Studies of P-glycoprotein and drug interactions

 描述(申请人提供)：P-糖蛋白(PGP)是一种高度动态的三磷酸腺苷结合盒(ABC)膜转运体，能将多种分子外排出细胞。它是决定药物在肠、肝、肾和脑中吸收、分布和排泄的主要因素，也是某些癌症、HIV和中枢神经系统(CNS)疾病化疗成功的障碍。据估计，大约一半的上市药物是Pgp的转运底物或抑制剂，候选药物的Pgp敏感性评估已成为制药行业开发新疗法的重要一步。然而，PGP结合分子表现出复杂的活性，作为ATPase的刺激物、抑制物、运输底物或不可运输的配体。尽管有广泛的研究，但我们对Pgp转运、抑制和逃避相关的复杂和多特异性Pgp-药物相互作用的机制仍知之甚少。本研究的目的是(1)确定配体的结构和化学性质如何影响其与PGP的相互作用；(2)表征PGP如何与不同类别的配体结合；(3)合理地进行化学合成以修饰现有药物以逃避PGP的转运。我们将使用化学、结构和功能相结合的方法来解决这些目标，这是基于文献中的几个主要进展，以及我们最近在PGP结构确定和配体相互作用方面所取得的进展。首先，我们建议在具有共同支架的配体集中文库中进行彻底的结构-活性关系研究。我们将使用一系列的功能分析，包括ATPase活性、详细的药物结合和竞争、基于细胞的转运、耐药性分析以及结构表征来评估这些化合物。其次，我们将使用几种互补的生物物理技术，包括X射线结晶学、单粒子电子显微镜(EM)和发光共振能量转移(LRET)，来确定PGP/配体的络合物结构、构象分布和构象变化的动力学。EM和LRET对PGP构象的表征，以及筛选出新的配体、洗涤剂和脂类，以及稳定PGP(和某些构象)的新结构，将有助于更高分辨率(&lt；3.0？)结构的研究，这是迄今为止PGP所未解决的关键障碍。第三，我们将在不降低药物效力的情况下，在已确定的位置上对几种PGP药物底物进行修饰，因此重点放在PGP相互作用的结果变化上。通过相关的三个目的的研究，我们将实现对多特异性Pgp与药物相互作用的详细和基本的了解，鉴于Pgp的药理学和临床意义，以及许多当前和研究中的药物对Pgp外排敏感，这将对药物开发产生深远的影响。

 DESCRIPTION (provided by applicant): P-glycoprotein (Pgp) is a highly dynamic ATP-binding cassette (ABC) membrane transporter that effluxes a diversity of molecules out of cells. It is a major determinant of drug absorption, distribution, and excretion in intestines, liver, kidney and brain, and also an impediment to successful chemotherapy in some cancers, HIV and central nervous system (CNS) diseases. About half of marketed drugs are estimated to be transport substrates or inhibitors of Pgp, and the evaluation of Pgp susceptibility of drug candidates has become an important step in the development of new therapeutics in the pharmaceutical industry. However, Pgp binding molecules exhibit complex activities, acting as ATPase stimulators, inhibitors, transport substrates, or non- transportable ligands. Despite extensive studies, we still have very limited understanding of the mechanisms for the complex and polyspecific Pgp-drug interactions pertaining to Pgp transport, inhibition and evasion. This study aims to (1) define how structural and chemical properties of a ligand affect its interactions with Pgp, (2) characterize how Pgp reacts upon the binding of different classes of ligands, and (3) rationalize chemical synthesis to modify existing drugs to evade Pgp transport. We will use an integrative chemistry, structural and functional approach to tackle these aims, which is based on several major advances in the literature and that we have most recently made on Pgp structural determination and ligand interactions. First, we propose to conduct a thorough structure-activity relationship study within a focused library of ligands bearing common scaffolds. We will use a battery of functional assays, including ATPase activity, detailed drug binding and competition, cell-based transport, and drug resistance assays, as well as structural characterizations to evaluate these compounds. Second, we will use several complementary biophysical techniques, including X- ray crystallography, single particle electron microscopy (EM), and luminance resonance energy transfer (LRET), for the determination of Pgp/ligand complex structures, conformational distributions, and the kinetics of conformational changes. The characterization of Pgp conformations by EM and LRET, together with the screen of novel ligands, detergents and lipids, as well as new constructs to stabilize Pgp (and certain conformations), will facilitate higher resolution (< 3.0 Å) structural studies, a critical barrier that has eluded Pgp thus far. Third, we will modify several Pgp drug substrates on positions that have been identified without diminution of drug potency, thus with a focus on resulting changes in Pgp interactions. By the related three aim studies we will achieve a detailed and fundamental understanding of polyspecific Pgp-drug interactions, which will have a far- reaching impact on drug discovery given the pharmacological and clinical significance of Pgp and that many current and investigational drugs are susceptible to Pgp efflux.