In vitro substrate assay for multi-drug resistance

描述（由申请人提供）：成功药物的一个标准是其到达目标部位的能力。候选药物无效的一个主要原因是这些化合物不能进入细胞或穿过存在于身体各个隔室之间的屏障，例如肠上皮、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从靶组织中泵出。

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.