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.

第一阶段的成功是第二阶段建议的基础。第二阶段的目标是引入反式氟体