MRP4 IN DRUG SENSITIVITY AND PHYSIOLOGY

MRP4 在药物敏感性和生理学中的应用

• 批准号: 6387100
• 负责人: JOHN D SCHUETZ
• 金额: $ 27.7万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387100
• 关键词: adenine analog; antiviral agents; cell line; drug metabolism; ganciclovir; gene expression; genetically modified animals; laboratory mouse; membrane model; membrane transport proteins; molecular cloning; multidrug resistance; northern blottings; protein structure function; recombinant proteins; western blottings; zidovudine

Achieving high intracellular concentrations of antiretroviral drugs, essential for effective therapy, may be prevented by the specific efflux of nucleoside monophosphates (not di- or triphosphates), however, no specific efflux transporter for these substances has been identified. Our preliminary studies have revealed that in cells resistant to azidothymidine (AZT) and (9-(2-phosphonylmethoxyethyl)adenine) (PMEA), a structural analog of dAMP and AMP, increased energy-dependent efflux of PMEA and AZT monophosphate occurs. Further studies utilizing gene specific reagents and antisera demonstrate only amplification and overexpression of MRP4 (multidrug resistance associated protein 4), a gene currently not linked to chemotherapeutic resistance. Somatic cell fusion studies demonstrated that the resistance could be conferred in a dominant fashion. Further studies, in patients have shown that high levels of MRP4 may contribute to therapeutic failure. To fully understand MRP4s' substrates, inhibitors and resistance properties, we propose to stably express both human and murine MRP4 in cells. . We will also use membrane vesicles prepared from these cells to evaluate transport in cases where anionic substrates are impermeant. We propose to characterize the murine mrp4 gene to facilitate development of the mrp4 nullizygous mouse. Both the mip4 nullizygous mouse and MRP4im~4 cell models will allow evaluation of the therapeutic efficacy and role of MRP4 in transport of antiretroviral drugs and other candidate anionic substrates. Further, the potential biological significance of the MRP4 gene has not escaped us. For a number of years an "efflux system" has been described for cAMP. The efflux is energy-dependent and a reported inhibitor of cAMP efflux is also an inhibitor of PMEA efflux. Our reagents will allow us to test the novel hypothesis that MRP4 transports cyclic nucleotides, a finding that would profoundly impact our understanding of cyclic nucleotide signaling. Thus, this proposal will elucidate not only the biological role ofMRP4 in cellular transport of organic substrates, but also facilitate the development of more effective therapeutic regimens by revealing MRP4 as a major determinant of intracellular concentration of antiretroviral drugs.

实现有效治疗所必需的抗逆转录病毒药物的高细胞内浓度可能会被单磷酸核苷（不是二磷酸或三磷酸）的特异性流出所阻止，但是，尚未鉴定出这些物质的特异性流出转运蛋白。我们的初步研究表明，在对叠氮胸苷 (AZT) 和 (9-(2-膦酰基甲氧基乙基)腺嘌呤) (PMEA)（dAMP 和 AMP 的结构类似物）具有抗性的细胞中，PMEA 和 AZT 单磷酸的能量依赖性外流会增加。利用基因特异性试剂和抗血清的进一步研究表明，仅 MRP4（多药耐药相关蛋白 4）的扩增和过度表达，该基因目前与化疗耐药性无关。体细胞融合研究表明，可以以显性方式赋予抵抗力。进一步的研究表明，患者中高水平的 MRP4 可能会导致治疗失败。为了充分了解 MRP4 的底物、抑制剂和耐药特性，我们建议在细胞中稳定表达人类和小鼠 MRP4。 。我们还将使用由这些细胞制备的膜囊泡来评估阴离子底物不渗透的情况下的转运。我们建议表征小鼠 mrp4 基因，以促进 mrp4 无效小鼠的发育。 mip4 无效小鼠和 MRP4im~4 细胞模型都可以评估 MRP4 在抗逆转录病毒药物和其他候选阴离子底物转运中的治疗功效和作用。此外，MRP4 基因的潜在生物学意义也并未被我们忽视。多年来，cAMP 的“外排系统”一直被描述。流出是能量依赖性的，据报道，cAMP 流出的抑制剂也是 PMEA 流出的抑制剂。我们的试剂将使我们能够测试 MRP4 转运环核苷酸的新假设，这一发现将深刻影响我们对环核苷酸信号传导的理解。因此，该提案不仅将阐明MRP4在有机底物细胞转运中的生物学作用，而且还将通过揭示MRP4作为抗逆转录病毒药物细胞内浓度的主要决定因素来促进更有效的治疗方案的开发。