In Phase I, as proposed, we successfully demonstrated proof of principle for the Flurosome-trans-abcg2 assay. This innovative assay characterizes how compounds interact with the important ATP-dependent drug efflux transporter, ABCG2 (BCRP, Breast Cancer Resistance Protein), i.e. the susceptibility of drug candidates to be extruded from their target tissue. A successful Phase I forms the basis for this Phase II proposal. The goal of Phase II is to bring the Fluorosome-trans-abcg2 assay to commercialization, providing a fully validated high-throughput solution for ABCG2 transporter 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-abcg2 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 ABCG2; 2) reconstitute the ABCG2 transporter in a functional form into lipid bilayers; 3) test and confirm the reconstituted ABCG2's ATPase activity; 4) use the reconstituted ABCG2 to construct lipid vesicles containing encapsulated drug sensor, Fluorosome-trans-abcg2; 5) demonstrate that ABCG2 is functional in the resulting Fluorosome-trans-abcg2 construct; 6) demonstrate the ability of Fluorosome-trans-abcg2 to detect the active transport of test substrates; and 7) demonstrate that the assay is sensitive to known inhibitors of ABCG2. In addition to succeeding in these initial goals, we have made a significant advance over what was proposed in Phase I, in that instead of relying on an external vendor for the purification and reconstitution of the ABCG2 protein, we have both improved these procedures and brought them entirely in house. For Phase II, we will bring the Fluorosome-trans-abcg2 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 ABCG2 transporter. The assay uses small amounts of test material and is simple to use. With respect to instrumentation, it 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 ABCG2 protein and its incorporation into our Flurosome-trans-abcg2 reagent; 2) the necessary extensive validation of the assay using drugs known to interact with ABCG2; 3) establishment of rigorous quality control methods required for reagent manufacture and storage; and 4) finally, the implementation of an extensive plan which includes an Early Adopter Program followed by product launch and revenue generation. Accomplishing these goals will result in a unique, easy to use, rapid, cost-effective and highly specific assay for characterizing the interaction of drugs and drug candidates with the ABCG2 transporter. The Fluorosome-trans-abcg2 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 current Fluorosome assays, those for the human pgp and BSEP, into the marketplace.