Developing new assays (Fluorosomes) for characterization of drug interactions of ABC multidrug transporters

Abstract

ABCB1/Pgp and ABCG2/BCRP multidrug transporters are important in developing multidrug resistance and significantly modify ADME‐Tox parameters of several therapeutic compounds. Regulatory agencies (EMA, FDA) state in their guidelines that drug candidates be tested for in vitro inhibition of Pgp and ABCG2. Only the application of different assays can reveal complex drug interactions of these polyspecific transporters. We investigate these interactions either in a native cell membrane environment or by using purified proteins reconstituted in a liposomal platform. ABC transporters extrude drugs at the expense of ATP hydrolysis, thus drug interactions can be followed in proteoliposomes (PL) by measuring ATPase activity or transport of labeled compounds. Providing appropriate lipid environment for ABCG2 was a key step in establishing new assays. PLs of ABCG2, in contrast to those containing ABCB1, have to contain cholesterol. In addition, phosphatidylethanolamine‐containing E.coli lipids support the ABCG2 activity better than phosphatidylcholine‐based lipids. In our international collaboration we are working on new assays similar to that developed earlier for the Pgp (the Fluorosome‐trans‐pgp assay). This method involves PLs that contain a fluorescent drug sensing probe in the interior of the PL, and measures in real time changes in fluorescence which reflect the Pgp‐ or ABCG2‐mediated transport. With Fluorosome‐trans‐abcg2/pgp assay, and direct liposomal assay observing the inhibition of uptake of fluorescently labeled substrate drugs we examine transporter‐drug interactions. The results are analyzed in correlation with the ATPase activity measurements.