Mechanism of Drug Transport by BCRP/ABCG2

BCRP/ABCG2 的药物转运机制

• 批准号: 7147533
• 负责人: QINGCHENG MAO
• 金额: $ 25.66万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147533
• 关键词: drug resistance; electron microscopy; human genetic material tag; membrane transport proteins; pharmacogenetics; pharmacokinetics; protein purification; protein structure; protein structure function; site directed mutagenesis; structural biology; yeasts

DESCRIPTION (provided by applicant): The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is a medically important ABC drug transporter. Overexpression of BCRP in cancer cells is associated with resistance to multiple anticancer drugs, including mitoxantrone, topotecan, and flavopiridol. Correlation between BCRP expression and drug resistance or patient survival has been observed with some hematological and solid tumors. In addition, BCRP is highly expressed in the placental syncytiotrophoblasts, in the apical membrane of the epithelium in the small intestine, and in the liver canalicular membrane. The tissue localization patterns, and results from recent studies, strongly suggest that BCRP functions as a protective efflux pump, and has the potential to limit oral absorption and increase biliary elimination of drugs that are BCRP substrates. At present, the molecular mechanism by which BCRP acts to transport drugs is unknown. Thus, the long-term goal of this proposal is to explore the molecular mechanism of BCRP. To achieve this goal, we propose 1 main specific aim, namely specific aim 1: structure and function analysis of BCRP. Site-directed mutagenesis and functional characterization of BCRP mutants, protein purification, and structure determination will be employed. The specific aim 1a is to purify BCRP overexpressed in the methylotrophic yeast Pichia pastoris. Purified protein will be functionally characterized and used for structural determination by electron microscopy. The specific aim 1b is mutational analyses to identify amino acid residues important for transport function and substrate selectivity of BCRP. The specific aim 1c is to analyze membrane topology of the transporter. Such studies will lead to a greater understanding of the molecular mechanism by which BCRP acts to transport drugs and provide the molecular basis for developing new ways to circumvent drug resistance in diseases such as cancers. Knowledge gained from the above studies will also aid in predicting potential drug-drug interactions and changes of pharmacokinetic properties of BCRP substrate drugs.

描述（申请人提供）：人乳腺癌耐药蛋白（BCRP，基因符号ABCG 2）是一种医学上重要的ABC药物转运蛋白。BCRP在癌细胞中的过表达与多种抗癌药物的耐药性相关，包括米托蒽醌、拓扑替康和flavopiridol。在一些血液肿瘤和实体瘤中观察到BCRP表达与耐药性或患者生存期之间的相关性。此外，BCRP在胎盘合体滋养层、小肠上皮细胞顶膜和肝小管膜中高度表达。组织定位模式和近期研究的结果强烈表明，BCRP作为保护性外排泵发挥作用，并有可能限制口服吸收并增加作为BCRP底物的药物的胆汁消除。目前，BCRP转运药物的分子机制尚不清楚。因此，本提案的长期目标是探索BCRP的分子机制。为了实现这一目标，我们提出了1个主要的具体目标，即具体目标1：BCRP的结构和功能分析。将采用BCRP突变体的定点诱变和功能表征、蛋白纯化和结构测定。具体目标1a是纯化在甲醇营养型酵母巴斯德毕赤酵母中过表达的BCRP。纯化蛋白将进行功能表征，并用于电子显微镜结构测定。具体目的1b是突变分析，以鉴定对BCRP的转运功能和底物选择性重要的氨基酸残基。具体目标1c是分析转运蛋白的膜拓扑结构。这些研究将使人们更好地了解BCRP转运药物的分子机制，并为开发新方法以规避癌症等疾病的耐药性提供分子基础。从上述研究中获得的知识也将有助于预测潜在的药物间相互作用和BCRP底物药物的药代动力学特性变化。