BIOCHEMICAL CHARACTERIZATION OF P-GLYCOPROTEIN

描述：(申请人摘要)P-糖蛋白(P-gp)由 MDR1基因，导致对无关天然药物的多药耐药 在人类肿瘤细胞中产生药物，并作为ATP依赖的功能 外排泵。整个项目的目标是(1)解开分子 P-gp药物结合部位的结构，(2)分子研究 MDR调制机制，以及(2)提供合理的途径 合成和开发多药耐药逆转剂。具体目标是 (1)紫杉醇与长春花碱相互作用模式的研究 (Vbl)环孢素A(CsA)类似物，他莫昔芬()和 与P-gp结合的相关试剂和其他MDR调节剂，以及 确定磷酸化是否影响这种结合；(2)基于 根据特定目标1的结果，评估以下各项的组合 两种MDR调节剂增强药物积累并增加 多药耐药细胞对紫杉醇和长春花碱的敏感性 (3)合成和使用紫杉醇的光亲和类似物， CsA和，表征了它们与P-gp的共价结合，并确定了 其他MDR调节剂是否抑制其与P-gp的结合； 紫杉醇、环孢素A和的多肽结合位点的鉴定和定位 测绘和定位抗体，并确定它们是否 与Vbl结合部位相互作用；和(5)鉴定氨基酸 紫杉醇、环孢素A和结合位点的多肽序列 Mdr1基因的测序、定点突变和3- 三维分子模型分析。MCF-7乳腺癌细胞 Mdr1基因或mdr1蛋白激酶C-α(PKC)的转染 将使用MDR基因或MDR变种MCF-7/ADR。动力学分析 将被执行以确定这些代理是否相互作用 竞争性或非竞争性，以及磷酸化是否影响 单独检测PKC、PKC对药物与P-gp结合的影响 激活剂、蛋白激酶C抑制剂与磷酸化磷酸酶 抑制剂。根据动力学数据，申请者将分析 紫杉醇和VBL蓄积的调控及细胞毒作用 使用有效的MDR调制器的组合，并确定是否 它们的相加或协同效应与它们的竞争力相关 或在细胞毒药物结合部位的非竞争性相互作用，以及 是否可以建立结构-活性关系。要确定 P-gp药物结合部位，(1)紫杉醇，Vbl， CsA和将被合成并用于表征其 与P-gp的共价结合，(2)光标记的P-gp将 免疫沉淀、消化和电泳法分解，以及 (3)P-gp定点抗肽抗体将用于 药物结合多肽的区域图谱。要解开 药物结合的分子结构和空间排列 结构域，(1)纯化的药物结合多肽片段将 测序并将放射性氨基酸的位置(S) (2)mdr1基因的定点突变将是 进行单一氨基酸的改变，并确定 这些突变对P-gp结合的影响不同 光亲和探针与亲本药物的比较；(3)分子模拟 将对药物结合部位进行分析。

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.