(487g) High Throughput Approach to Drug Discovery: Sars Coronavirus - a Case Study

Authors: 
Gosalia, D. N., University of Pennsylvania
Diamond, S. L., University of Pennsylvania
Simmons, G., Department of Microbiology
Bates, P., University of Pennsylvania


Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots/cm2. Using aerosol deposition, subsequent reagents and water were metered into each reaction center in order to rapidly assemble diverse multicomponent reactions without cross-contamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease-substrate interactions, and high throughput screening reactions. A library of pharmacologically active compounds was microarrayed in triplicates on 100 slides and screened against 40 different proteases. Novel inhibitors were detected for a protease target implicated in the mechanism of action for entry of SARS-CoV (Severe Acute Respiratory Syndrome-associated Coronavirus), for which there is currently no effective treatment. The best protease inhibitor had an IC50 of 2.5 nM in solution. Inhibition of infection mediated by SARS-CoV Spike glycoprotein was quantified using single round lentiviral-based pseudotype entry assays into 293T target cells with luciferase reporter gene as a read-out. The best inhibitor had an IC95 of 2 mM. Similar results were seen on Vero E6 cells. Our method shows that from one printing run that consumes <1 nanomole of compound, large combinatorial libraries can be subjected to numerous separation-free, homogeneous assays at volumes 103 to 104 smaller than current high throughput methods.