BIOCHEMICAL CHARACTERIZATION OF P-GLYCOPROTEIN

描述：（申请人的摘要）P-糖蛋白（P-gp）由 MDR 1基因，导致对无关的自然 产品药物在人类肿瘤细胞，并作为一个ATP依赖的功能， 外排泵 总体项目目标是（1）解开分子 P-gp药物结合位点的分子结构，（2）研究P-gp药物结合位点的分子结构， MDR调节机制，（2）提供合理的方法 合成和开发MDR逆转剂。 具体目标是 (1)探讨紫杉醇与长春碱的相互作用方式 (VBL)与环孢菌素A（CsA）类似物、他莫昔芬（TAM）和 相关药物和其他MDR调节剂，用于结合P-gp，和 确定磷酸化是否影响这种结合;（2）基于 根据具体目标1的结果，评估以下组合是否 两种MDR调节剂增强药物蓄积， MDR细胞对紫杉醇和VBL的敏感性相加或 协同作用;（3）紫杉醇光亲和类似物的合成和应用， CsA和TAM，表征其与P-gp的共价结合，并确定 其他MDR调节剂是否抑制其与P-gp的结合;（4） 用肽段法对紫杉醇、CsA和TAM的结合位点进行鉴定和定位 定位和定点抗体，并确定它们是否 与VBL结合位点相互作用;和（5）鉴定氨基酸 紫杉醇、CsA和TAM结合位点的肽序列 测序、MDR 1基因的定点突变和3- 三维分子模拟分析 MCF-7乳腺癌细胞 用MDR 1基因或MDR 1蛋白激酶C-α（PKC）转染 将使用MDR基因或MDR变体MCF-7/ADR。 动力学分析 将被执行，以确定是否 这些代理人相互作用 竞争性或非竞争性，以及磷酸化是否影响 通过检查单独PKC、PKC 激活剂、PKC抑制剂和磷酸化磷酸酶 抑制剂的 根据动力学数据，申请人将分析 紫杉醇和VBL积累和细胞毒性调节 使用有效MDR调节剂的组合，并确定是否 它们的加和或协同效应与它们的竞争性相关， 或在细胞毒性药物结合位点的非竞争性相互作用，和 是否可以建立结构-活性关系。 以识别 P-gp药物结合位点，（1）紫杉醇的光敏类似物，VBL， CsA和TAM将被合成并用于表征它们的结构。 与P-gp共价结合，（2）光标记的P-gp将 免疫沉淀、消化和电泳分离，和 (3)P-gp定点抗肽抗体将用于 药物结合肽的结构域作图。 解开 药物结合的分子结构和空间排列 结构域，（1）纯化的药物结合肽 片段将是 测序并 放射性氨基酸的位置将是 鉴定，（2）MDR 1基因的定点诱变将被 进行单个氨基酸的变化，并确定是否 这种突变不同地影响P-gp结合， 光亲和探针与母体药物，以及（3）分子建模 将进行药物结合位点的分析。

DESCRIPTION: (Applicant's Abstract) P-glycoprotein (P-gp) encoded by the MDR1 gene, causes multidrug resistance (MDR) to unrelated natural product drugs in human tumor cells, and functions as an ATP-dependent efflux pump. The overall project goals are (1) to unravel the molecular structure of P-gp drug binding sites, (2) to investigate the molecular mechanisms of MDR modulation, and (2) to provide rational approaches to synthesize and develop MDR reversing agents. The specific aims are (1) investigate the modes of interaction of taxol and vinblastine (VBL) with cyclosporin A (CsA) analogs, tamoxifen (TAM) and related agents, and other MDR modulators for binding to P-gp, and determine whether phosphorylation affects this binding; (2) based on the results of Specific Aim 1, evaluate whether combinations of two MDR modulators enhance drug accumulation and increase the sensitivity of MDR cells to taxol and VBL additively or synergistically; (3) synthesize and use photoaffinity analogs of taxol, CsA and TAM, characterize their covalent binding to P-gp, and determine whether other MDR modulators inhibit their binding to P-gp; (4) identify and map the binding sites of taxol, CsA and TAM by peptide mapping and site-directed antibodies, and determine whether they interact with the VBL binding site; and (5) identify the amino acid sequences of the taxol, CsA and TAM binding sites by peptide sequencing, site-directed mutagenesis of the MDR1 gene, and 3- dimensional molecular modeling analysis. MCF-7 breast cancer cells transfected with the MDR1 gene or MDR1 protein kinase C-alpha (PKC) genes, or an MDR variant, MCF-7/ADR, will be used. Kinetic analysis will be performed to determine whether these agents interact competitively or noncompetitively, and whether phosphorylation affects drug binding to P-gp by examining the effects of PKC alone, PKC activators, PKC inhibitors and phosphorylation phosphatase inhibitors. Based on the kinetic data, the applicant will analyze the modulation of taxol and VBL accumulation and cytotoxicity using combinations of potent MDR modulators, and determine whether their additive or synergistic effects correlate with their competitive or noncompetitive interaction at the cytotoxic drug binding sites, and whether structure-activity relationships can be made. To identify the P-gp drug binding sites, (1) photoactive analogs of taxol, VBL, CsA and TAM will be synthesized and used to characterize their covalent binding to P-gp, (2) the photolabeled P-gp will be immunoprecipitated, digested, and resolved by electrophoresis, and (3) P-gp site-directed anti-peptide antibodies will be used for domainal mapping of the drug-bound peptides. To unravel the molecular architecture and spatial arrangements of the drug binding domains, (1) purified drug-bound peptide fragments will be sequenced and the position(s) of radioactive amino acids will be identified, (2) site-directed mutagenesis of the MDR1 gene will be performed to make single amino acid changes and determine whether such mutations differently affect the P-gp binding of the photoaffinity probes vs. parent drugs, and (3) molecular modeling analysis of the drug binding sites will be performed.