Rapid in vitro substrate assay for the multi-drug resistance p-glycoprotein

描述(由申请人提供)： 药物到达并穿透其体内预定靶点的能力是其成功治疗疾病的关键。然而，药物外排蛋白如P-糖蛋白(Pgp)主动将疏水性药物泵离靶组织，并与化疗中的低口服吸收和多药耐药有关。蛋白质泵越来越受到制药行业的关注，最重要的是基于FDA新的指南草案，该指南要求了解候选药物是Pgp的底物还是抑制剂。目前的PGP检测方法繁琐、昂贵且不可靠。我们的总体目标是制备和验证一种新型的分析试剂-荧光体-反式PGP-它利用我们开发的荧光体技术来测量小分子通过脂膜的被动通透性。荧光体-反式-Pgp将提供一种直接、可靠、简单、快速和廉价的方法来确定药物是Pgp底物还是抑制剂。为了实现这一目标，在本项目的第一阶段，我们克隆并在HEK293细胞中表达了含有10His标签的人PGP构建体。分离纯化脂胶束中的PGP 10His蛋白。在纯化的PGP 10His脂胶束中证实了维拉帕米刺激的ATPase活性。将纯化的PGP 10His重组为脂质体。建立了荧光体-反式-PGP的制备和检测方法。验证了将用于筛选潜在PGP底物和抑制剂的测试系统。该项目的第二阶段将使荧光体-反式-PGP分析达到重要的商业产品的状态。为了完成这一开发，第二阶段的具体目标是：1.完成荧光体-反式-PGP的生产和验证。2.将荧光体-反式Pgp的生产扩大到商业水平。3.用具有代表性的Pgp底物和抑制剂展示荧光体-反式-Pgp的有效性。4.优化荧光体-反式-PGP系统的性能。5.开发数据分析软件。我们在第二阶段的首要目标是将Fluorosome-Trans-PGP及其配套软件推向市场。第二阶段的成功完成将满足对一种可靠和经济的方法的日益增长的需求，以测量药物对PGP泵外排的敏感性或作为PGP抑制剂。该项目还为基于荧光体的系统的设计奠定了基础，以测试候选药物对其他药物转运蛋白的敏感性。市场包括制药和生物技术公司、合同研究机构和内部收费服务化验。该项目完成了一种测试的开发，该测试确定药物是否会通过生物泵从目标组织中挤出。因此，该测试使制药业能够在早期阶段评估候选药物是否适合继续开发。这种测试可靠、简单、快速、廉价，而且易于接受机器人技术。

DESCRIPTION (provided by applicant): The ability of a drug to reach and penetrate its intended target within the body is critical to its success in treating disease. However, drug efflux proteins such as p-glycoprotein (pgp) actively pump hydrophobic drugs away from target tissues and are linked to low oral absorption and multidrug resistance in chemotherapy. Protein pumps are of increasing interest to the pharmaceutical industry, most importantly based on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate or inhibitor of pgp. Current pgp assays are cumbersome, expensive and unreliable. Our overall goal is to prepare and validate a novel assay reagent - Fluorosome-trans-pgp - which capitalizes on the Fluorosome Technology that we have developed for measuring passive permeability of small molecules through lipid membranes. Fluorosome-trans-pgp will provide a direct, reliable, simple, rapid and inexpensive assay to determine if a drug is a pgp substrate or inhibitor. Toward this goal, in phase I of this project we have: cloned and expressed the human pgp construct containing a 10His tag in HEK293 cells. isolated and purified the protein "pgp 10His" in lipid micelles. demonstrated verapamil-stimulated ATPase activity in the purified pgp 10His lipid micelles. reconstituted the purified pgp 10His into liposome membranes. developed the methodology for the manufacture and assay of Fluorosome-trans-pgp. validated the test systems that will be used to screen potential pgp substrates and inhibitors. Phase II of this project will bring the Fluorosome-trans-pgp assay to the state of an important commercial product. To complete this development, the Specific Aims of phase II are to: 1. complete Fluorosome-trans-pgp production and validation. 2. scale up Fluorosome-trans-pgp production to commercial levels. 3. demonstrate the utility of Fluorosome-trans-pgp with representative pgp substrates and inhibitors. 4. optimize the properties of the Fluorosome-trans-pgp system. 5. develop data analysis software. Our overriding goal in Phase II is to bring Fluorosome-trans-pgp and accompanying software to market. Successful completion of phase II will satisfyi the increasing need for a reliable and economical method to measure a drug's susceptibility to efflux by the pgp pump or to act as a pgp inhibitor. This project also lays the foundation for the design of Fluorosome-based systems to test for the susceptibility of drug candidates to other drug transport proteins. Markets include pharmaceutical and biotechnology companies, contract research organizations, and in-house fee-for service assays. This project completes the development of a test which determines if drugs will be extruded from their target tissue by biological pumps. The test thereby allows the pharmaceutical industry to evaluate, at an early stage, the suitability of drug candidates for continued development. The test is reliable, simple, rapid, inexpensive and amenable to robotics.