FOLYLPOLYGLUTAMATE SYNTHETASE IN CANCER CHEMOTHERARY

癌症化疗中的酰基聚谷氨酸合成酶

• 批准号: 2091185
• 负责人: JOHN J MCGUIRE
• 金额: $ 14.83万
• 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 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2091185
• 关键词: FOLYLPOLYGLUTAMATE; SYNTHETASE; CANCER; CHEMOTHERARY

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.

拟议研究的长期目标是开发改进的 利用叶酰-或的某些方面治疗人类癌症 抗叶聚(γ-谷氨酸)合成。叶基多谷氨酸盐是 对细胞增殖至关重要，而经典的多谷氨酸 抗叶酸与它们的细胞毒性有关，而且常常是关键的。 行动。聚谷氨酸代谢物的合成在药物中的作用 抗性，并可能在选择性方面发挥作用。全生化 对叶酰-AND的合成和功能的认识 因此，抗叶基多谷氨酸盐可能允许开发新的试剂或 旨在利用这一关键过程的战略。这个长期目标 将在这一批款期内通过以下具体目标加以解决 其中包含了本实验独有的元素： 1.人叶基聚谷氨酸的设计、合成及表征 合成酶(FPGS)抑制剂。合理设计缓蚀剂的基础是 在本实验室中使用纯化的、良好的 具有人类FPGS的特征。新型抗叶酸盐，可能潜在地抑制 FPGS(基于结构)或将添加到FPGS结构-活动 并对数据库进行了研究。合成由公认的专家进行 在对抗敌化学方面，主要是M.G.奈尔博士和J.R.派珀博士。全 在这个实验室里，我们将对防腐药物进行全面的研究。感兴趣的是第二位 产生第一个类似物，人的FPGS特异性抑制物， 5，8-二氮-蝶酰-鸟氨酸(Dr.Piper)，其设计和 在这个实验室里表现出来的特征。将在调查中使用FPGS抑制剂 正常人白血病多谷氨酸代谢及其调节的研究 细胞系。从这类研究中获得的基本知识可能会带来更多 有效使用现有的抗病毒药物或提出新的治疗靶点 干预。 2.人类白血病的发生发展和特性的特征 对甲氨蝶呤(MTX)耐药的细胞株 MTX聚谷氨酸合成。在第一个也是唯一一个具有 获得的MTX抗性减少了MTX聚谷氨酸的合成，因为 唯一的来源，抗性被追溯到降低的FPGS活性。类似 已经在临床上发现了耐药性。这在临床上是相关的 将对抗性表型进行表征。进一步核实这一点 将获得抵抗的唯一机制，以及 交叉阻力数据。酶学性质和物理性质的比较 对敏感和耐药细胞的研究可能提供对 变化的性质和对设计FPGS有用的信息 抑制剂(特定目标1)。我们还建议研究其动力学。 这种表型的发生和影响频率的因素 在体外发生。 3.人FPGS相关酶分子探针的研制 用于合成聚谷氨酸。将分离出一个人的FPGS基因，用于 该酶的调节和分子药理学研究。这 探头也将用来表征缺陷的性质 多聚谷氨酰化缺失的抗甲氨蝶呤品系(特定目标2)。一个 将准备表达载体以使该酶大量可用 用于酶特性和抑制剂设计研究的数量。在……里面 此外，还将准备针对FPGS蛋白的抗体进行研究 在蛋白质水平上的调控。