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-糖蛋白泵药物，外源性物质和营养物质的细胞通过部分特征 ATP依赖机制。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和药物间相互作用。P-gp可能是 由于其在几种癌细胞中的过表达， 癌的本提案的目的是填补三个不同方面的P-gp的知识空白 机制每一个目标都具有构象动力学的共同机制要素。 第一个目的是定义低ATP占用率下的P-gp构象，以了解 它们如何控制对底物流出至关重要的下游构象变化，以及 它们在有底物和无底物的情况下是如何不同的。这些构象 假设差异是必要的，以尽量减少浪费支出的ATP在 缺乏可转运底物，并控制下游的适当构象通路 步这些差异将通过快速，预平衡，动力学方法与状态- 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。的 脂质纳米盘平台提供了对P-gp所在的脂质双层的精细控制。P-gp将 可以掺入具有不同头部基团或酰基的脂质的不同比例的纳米盘中 提供双层流动性的不同表面电荷的链。基底和基质 不同脂质环境中依赖ATP酶活性的变化与 通过H/DX确定构象。 第三个目的是确定P-gp能够与如此广泛的细胞相互作用的机制。 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 在整个生物学中，许多蛋白质都是如此，但分子基础却很难确定。给你，普雷 平衡结合动力学和单分子荧光方法将用于比较 P-gp集合与单个P-gp纳米盘中结合参数的分布， 确定P-gp是否利用相互转换构象或持久的独特构象， 构象以募集和保留不同结构的药物。