(474d) Engineered Fusion Proteins Containing Full-Length Substrates for Protease Mechanism Studies and High-Throughput Screening for Pharmaceutical Lead Compounds

Proteases are critically important to human health where they have active roles in numerous cellular regulatory pathways, pathogen life cycles, and bacterial toxins. The large number of proteases of interest has led to diverse experimental approaches to study proteases and to screen for small molecule inhibitors of protease activity. As many proteases have been demonstrated to require protease/substrate interactions at sites distant from the cleavage site for optimal activity, there is a real need to include full-length substrates in mechanistic studies of protease activity and screens for pharmaceutically relevant protease inhibitors. Therefore, we have engineered fluorescent fusion proteins that incorporate full-length substrates with a high affinity binding tag on the amino terminus and a green fluorescent protein on the carboxyl terminus. We have designed custom fusion proteins to serve as protease substrates for the light chain of Botulinum neurotoxin type A (BoNTA LC), the Lethal Factor protease of B. anthracis, the human Factor Xa protease, and the NS2B/NS3 protease of Dengue virus. All of these proteases have important ramifications for human health, but have primarily been investigated using short FRET peptides. Here, we will show the use of our fluorescent fusion proteins in multiplexed microsphere assays (analyzed via flow cytometry) and in a unique full-length substrate solution based FRET based assay. Additionally, we have demonstrated the utility of these assays via: 1. Kinetic studies of protease mechanism for the above mentioned proteases 2. Evaluation of the contribution of distal interaction sites to protease activity of the BoNTA LC. 3. A novel multiplexed microsphere based protease assay that we have combined with high throughput flow cytometry to screen for inhibitors of BoNTA LC, which screened 880 off patent drugs and bio-available compounds to identify a potent inhibitor of BoNTA LC. Using these assays, we have demonstrated the validity of our full-length substrate assays for protease mechanism studies and illustrated the potential that this approach has for high throughput screening for protease inhibitors.