FOLYLPOLYGLUTAMATE SYNTHETASE IN CANCER CHEMOTHERAPY

描述：(申请者摘要)该项目的长期目标是 通过利用叶基-或的某些方面来改善人类癌症的治疗 抗叶聚(γ-谷氨酸)合成。叶基多谷氨酸盐是必不可少的 对于细胞生长，而经典的抗叶酸的多谷氨酸是 与它们的细胞毒作用有关，而且往往是关键的。一种新的合成方法 多聚谷氨酸代谢物也在耐药性中发挥作用。详细 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 将在本申请中通过以下具体目标加以解决。 具体目标1将探索叶基多谷氨酸合成酶(FPGS)， 作为药物靶标的负责合成聚谷氨酸的酶 发展。由于FPGS活性的突变缺失是致命的，所以FPGS是 癌症化疗的潜在治疗靶点。合理的设计 基于酶机制和构效关系的FPGS抑制剂 在申请人的实验室中使用同质、重组 人类的FPGS。令人感兴趣的是潜在的基于机制的抑制剂和 增强公认的基于叶酸的效力和/或摄取的修饰 含鸟氨酸的FPGS抑制剂。预期的生物效应 将在一个模型系统中探索来自FPGS抑制的 叶基多谷氨酸缺乏症。这些研究应该确定FPGS是否是 一个可行的药物靶点。具体目标2将界定 人白血病和人实体细胞对甲氨蝶呤和甲氨蝶呤的耐药性 肿瘤模型系统。第一次发现的FPGS活性降低 涉及申请者实验室的合作研究，现在是 在体外和体内被确定为抗叶酸的一种机制， 在诊所里。这种临床相关的耐药表型将是 进一步表现为其在临床前的频率和演变 白血病和实体瘤的模型。一个多细胞椭球系统将 人实体瘤抗叶酸模型的研制与鉴定 抵抗。特定目标3将定义人类的分子药理学 FPGS，特别是与抗叶酸抗性有关的。一种人FPGS基因的克隆 将用于FPGS的进一步分子药理学研究和 鉴定甲氨蝶呤抗性品系缺陷的性质 多聚谷氨酰化(特定目标2)。针对FPGS的抗肽抗体将是 用于在蛋白质水平上研究FPGS的调控及其在植物中的地位 耐甲氨喋呤的细胞系。抗人FGS蛋白全长的多克隆抗体 也将被准备和使用。

DESCRIPTION: (Applicant's Abstract) The long-term goal of this program is to improve human cancer treatment by exploiting aspects of folyl- or antifolylpoly(gamma-glutamate) synthesis. Folylpolyglutamates are essential for cell growth, while polyglutamates of classical antifolates are implicated in, and often critical for, their cytotoxic action. Synthesis of polyglutamate metabolites also plays a role in drug resistance. Detailed understanding of the synthesis and function of folyl- and antifolylpolyglutamates may thus allow development of new agents or strategies designed to exploit this critical process. This long term goal will be addressed in this application through the following Specific Aims. Specific Aim 1 would explore folylpolyglutamate synthetase (FPGS), the enzyme responsible for synthesis of polyglutamates, as a target for drug development. Since mutational deletion of FPGS activity is lethal, FPGS is a potential therapeutic target for cancer chemotherapy. Rational design of FPGS inhibitors will be based on enzyme mechanism and structure-activity data generated in the applicant's laboratory using homogeneous, recombinant human FPGS. Of interest are potential mechanism-based inhibitors and modifications to enhance potency and/or uptake of recognized folate-based ornithine-containing FPGS inhibitors. The biological effects to be expected from FPGS inhibition will be explored in a model system for folylpolyglutamate deficiency. These studies should define whether FPGS is a viable drug target. Specific Aim 2 would define the relationship between FPGS and methotrexate (MTX) resistance in human leukemia and human solid tumor model systems. Decreased FPGS activity, as first identified in collaborative studies involving the applicant's laboratory, is now established as a mechanism of resistance to antifolates in vitro, in vivo, and in the clinic. This clinically relevant resistance phenotype will be further characterized as to its frequency and evolution in preclinical models of leukemia and solid tumors. A multicellular spheroid system will be developed and characterized as a model for human solid tumor antifolate resistance. Specific Aim 3 would define the molecular pharmacology of human FPGS, especially in relation to antifolate resistance. A human FPGS cDNA will be used in further studies of the molecular pharmacology of FPGS and to characterize the nature of the defect in MTX-resistant lines deficient in polyglutamylation (Specific Aim 2). An antipeptide antibody to FPGS will be used to study regulation of FPGS at the protein level and its status in MTX-resistant cell lines. A polyclonal antibody to holo human FPGS protein will also be prepared and utilized.