Production and inhibition assay for bile salt export protein

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 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.