A novel cell-based 384-well, label-free assay for discovery of inhibitors of influenza A virus

Abstract

A novel cell-based 384-well, label-free assay for discovery of inhibitors of influenza A virus Fuli Jia1, Clinton Maddox2, Alice Gao3, Louie Tran3, William Severson1, E Lucile White2, Lynn Rasmussen2, Anju Dang3, Colleen B Jonsson1,41Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, AL, USA; 2High-Throughput Screening Center, Southern Research Institute, Birmingham, AL, USA; 3Corning Life Science, Corning, NY, USA; 4Department of Microbiology and Immunology, University of Louisville, Louisville, KY, USAAbstract: Resonance wave-guide (RWG) biosensor technology allows label-free measurements of global cellular responses of the dynamic mass redistribution (DMR). We hypothesized that the DMR signals to extracellular stimulations occurring upon entry of a virus, could provide a new approach for the development of physiologically relevant cell-based assays for screening of small molecules. We explored this technology with influenza virus (A/Udorn/72, H3N2) using MDCK and Vero E6 cell lines in a 384-well format. The MDCK cell line assay was optimized with a fibronectin-coated surface microplate with 6000 cells per well that were infected at a multiplicity of infection (MOI) of 1. Under this set of optimized conditions, for the vero E6 cells, an assay window of 1130 pm shift were obtained at 24 hours. The Vero E6 cell line assay was optimized using a poly-D-lysine-coated surface with seeding density of 6000 cells per well that were infected at a MOI of 5. Under this set of optimized conditions for the MDCK cells, an assay window of >600 units and Z values of 0.6–0.7 were obtained at 24 h. A small library of 1120 compounds was screened using the MDCK, which demonstrates the feasibility of the approach for high-throughput screening.Keywords: small molecule screening, label-free technology, antivirals