Development of a FRET based Bile Salt Export Pump Inhibition Liposomal Assay

成功的第一阶段构成了第二阶段提案的基础。第二阶段的目标是将荧光体-反式- BSEP检测走向商业化，为药物诱导的BSEP提供了一个完全有效的高通量解决方案 转运蛋白抑制研究到世界各地的药物开发组织。我们的最终目标是将一种 适用于FDA和EMA授权的所有转运体的全套分析试剂，使制药行业能够 有效评估候选药物持续开发的适宜性。“荧光体-反式-bsep” 试剂将是这套试剂中的一种。 我们能够成功地实现我们第一阶段提案的所有原则证明目标。简而言之，我们能够 为了：1)生产纯化的人BSEP，2)将BSEP转运体以功能形式重组到脂质双层中，并 确定其ATPase活性，3)在BSEP内部封装传感器制备荧光体-反式BSEP 蛋白脂质体，4)证明了BSEP的转运蛋白在所得到的荧光体-反式BSEP构建中的功能； 演示荧光体-反式BSEP检测测试底物主动转运的能力；以及7)演示 该检测方法对已知的BSEP抑制剂敏感。我们已经取得了重大进展，比起 第一阶段，我们已经制定了内部生产纯化和重组BSEP的方案 规避对外部供应商的任何依赖。其次，我们已经提纯了一种直接响应的胆汁酸传感器 对BSEP的生理底物胆汁酸。因此，BSEP介导的ATP驱动的胆汁酸转运及其对胆汁酸转运的抑制 药物的这种BSEP功能可以在荧光体-反式BSEP结构中直接测量。 对于第二阶段，我们将把荧光体-反式-BSEP检测带入商业阶段。当成功时，这种检测将是 第一个对BSEP具有明确特异性的现成的高通量分析，可供药物开发人员使用 转运蛋白抑制。该分析使用少量的测试材料，使用简单，只需要一个标准 注入多井荧光板读取器，在一分钟内产生实时数据。 为了将这一检测方法商业化，我们建议：1)开发各种内部工艺/技术 使纯化的BSEP和胆汁酸传感器蛋白的大规模生产和成功掺入成为可能 进入我们的氟尿体-反式-bsep试剂；2)使用已知的药物对该分析进行必要的广泛验证 与BSEP互动；3)建立试剂制造所需的严格质量控制方法 存储；4)最后，实施早期采用者计划。 荧光体-反式-BSEP检测将作为一种极具竞争力和实用的产品进入市场。这 将极大地方便我们带来另外两种荧光体分析方法，即人类P-糖蛋白和乳房 抗癌蛋白，进入市场。

A successful Phase I forms the basis for this Phase II proposal. The goal of Phase II is to bring the Fluorosome-trans- bsep assay to commercialization, providing a fully validated high-throughput solution for drug-induced BSEP transporter inhibition studies to drug development groups worldwide. Our ultimate goal is to commercialize a complete suite of assay reagents for all FDA and EMA mandated transporters, enabling the pharmaceutical industry to efficiently evaluate the suitability of drug candidates for continued development. The “Fluorosome-trans-bsep” reagent will be one of this suite of reagents. We were able to successfully accomplish all the proof-of-principle goals of our Phase I proposal. In brief, we were able to: 1) produce purified human BSEP, 2) reconstitute the BSEP transporter in a functional form into lipid bilayers and confirm its ATPase activity, 3) prepare Fluorosome-trans-bsep by encapsulating a sensor in the interior of the BSEP proteoliposomes, 4) demonstrate BSEP’s transporter function in the resulting Fluorosome-trans-bsep construct; 6) demonstrate the ability of Fluorosome-trans-bsep to detect the active transport of test substrate; and 7) demonstrate that the assay is sensitive to known inhibitors of BSEP. We have made significant advances over what was proposed in Phase I. We have established protocols for production of purified and reconstituted BSEP in-house thereby circumventing any reliance on an external vendor. Secondly, we have purified a Bile Acid Sensor that responds directly to bile acid, the physiological substrate of BSEP. Thus, ATP-driven bile acid transport mediated by BSEP and inhibition of this BSEP function by drugs can be directly measured in the Fluorosome-trans-bsep construct. For Phase II, we will bring the Fluorosome-trans-bsep assay to a commercial stage. When successful, this assay will be the first off-the-shelf high throughput assay available to drug developers that is unambiguously specific for the BSEP transporter inhibition. The assay uses small amounts of test material, is simple to use and requires only a standard injecting multiwell fluorescence plate reader, which generates real time data in under a minute. To bring this assay to commercialization, we propose: 1) the development of various in-house processes/technologies that enable the large scale production of purified BSEP and Bile Acid Sensor proteins and their successful incorporation into our Flurosome-trans-bsep reagent; 2) the necessary extensive validation of the assay using drugs known to interact with BSEP; 3) establishment of rigorous quality control methods required for reagent manufacture and storage; and 4) finally, the implementation of an Early Adopter Program. The Fluorosome-trans-bsep assay will be poised to enter the market as a highly competitive and useful product. This will greatly facilitate us in bringing our other two Fluorosome assays, those for the human P-glycoprotein and Breast Cancer Resistance Protein, into the marketplace.