methotrexate (MTX) because of decreased MTX polyglutamate synthesis. In the first and only cell lines with acquired MTX resistance having reduced MTX polyglutamate synthesis as the sole source, resistance was traced to decreased FPGS activity. Similar resistance has been identified in the clinic. This clinically relevant resistance phenotype will be characterized. Further verification that this is the sole mechanism of resistance will be obtained, along with cross-resistance data. Comparison of the enzymatic and physical properties of the sensitive and resistant cell FPGS may provide both insight into the nature of the change and information useful for the design of FPGS inhibitors (Specific Aim 1). We also propose to study the kinetics of occurrence of this phenotype and factors affecting the frequency of occurrence in vitro. 3. Development of molecular probes for human FPGS, the enzyme responsible for polyglutamate synthesis. A human FPGS cDNA will be isolated to use in studies of the regulation and molecular pharmacology of this enzyme. This probe would also be used to characterize the nature of the defect in the MTX-resistant lines deficient in polyglutamylation (Specific Aim 2). An expression vector will be prepared to make the enzyme available in large quantity for enzyme characterization and inhibitor design studies. In addition, antibodies to the FPGS protein will be prepared to study regulation at the protein level.

该研究的长期目标是开发改进的 利用叶酸的某些方面治疗人类癌症 antifolylpoly（gamma-glutamate）合成。 Folylpolyglutamates是 细胞增殖所必需的，而经典的聚谷氨酸 抗叶酸剂与它们的细胞毒性有关，并且通常是至关重要的。 行动上 聚谷氨酸代谢产物的合成在药物中起作用 电阻，并可能具有选择性的作用。 全生化 了解叶酸的合成和功能， 因此，抗叶酸聚谷氨酸盐可以允许开发新的药剂， 旨在利用这一关键过程的战略。 这个长期目标 将通过以下具体目标在本赠款期内予以解决 其中包含了本实验室独有的元素： 1. 人叶酸聚谷氨酸酯的设计、合成及表征 合成酶（FPGS）抑制剂。 缓蚀剂的合理设计是基于 本实验室使用纯化的良好- 人的FPGS。 可能抑制叶酸代谢的新型抗叶酸剂 FPGS（基于结构）或将添加到FPGS结构-活性 数据库进行了研究。 合成由公认的专家进行 在抗真菌化学方面，主要是M.G.博士。Nair和J.R.派珀 所有 antifols将在本实验中得到充分表征。 感兴趣的是第二 第一代人FPG特异性抑制剂的类似物， 5，8-二脱氮-蝶酰-鸟氨酸（Piper博士），其被设计并 在这个实验室里。 FPGS抑制剂将用于研究 正常人白血病多聚谷氨酸代谢及其调节 细胞系 从这些研究中获得的基本知识可能会使更多的 有效使用现有抗叶酸剂或建议新的治疗靶点 干预 2. 人类白血病的发生和性质的表征 细胞系是耐甲氨蝶呤（MTX），因为减少 MTX聚谷氨酸合成。 在第一个也是唯一一个 获得的MTX抗性具有减少的MTX聚谷氨酸盐合成， 唯一来源，抗性可追溯至FPGS活性降低。 类似 在临床上已经发现了耐药性。 到临床相关 将表征抗性表型。 进一步证实， 是唯一的抵抗机制，将获得，沿着 交叉抗性数据。 酶性质和物理性质的比较 敏感细胞和耐药细胞的FPGS可以提供对 变更的性质和对FPGS设计有用的信息 抑制剂（具体目标1）。 我们还建议研究的动力学 这种表型的发生和影响频率的因素 发生在体外。 3. 人类FPGS分子探针的开发， 用于聚谷氨酸合成。 将分离人FPGS cDNA以用于 研究这种酶的调节和分子药理学。 这 探针也将用于表征缺陷的性质， 多聚谷氨酰化缺陷的MTX抗性系（特异性目标2）。 一个 将制备表达载体以使酶可大量获得。 用于酶表征和抑制剂设计研究的数量。 在 此外，将制备针对FPGS蛋白的抗体用于研究 蛋白质水平的调控。