Development of a FRET based Bile Salt Export Pump Inhibition Liposomal Assay - 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.
