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靶部位的能力。候选物无效的一个主要原因是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之外，许多膜含有能量（ATP）驱动的外排蛋白，“多药耐药蛋白”（MDRP），其主动地将许多药物泵离其靶组织。 在这个项目中，我们建议开发一种创新的，简单的和有效的测定，以确定候选药物的MDRP家族的主要成员，P-糖蛋白（pgp）的敏感性。这种独特的检测方法应该为药物发现提供一种有价值的工具。针对一种MDRP的该检测方法的开发也将为未来针对MDRP家族其他成员的检测方法提供基础。我们建议开发一种新形式的荧光体，荧光体-frans-pgp，部分基于GLSynthesis现有的荧光体技术。具体来说，我们建议克隆和表达人pgp，并将其纳入荧光体-frans的膜双层。Fluorosome-transis是GLSynthesis开发的脂质体纳米颗粒，用于测量药物通过膜双层的被动渗透性。将通过测量供试底物的ATP酶活性和ATP依赖性通量来确认pgp功能性掺入荧光体-反式-pgp。底物依赖性运输的动力学和平衡结果的预测将通过测试化合物的实验进行验证。将建立潜在底物的检测方案，并采集一系列已知pgp底物的ATP依赖性转运数据。 荧光体-frans-pgp将提供一种方便的体外检测方法，用于确定候选药物对多药耐药蛋白家族主要成员pgp排斥的敏感性。Fluorosome-f-ans-Pgp检测将适用于广泛的化合物，执行简单且具有成本效益，并且仅需要标准荧光光度计。Fluorosome-frans-pgp检测适用于多孔板格式和机器人技术，用于候选药物文库的最终中到高通量筛选。

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.