Production and inhibition assay for bile salt export protein

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 ABCB 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 ABCB 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 PUBLIC HEALTH RELEVANCE This project will result in a unique technique to determine if drugs under development inhibit the bile salts export protein BSEP pump This pump is responsible for the transfer of bile salts from the liver to allow normal bile formation and flow Inhibition of this pump by a drug can lead to drug induced liver injury which is a serious cause of illness in humans