MECHANISM OF ATP DEPENDENT TRANSPORT BY MRP1 PROTEIN

MRP1 蛋白依赖 ATP 的转运机制

• 批准号: 6489403
• 负责人: Xiu-Bao Chang
• 金额: $ 24.1万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6489403
• 关键词: P glycoprotein; active sites; adenosine triphosphate; adenosinetriphosphatase; binding sites; cell line; chemical binding; enzyme activity; enzyme inhibitors; monoclonal antibody; multidrug resistance; phosphoproteins; protein kinase; site directed mutagenesis

Overexpression of P-glycoprotein (P-gp) and/or multidrug resistance-associated protein (MRP1) is associated with resistance of tumors to a wide range of chemotherapeutic drugs. Many cancers initially respond well to chemotherapeutic treatment, but these tumors eventually become resistant to those cytotoxic drugs. Some other cancers are resistant to anticancer drugs, even at the beginning of treatment. Therefore, no matter whether the resistance is "acquired" or "intrinsic," multidrug-resistance is one of the major obstacles to the successful treatment of many types of cancers. The mechanism of multidrug resistance conferred by either P-gp or MRP1 involves extrusion of the drugs out of the tumor cells. Both P-gp and MRP1 are members of the ABC superfamily of transport proteins, typically containing two membrane-spanning domains and two nucleotide binding domains. However, MRP1 differs from P-gp in that it contains an extra membrane-spanning domain at the N-terminus. ATP (binding and hydrolysis) is required in both cases. However, the transport steps differ in that P-gp extrudes the unmodified drugs directly, while the drugs transported by MRP1 protein require the presence of a hydrophilic compound, e.g., glutathione- or glucuronide-conjugates. Since the mechanism of multidrug resistance conferred by P-gp or MRP1 is to reduce the level of drug accumulation inside of the cell, inhibiting of the function of P-gp or MRP1 may reverse the drug resistance. Therefore, much effort is being devoted to discover specific inhibitors of these pumps. Present candidates as modulators of the process include verapamil, cyclosporin A, Cremophor, ardeemins, PSC833, rifamycins, RU486, MS-209, non-steroidal inflammatory drugs, acrolein, pyridine analogues, ONO-1078, chloroacetaldehyde, imidazothiazole derivatives, and even some protein kinase inhibitors. However, in order to develop well-designed specific modulators, it is important to understand the mechanisms of drug transport carried out by each individual protein. The first specific aim is to further characterize the ATP finding/hydrolysis and substrate transport by MRP1 protein. The second aim is to determine whether MRP1 protein phosphorylates itself or is a substrate for other protein kinases. This is based on our hypotheses that the phosphorylated MRP1 protein is an intermediate (i.e., there is an autophosphorylation) during ATP hydrolysis and substrate transport. The third aim is to define the substrate binding site(s). New insights gained from these aims should provide the basis for novel means of combating multidrug resistance.

P-糖蛋白 (P-gp) 和/或多药的过度表达 耐药相关蛋白（MRP1）与肿瘤的耐药性相关 多种化疗药物。许多癌症最初反应良好 化疗，但这些肿瘤最终会对化疗产生耐药性 那些细胞毒性药物。其他一些癌症对抗癌药物有抵抗力， 即使在治疗开始时。因此，无论是否抵抗 是“获得性”还是“内在”，多重耐药性是主要的因素之一 成功治疗多种癌症的障碍。机制 P-gp 或 MRP1 赋予的多药耐药性涉及挤出 将药物从肿瘤细胞中排出。 P-gp 和 MRP1 都是 ABC 的成员 转运蛋白超家族，通常包含两个跨膜蛋白 域和两个核苷酸结合域。然而，MRP1 与 P-gp 的不同之处在于 它在 N 末端含有一个额外的跨膜结构域。 ATP 这两种情况都需要（结合和水解）。然而，运输 步骤的不同之处在于，P-gp 直接挤出未修饰的药物，而 MRP1 蛋白转运的药物需要亲水性的存在 化合物，例如谷胱甘肽-或葡糖苷酸-缀合物。由于机制 P-gp或MRP1赋予的多药耐药性是降低药物水平 细胞内积累，抑制 P-gp 或 MRP1 的功能可能 逆转耐药性。因此，我们正在投入大量精力 发现这些泵的特定抑制剂。将候选人介绍为调制者 该过程包括维拉帕米、环孢菌素 A、Cremophor、ardeemins、PSC833、 利福霉素、RU486、MS-209、非甾体抗炎药、丙烯醛、吡啶 类似物、ONO-1078、氯乙醛、咪唑并噻唑衍生物，甚至 一些蛋白激酶抑制剂。然而，为了开发出精心设计的 特定的调节剂，了解药物的机制很重要 由每个单独的蛋白质进行的运输。第一个具体目标是 进一步表征 MRP1 的 ATP 发现/水解和底物运输 蛋白质。第二个目的是确定MRP1蛋白是否磷酸化 本身或者是其他蛋白激酶的底物。这是基于我们的 假设磷酸化的 MRP1 蛋白是中间体（即，有 是 ATP 水解和底物运输过程中的自磷酸化）。这 第三个目标是确定底物结合位点。从中获得的新见解 这些目标应为打击多种药物的新手段奠定基础 反抗。