(402g) Microspheres and Flow Cytometry as a Platform for Protease Assays in High-Throughput Screening and Protease Kinetic Analysis

The ability to multiplex microspheres while simultaneously analyzing fluorescent biochemical reactions on the surface makes microsphere based assays a valuable tool for biochemical analysis and high-throughput screening (HTS). Previously, we described development of a microsphere based flow cytometry assay for multiplexed analysis of proteases and substrates and its application in HTS to detect selective protease inhibitors. Here, we report recent screening results and advances by which the capabilities of this platform have been significantly extended through the use of increased multiplexing levels and biomimetic surfaces. In this talk we will describe our approach where multiple protease substrates are biotinylated at the N-terminus and fused with green fluorescence protein at the C terminus. Such substrates are attached to multiplex streptavidin coated microsphere sets that are multiplexed using varying loading of fluorescent dyes. Test compounds, protease and substrate microspheres are combined in 384 well plates and analyzed for protease activity using the HyperCyt flow cytometry HTS platform. Protease activity is detectable as loss of GFP fluorescence from microspheres due to substrate cleavage. Our approach incorporates three important assay features: 1) Use of full-length substrates to detect compounds that act at distal interaction sites. 2) Parallel analysis of multiple proteases in a well (multiplexing) for immediate reporting of compound specificity. 3). Ability to use biomimetic surfaces to present screening substrates that have significant biological relevance. We have developed assays for the proteases Factor Xa, Bacillus anthracis Lethal Factor (LF), Clostridium botulinum type A Light Chain (BoNT/A LC) and are developing this platform for other proteases of medical relevance such as NS2/NS3B protease that cleaves the polyprotein of Dengue virus during viral maturation. We will present results of HTS using these protease assays that have led to the discovery of several new inhibitors. These include novel LF inhibitors and, most notably, ebselen, an inhibitor of BoNT/A LC that is bioavailable and shows little to no known side effects in animal models. We have also developed novel techniques for the study of protease/substrate kinetics that take advantage of the inherently low substrate concentrations in these assays, allowing determination of the specificity constant of a protease. Finally, we will present the development of biomimetic surfaces consisting of substrates in suspended lipid bilayers on the microsphere surface. These substrates will enable accurate polyprotein substrates to be created and screened using NS2/NS3B protease from Dengue. This work has demonstrated that microsphere based protease assays in conjunction with high throughput flow cytometry can simultaneously analyze multiple proteases at once for identification of both inhibitors and compounds which increase the activity of a protease of interest. The platform has now been extended to quantitative analysis of protease/substrate kinetics, solution based assays have been developed for kinetic parameter comparison and validation, and biomimetic surfaces have been developed to increase the range of proteases that can be screened using our approach. In this manner we have created a generally applicable HTS platform that can be applied to virtually any protease of interest.