In vitro substrate assay for multi-drug resistance

描述(由申请人提供)：一种成功的药物的一个标准是它到达目标位置的能力。候选药物无效的一个主要原因是，化合物不进入细胞或跨越存在于身体不同部分之间的屏障，例如肠道上皮、血脑屏障、胎盘。除了细胞膜的被动屏障外，许多细胞膜还含有能量(ATP)驱动的外排蛋白，即“多药耐药蛋白”(MDRP)，它们主动将许多药物泵离目标组织。 在这个项目中，我们建议开发一种创新的、简单而有效的方法来确定候选药物对MDRP家族的一个主要成员--P-糖蛋白(Pgp)的敏感性。这种独特的分析方法应该为药物发现提供一个有价值的工具。一种MDRP检测方法的发展也将为未来MDRP家族其他成员的检测奠定基础。我们计划开发的是一种新形式的荧光体-Frans-PGP，部分基于GL合成公司现有的荧光体技术。具体地说，我们建议克隆和表达人Pgp，并将其整合到荧光体-Frans的膜双层中。荧光体-反式微球是GL合成公司开发的一种脂质体纳米粒子，用于测量药物通过膜双层的被动通透性。通过测试底物的ATPase活性和ATP依赖的通量将证实PGP功能性地掺入到F-Frans-PGP中。依赖于底物的传输的动力学和平衡结果的预测将通过测试化合物的实验来验证。将建立测试潜在底物的协议，并将获得一系列已知PGP底物的依赖于ATP的运输数据。 Folorosome-Frans-Pgp是多药耐药蛋白家族中的一个重要成员，它为确定候选药物对Pgp排斥的敏感性提供了一种简便的体外检测方法。荧光小体-f/-ANS-PGP分析将适用于广泛的化合物，操作简单，成本效益高，只需要标准的荧光分光光度计。该荧光体-Frans-PGP分析适用于多孔板形式和机器人技术，最终用于药物候选库的中高通量筛选。

DESCRIPTION (provided by applicant): One criterion of a successful drug is its ability to reach its target site. A major reason that candidates are ineffective is that the compounds do not enter cells or cross the barriers that exist between the various compartments of the body, e.g. intestinal epithelium, blood-brain barrier, placenta. In addition to the passive barrier of the cell membrane, many membranes contain energy (ATP) driven efflux proteins, "multi-drug resistance proteins" (MDRP), which actively pump many drugs away from their target tissue. In this project, we propose to develop an innovative, simple and effective assay to determine the susceptibility of drug candidates to a major member of the MDRP family, p-glycoprotein (pgp). This unique assay should provide a valuable tool in drug discovery. The development of this assay for one MDRP will also provide the groundwork for future assays for other members of the MDRP families. What we propose to develop is a new form of Fluorosome, Fluorosome-frans-pgp, based in part on GLSynthesis' existing Fluorosome Technology. Specifically, we propose to clone and express human pgp and incorporate it into the membrane bilayer of Fluorosome-frans. Fluorosome-trans are liposomal nano-particles developed by GLSynthesis to measure the passive permeabilities of drugs through membrane bilayers. Functional incorporation of pgp into Fluorosome-frans-pgp will be confirmed by measuring ATPase activity and ATPdependent flux of test substrates. Predictions of kinetic and equilibrium results for substrate-dependent transport will be verified by experiments with test compounds. Protocols for the testing of potential substrates will be established, and data for ATP-dependent transport of a series of known pgp substrates will be acquired. Fluorosome-frans-pgp will provide a convenient in vitro assay for determining the susceptibility of drug candidates to exclusion by pgp, a major member of the multi-drug resistance protein family. The Fluorosome-f/-ans-pgp assay will be applicable to a wide range of compounds, simple in execution and costeffective, and will require only standard fluorescence spectrophotometers. The Fluorosome-frans-pgp assay is adaptable to multiwell plate formats and robotics, for eventual moderate to high throughput screening of drug candidate libraries.