Drug, Nucleotide, and Lipid Interactions with P-glycoprotein

药物、核苷酸和脂质与 P-糖蛋白的相互作用

• 批准号: 10672242
• 负责人: WILLIAM M ATKINS
• 金额: $ 31.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10672242
• 关键词: ABCB1 gene; ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Behavior; Binding; Binding Sites; Cell membrane; Cells; Charge; Cholesterol; Coupled; Coupling; Deuterium; Disease; Drug Interactions; Drug resistance; Elements; Environment; Equilibrium; Exhibits; Expenditure; Fluorescence; Fluorescence Spectroscopy; Food-Drug Interactions; Goals; Health; Heterogeneity; Human; Hydrogen; Kinetics; Knowledge; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Literature; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Membrane; Membrane Fluidity; Membrane Lipids; Methods; Micelles; Molecular; Molecular Biology; Molecular Conformation; Nucleotides; Nutrient; Pharmaceutical Preparations; Play; Proteins; Pump; Reaction; Role; Series; Sphingomyelins; Structure; Suggestion; Surface; Theoretical model; Tissues; Tryptophan; Vesicle; Xenobiotics; cancer cell; detoxication; efflux pump; flexibility; improved; inhibitor; kinetic model; nanodisk; overexpression; preference; recruit; residence; response; single molecule; small molecule; stopped-flow fluorescence

Project Summary P-glycoprotein pumps drugs, xenobiotics and nutrients out of cells via a partially characterized ATP-dependent mechanism. Due to the extreme substrate promiscuity of P-gp, it contributes to the disposition of nearly all small molecule drugs and to drug-drug interactions. P-gp may be particularly important in cancer cell drug resistance due to its over expression in several cancers. The aims of this proposal are to fill knowledge gaps in three distinct aspects of P-gp mechanism. Each aim shares the common mechanistic element of conformational dynamics. The first aim is to define the P-gp conformations at low ATP occupancy in order to understand how they control downstream conformational changes that are essential for substrate efflux, and how they are different in the presence vs. the absence of substrates. These conformational differences are hypothesized to be essential for minimizing wasteful expenditure of ATP in the absence of transportable substrate and to control proper conformational access in downstream steps. These differences will be identified by rapid, pre-equilibrium, kinetic methods with state- of-the-art kinetic modeling and Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX) with P-gp in lipid nanodiscs. The second aim is to understand the extreme sensitivity of P-gp to its lipid environment. The lipid nanodisc platform provides fine control of the lipid bilayer in which P-gp resides. P-gp will be incorporated in nanodiscs with varying ratios of lipids with different head groups or acyl chains that provide different surface charge of bilayer fluidity. The basal- and substrate dependent ATPase activity in the different lipid environments will be correlated with changes in conformation determined by H/DX. The third aim is to determine the mechanism by which P-gp is able to interact with such a wide range of substrates and inhibitors. Substrate promiscuity is an increasingly appreciated behavior for many proteins throughout biology but the molecular basis is poorly defined. Here, pre equilibrium binding kinetics and single molecule fluorescence methods will be used to compare the distribution of binding parameters in P- gp ensembles vs. single P-gp nanodiscs in order to determine whether P-gp exploits interconverting conformations or persistent distinct conformations to recruit and retain drugs of varying structure.

项目摘要 p-糖蛋白通过部分表征的细胞中的细胞泵送药物，异种生物和营养 依赖ATP的机制。由于P-gp的极端底物滥交，它有助于 几乎所有小分子药物的处置和药物相互作用。 P-gp可能是 由于癌细胞耐药性在几个 癌症。该建议的目的是填补P-gp的三个不同方面的知识空白 机制。每个目标都具有构象动力学的共同机械元素。 第一个目的是在低ATP占用率下定义P-gp构象以了解 他们如何控制下游构象变化，这对于底物排出至关重要， 在存在的情况下，它们是如何不同的。这些构象 假设差异对于最大程度地减少ATP的浪费支出至关重要 没有可运输的基材并控制下游的适当构象接入 步骤。这些差异将通过具有状态 - 与艺术动力学建模和氢/氘交换质谱法（H/DX）一起 脂质纳米盘中的P-gp。 第二个目的是了解P-gp对脂质环境的极端敏感性。这 脂质纳米轴平台可很好地控制P-gp所在的脂质双层。 P-gp会 并入具有不同头部或酰基的脂质比的纳米盘中 提供双层流动性的不同表面电荷的链。基础和基材 不同脂质环境中的依赖性ATPase活性将与变化相关 由H/DX确定的构象。 第三个目的是确定p-gp能够与如此宽的机制相互作用的机制 底物和抑制剂范围。底物滥交是一种越来越多的行为 对于整个生物学的许多蛋白质，但分子基础的定义很差。在这里，pre 平衡结合动力学和单分子荧光方法将用于比较 在P-GP集合中与单个P-gp纳米盘中结合参数的分布，以便 确定P-gp是利用互换构象还是持续不同的不同 构成招募和保留不同结构的药物。