FOLYLPOLYGLUTAMATE SYNTHETASE IN CANCER CHEMOTHERAPY

描述：（申请人的摘要）该计划的长期目标是 通过利用叶酰-（或 抗叶酰-）聚谷氨酸合成。Folylpolyglutamates是细胞生长所必需的 生长，而经典抗叶酸剂的聚谷氨酸盐涉及其 细胞毒性作用和抗性，并可能具有选择性的作用。详细 对（抗）叶酰聚谷氨酸盐的合成和功能的理解可以 从而允许设计新的代理或策略来利用这一关键过程。 这一长期目标将通过三个具体目标来实现：1. 叶酸聚谷氨酸合成酶（FPGS）的探索， 聚谷氨酸合成，作为药物靶点。FPGS的突变失活是 因此，FPGS是潜在的癌症化疗靶点。抑制剂设计 基于申请人的酶机制和结构-活性数据，使用 重组人FPGS。经过专家叶酸化学家的合成， 合作实验室，抗叶酸剂将在申请人的 实验室主要的兴趣将是基于机制的优化 含磷抑制剂发现在最后一个赠款期间。类似物 其抑制完整细胞中纯化的FPGS和多聚谷氨酰胺化（即， 将被研究，以澄清其特异性和细胞 方面的影响.有希望的药物将进行初步的毒性和治疗测试 以确定FPGS是否是有用的治疗靶点。2.确定 表观物理化学差异的功能意义 线粒体（mFPGS）和胞质FPGS（cFPGS）。虽然由一个基因编码， mFPGS和cFPGS在SDS-PAGE迁移率方面不同，这可能反映了功能性的差异。 差异，特别是与抗叶酸剂抗性有关（目的3）。的 申请人将探索这种物理化学变化的假设， 在这项授权下发现的，反映在动力学，底物特异性， 和/或法规变更。此外，由于结构差异可能是 反映的功能，他将审查的假设，这是造成的， 线粒体前导序列截短或翻译后修饰。 3.阐明cFPGS和mFPGS在MTX耐药中的作用。临床上，MTX是 通常以大剂量推注或短时间（24小时）输注给药。申请人 已经表明该方案通过FPGS缺陷选择抗性。FPGS是 在细胞质和线粒体中表达。MTX不能进入线粒体， 而还原叶酸单谷氨酸盐可以。已知（Shane et al.）的 单独表达mFPGS可以在两种细胞中建立叶酸聚谷氨酸池， 细胞质和线粒体并允许正常细胞生长。因此，他假设 cFPGS的差异性下降，而不是两者的平行下降， 同种型，可以有助于抵抗脉冲MTX暴露。这也许可以解释 为什么高水平的耐药性脉冲甲氨蝶呤可以发生在没有大的 总FPGS活性降低或谷氨酰水解酶活性升高。

DESCRIPTION: (Applicant's Abstract) The long-term goal of this program is to improve treatment of human cancer by exploiting aspects of folyl- (or antifolyl-) polyglutamate synthesis. Folylpolyglutamates are essential for cell growth, while polyglutamates of classical antifolates are implicated in their cytotoxic action and resistance and may have a role in selectivity. Detailed understanding of the synthesis and function of (anti)folylpolyglutamates may thus allow design of new agents or strategies to exploit this critical process. This long-term goal will be addressed through three specific aims: 1. Exploration of folylpolyglutamate synthetase (FPGS), the enzyme responsible for polyglutamate synthesis, as a drug target. Mutational inactivation of FPGS is lethal, thus FPGS is a potential cancer chemotherapy target. Inhibitor design is based on enzyme mechanism and structure-activity data of the applicant using recombinant human FPGS. After synthesis by expert folate chemists in collaborating laboratories, antifolates will be studied in the applicant's laboratory. Of primary interest will be the optimization of mechanism-based phosphorous-containing inhibitors found during the last grant period. Analogs that inhibit both purified FPGS and polyglutamylation in intact cells (i.e., are transported) will be studied to clarify their specificity and cellular effects. Promising drugs will undergo initial toxicity and therapeutic testing in vivo to define whether FPGS is a useful therapeutic target. 2. Determine the functional significance of the apparent physico-chemical difference between mitochondrial (mFPGS) and cytosolic FPGS (cFPGS). Although encoded by one gene, mFPGS and cFPGS differ in SDS-PAGE mobility, which may reflect a functional difference, particularly as related to antifolate resistance (Aim 3). The applicant will explore the hypothesis that this physico-chemical variance, discovered under this grant, is reflected in kinetic, substrate specificity, and/or regulation alterations. Also, since the structural difference may be reflective of function, he will examine hypotheses that it is caused by mitochondrial leader sequence truncation or by post-translational modification. 3. Elucidate the role of cFPGS and mFPGS in MTX resistance. Clinically, MTX is often given as a bolus or short (24 hr) infusion at high dose. The applicant has shown that this regimen selects for resistance via FPGS deficiency. FPGS is expressed in both cytosol and mitochondria. MTX cannot enter mitochondria, while reduced folate monoglutamates can. It is known (Shane et al.) that expression of mFPGS alone can establish folylpolyglutamate pools in both cytosol and mitochondria and allow normal cell growth. Thus, he hypothesizes that a differential decrease in cFPGS, rather than a parallel decrease in both isoforms, can contribute to resistance to pulse MTX exposure. This may explain why high-level resistance to pulse MTX can occur in the absence of a large decrease in total FPGS activity or a rise in glutamyl hydrolase activity.