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

第一阶段的成功是第二阶段提案的基础。第二阶段的目标是将荧光体-反式- bsep检测商业化，为药物诱导的BSEP提供完全验证的高通量解决方案 转运蛋白抑制研究提供给世界各地的药物开发小组。我们的最终目标是将 适用于所有FDA和EMA授权运输商的全套检测试剂，使制药行业能够 有效地评估候选药物的持续开发的适用性。“荧光体-反式-BSEP” 试剂将是这套试剂中的一种。 我们成功地实现了第一阶段提案的所有原理验证目标。简而言之，我们能够 以：1）产生纯化的人BSEP，2）将功能性形式的BSEP转运蛋白重建为脂质双层， 3）通过在BSEP内部封装传感器来制备Fluorosome-trans-bsep 蛋白脂质体，4）证明BSEP在所得荧光体-trans-bsep构建体中的转运蛋白功能; 6） 证明Fluorosome-trans-bsep检测测试底物的主动转运的能力;和7）证明 该试验对已知的BSEP抑制剂敏感。我们已经取得了重大进展， 一阶段我们已经建立了内部生产纯化和复溶BSEP的方案， 避免对外部供应商的任何依赖。其次，我们纯化了一种胆汁酸传感器， 胆汁酸，BSEP的生理底物。因此，BSEP介导的ATP驱动的胆汁酸转运和BSEP对胆汁酸转运的抑制作用， 药物的这种BSEP功能可以在荧光体-反式-BSEP构建体中直接测量。 对于第二阶段，我们将使荧光体-反式-bsep测定进入商业阶段。成功后，该试验将 第一个现成的高通量测定可用于药物开发人员，明确特异性为BSEP 转运蛋白抑制该检测方法使用少量的测试材料，使用简单，只需要一个标准 注射多孔荧光板阅读器，在一分钟内生成真实的时间数据。 为了使该测定商业化，我们建议：1）开发各种内部工艺/技术 其使得能够大规模生产纯化的BSEP和胆汁酸传感器蛋白并成功掺入 我们的荧光体-反式-bsep试剂; 2）使用已知的药物进行必要的广泛验证， 与BSEP相互作用; 3）建立试剂生产所需的严格质量控制方法， （4）最后，实施一项早期的再生产计划。 Fluorosome-trans-bsep检测将作为一种极具竞争力和实用的产品进入市场。这 这将极大地促进我们将我们的其他两个荧光体检测，即人类P-糖蛋白和乳腺癌 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.