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

基于 FRET 的胆汁盐输出泵抑制脂质体测定的开发

• 批准号: 10016337
• 负责人: Praveen Bansal
• 金额: $ 68.48万
• 依托单位: TFC BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2022-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10016337
• 关键词: ABCG2 gene; ATP phosphohydrolase; Active Biological Transport; Bile Acids; Bile fluid; Biliary; Binding; Biological Assay; Bioreactors; Biotechnology; Blood; Carrier Proteins; Cell Culture Techniques; Cells; Clinical; Column Chromatography; Data; Data Analyses; Detergents; Development; Documentation; Drug Industry; Drug usage; Economic Burden; Encapsulated; Equipment and supply inventories; Excision; Fluorescence; Fluorescence Resonance Energy Transfer; Gel Chromatography; Generations; Goals; Health Care Costs; Healthcare Systems; Hepatic; Hepatobiliary; Hepatocyte; Hepatotoxicity; Human; In Vitro; Infection; Insecta; Kinetics; Legal patent; Letters; Life; Lipid Bilayers; Lipids; Liposomes; Literature; Liver; Longevity; Materials Testing; Measures; Mediating; Membrane; Methods; Mutation; P-Glycoprotein; Patients; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Preparation; Procedures; Process; Production; Progressive intrahepatic cholestasis; Proteins; Protocols documentation; Pump; Quality Control; Reader; Reagent; Robotics; Role; Safety; Science; Services; Severities; Site; System; Techniques; Technology; Testing; Time; Validation; Vendor; Work; base; bile salts; commercialization; drug candidate; drug development; drug withdrawal; experimental study; high throughput screening; in vitro Assay; inhibitor/antagonist; instrumentation; large scale production; liver injury; new technology; novel; programs; protein function; protein purification; proteoliposomes; reconstitution; sensor

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.

成功的I阶段构成了该II期建议的基础。第二阶段的目的是带来荧光体转 - BSEP分析用于商业化，为药物诱导的BSEP提供了完全验证的高通量解决方案 向全球药物开发组的运输抑制作用研究。我们的最终目标是商业化 所有FDA和EMA强制运输商的完整套件，使制药行业能够 有效评估候选药物对持续发展的适用性。 “荧光体反式-BSE” 试剂将是这套试剂套件之一。 我们能够成功实现I阶段提案的所有原理目标。简而言之 TO：1）生产纯化的人BSEP，2）以功能形式重构BSEP转运蛋白，以脂质双层和 确认其ATPase活性，3）通过将传感器封装在BSEP的内部中来制备荧光体反式-BSEP 蛋白脂质体，4）在所得的荧光体 - 传播BSEP构建体中展示了BSEP的转运蛋白功能； 6） 证明了荧光体 - 民-BSEP检测测试底物的主动转运的能力； 7）演示 该测定对BSEP的已知抑制剂敏感。我们已经对提出的建议取得了重大进步 第1阶 规避对外部供应商的任何依赖。其次，我们纯化了一个直接响应的胆汁酸传感器 到胆汁酸，BSEP的物理底物。那是由BSEP介导的ATP驱动的胆汁酸转运和抑制 该药物的BSEP功能可以直接在荧光体TRANS-BSEP构建体中进行测量。 对于第二阶段，我们将将荧光体TRANS-BSEP测定法带到商业阶段。成功后，该测定法会 毒品开发人员可用的第一个现成的高吞吐量，对BSEP明确特定 转运蛋白抑制。该测定使用少量测试材料，易于使用，仅需要标准 注射多井荧光板读取器，该读取器在一分钟内生成实时数据。 为了将此评估带入商业化，我们建议：1）开发各种内部流程/技术 这使纯化的BSEP和胆汁酸传感器蛋白及其成功的保险可以大规模生产 进入我们的Flurosome-Trans-BSEP试剂； 2）使用已知的药物对测定的必要广泛验证 与BSEP相互作用； 3）建立试剂制造所需的严格质量控制方法和 贮存; 4）最后，实施早期采用者计划。 荧光体Trans-BSEP测定法将被毒化，以作为一种高度竞争和有用的产品进入市场。这 将极大地促进我们带来其他两个荧光体测定 抗癌蛋白进入市场。