IMPROVED CANCER THERAPY VIA MOLECULAR STUDIES OF THE PGP

通过 PGP 的分子研究改善癌症治疗

• 批准号: 2099169
• 负责人: GENE A SCARBOROUGH
• 金额: $ 14.52万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2099169
• 关键词: Baculoviridae; P glycoprotein; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; antineoplastics; cell free system; chemical structure; crystallization; drug screening /evaluation; gene expression; high performance liquid chromatography; liposomes; membrane proteins; multidrug resistance; neoplastic cell; protein reconstitution; recombinant proteins

The development of multidrug resistance (MDR) in tumor cells is probably responsible for many clinical failures of cancer chemotherapy. The molecular basis of MDR is overexpression of the MDR1 gene, which codes for a 170 kDa integral membrane glycoprotein known as the P-glycoprotein, or Pgp. The Pgp is an ATP-dependent drug pump, capable of transporting chemotherapeutic drugs out of cells against a concentration gradient and thereby minimizing their cytotoxic effects. We have recently discovered a previously undetected high capacity, drug-stimulated, membrane-bound ATPase activity associated with the human Pgp overexpressed via a recombinant baculovirus in cultured insect cells, which makes possible a variety of new experimental approaches to understanding the properties of this clinically important enzyme. Utilizing this system for manipulating the Pgp and measuring its activity, the studies proposed in this application are designed to identify effective agents for interfering with Pgp function in a two part plan. First, we shall investigate several different aspects of the Pgp-ATPase function in isolated membranes, including its drug specificity, drug transport activity, possible physiological role, and possible regulation by protein kinases and phosphatases. Second, we shall investigate a variety of aspects of the molecular structure and mechanism of the Pgp. In these studies, we shall investigate the roles of the two proposed nucleotide binding sites in the Pgp by site-directed mutagenesis, establish conditions for detergent solubilization and purification of the Pgp in active form, and develop a reliable procedure for reconstituting the Pgp into artificial phospholipid vesicles. With the reconstituted Pgp-proteoliposomes, we shall attempt to develop a liposomal drug transport assay, attempt to establish the minimum functional unit of the Pgp, and explore the transmembrane topography of the Pgp. And finally, we shall attempt to prepare large, three-dimensional crystals of the Pgp utilizing our newly developed methodology for crystallizing integral membrane proteins. It is anticipated that better drugs and drug combinations, and possibly even new therapeutic approaches, for the treatment of cancer could emerge from the information obtained.

肿瘤细胞中多药耐药性（MDR）的发展可能是 负责癌症化疗的许多临床失败。 这 MDR的分子基础是MDR1基因的过表达，该基因代码 对于170 kDa的整合膜糖蛋白，称为P-糖蛋白， 或PGP。 PGP是一个依赖ATP的药物泵，能够运输 针对浓度梯度和 从而最大程度地减少其细胞毒性作用。 我们最近发现了 先前未发现的高容量，药物刺激的膜结合 与人类PGP相关的ATPase活性通过A过表达 培养的昆虫细胞中的重组杆状病毒，这使得成为可能 了解了解特性的各种新实验方法 该临床上重要的酶。 利用此系统来操纵 PGP并衡量其活性，这是在此提出的 应用旨在识别有效的代理 在两部分计划中使用PGP功能。 首先，我们将调查 孤立的PGP-ATPase函数的几个不同方面 膜，包括其药物特异性，药物运输活性， 可能的生理作用，并可能由蛋白激酶调节 和磷酸酶。 其次，我们将研究 PGP的分子结构和机制。 在这些研究中，我们 应研究两个提出的核苷酸结合位点的作用 在PGP中按位置定向诱变，建立条件 PGP以活性形式的洗涤剂溶解和纯化，并且 制定可靠的程序，以将PGP重新建立为人造 磷脂囊泡。 与重构的PGP-蛋白质体，我们 应尝试开发脂质体药物运输测定法，尝试 建立PGP的最低功能单元，并探索 PGP的跨膜地形。 最后，我们将尝试 准备使用我们新的PGP的大型三维晶体 开发了结晶整体膜蛋白的方法。 它 预计更好的药物和药物组合，甚至可能 用于治疗癌症的新治疗方法可能会从 获得的信息。