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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 P-糖蛋白通过部分表征的方式将药物、外源物质和营养物质从细胞中泵出 ATP依赖性机制。由于 P-gp 的极端底物混杂性，它有助于几乎所有小分子药物的处置以及药物间相互作用。 P-gp 可能是由于其在多种细胞中过度表达，因此在癌细胞耐药性中特别重要癌症。该提案的目的是填补 P-gp 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制要素。第一个目标是定义低 ATP 占用率下的 P-gp 构象，以便了解它们如何控制底物流出所必需的下游构象变化，以及它们在存在底物和不存在底物时有何不同。这些构象假设差异对于最大限度地减少 ATP 的浪费支出至关重要缺乏可运输底物并控制下游适当的构象访问步骤。这些差异将通过快速、预平衡、动态方法和状态- 最先进的动力学建模和氢/氘交换质谱 (H/DX) 脂质纳米盘中的 P-gp。第二个目的是了解 P-gp 对其脂质环境的极端敏感性。这脂质纳米圆盘平台可对 P-gp 所在的脂质双层进行精细控制。 P-gp将可以掺入具有不同比例的具有不同头基或酰基的脂质的纳米圆盘中提供双层流动性的不同表面电荷的链。基础和底物不同脂质环境中依赖的 ATP 酶活性将与由H/DX确定构象。第三个目标是确定 P-gp 能够与如此广泛的相互作用的机制。底物和抑制剂的范围。基质混杂是一种越来越受到赞赏的行为对于整个生物学中的许多蛋白质来说，但其分子基础尚不清楚。在这里，预将使用平衡结合动力学和单分子荧光方法来比较 P-gp 整体与单个 P-gp 纳米圆盘中结合参数的分布，以便确定 P-gp 是否利用相互转换的构象或持久的不同构象招募和保留不同结构的药物的构象。