Production and inhibition assay for bile salt export protein

 描述(申请人提供)：该项目的目标是设计和构建一个高特异性和高通量的体外生物检测系统，以表征药物和候选药物与人BSEP的相互作用。胆盐输出蛋白(BSEP/ABCB11)是定位于肝小管膜上的一种活性的、依赖于ATP的转运蛋白。它介导胆盐从肝细胞外流到胆汁中，对正常的胆汁形成和流动是必不可少的。由于外源生物或ABCB11基因突变导致的BSEP活性抑制可能会导致细胞毒性胆汁成分在细胞内积聚而导致肝损伤。鉴于药物引起的肝损伤(DILI)是导致人类严重疾病、停药、上市后、监管失败以及临床前和临床开发过程中的损耗的主要原因，监管机构建议对候选药物的BSEP抑制潜力进行体外测量。该检测系统将由一个纯化的、具有功能的BSEP组成，该BSEP集成到新的检测平台--荧光体(R)平台中。这种检测系统将被称为荧光体-反式-BSEP，将作为现有唯一一种使用从过度表达BSEP的细胞膜产生的倒置小泡的方法的优越替代品而上市。实现这些目标的具体目标包括生产、纯化和重组活性人BSEP[这些步骤将在分包公司P.I.博士Balazs Sarkadi博士的实验室完成，他是ABC转运体领域的已知专家]，鉴定最佳测试底物，暂定为长春花素，即将由转运体主动运输并显示显著猝灭的底物 用于荧光体反式的传感器，重组BSEP底物和抑制剂的BSEP ATPase活性的测定，以及由重组BSEP形成荧光体反式BSEP，药物转运及其抑制的测量，伴随着平行ATPase活性的测量。这个项目的结果将是一个简单的商业分析，这是明确的BSEP特异性。它具有快速、高信噪比、提供实时结果，并将适用于非常少量的广泛的化合物。它将只需要一个荧光平板阅读器，而不是现有的分析方法所要求的LC-MS或放射性。新的检测方法将适用于中、高通量的筛查。它将作为试剂和内部服务在市场上提供。

 DESCRIPTION (provided by applicant): The objective of the proposed project is to design and construct a highly-specific and high throughput in vitro biological assay system to characterize the interaction of drugs and drug candidates with human BSEP. Bile Salt Export Protein (BSEP/ABCB11) is an active, ATP-dependent transporter localized in the liver canicular membrane. It mediates efflux of bile salts from hepatocytes into bile and is essential for normal bile formation and flow. Inhibition of BSEP activity due to an administered xenobiotic or a mutation in the ABCB11 gene can lead to liver injury caused by intracellular accumulation of cytotoxic bile constituents. Given that drug- induced liver injury (DILI) is a major cause of serious illness in humans, drug withdrawal, post marketing, regulatory failure, and attrition during preclinical and clinical development, regulatory agencies recommend in vitro measurement of the BSEP inhibitory potential of drug candidates. The assay system will consist of a purified and functionally-competent BSEP integrated into the novel assay platform, the Fluorosome(r) Platform. This assay system, to be referred to as Fluorosome-trans-bsep, will be marketed as a superior substitute for the only existing method which uses inverted vesicles generated from membranes of cells overexpressing BSEP. The specific aims meant to achieve these goals include Production, purification and reconstitution of active human BSEP [these steps will be accomplished in the lab of subcontract P.I. Dr. Balazs Sarkadi, a known expert in the field of ABC transporters], identification of an optimal test substrate, tentatively vinblastin, i.e. one that will be actively transported by the transporter and show significant quenching of the sensor employed in Fluorosome-trans, determination of reconstituted BSEP ATPase activities for demonstrated BSEP substrates and inhibitors, and formation of Fluorosome-trans-bsep from reconstituted BSEP, measurement of drug transport and its inhibition, accompanied by parallel ATPase activity measurements. The result of this project will be a simple to use commercial assay that is unambiguously specific for BSEP. It will be rapid with a high signal to noise ratio, provide real time results, and will be applicable to a wide range of compounds at very small amounts. It will only require a fluorescence plate reader rather than LC-MS or radioactivity as is required for an existing assay. The new assay will be amenable to moderate and high throughput screening. It will be offered on the market both as a reagent and as an in-house service.