The Epic cell assay technology (Corning Inc., Corning, NY) uses a resonant waveguide grating optical biosensor to measure cellular response to ligands manifested through dynamic mass redistribution (DMR) of cellular contents. The DMR measurement is a noninvasive, label-free assay that can be used to assess the pharmacological properties of compounds. In this study, a panel of 12 compounds was evaluated against two G protein-coupled receptor (GPCR) targets in recombinant expressed cell lines using the Corning Epic system in 384-well microplates. The evaluation was performed in a double-blinded fashion such that the identity and properties of both the GPCR targets and compounds were unknown to the researchers at the time of the study. Analysis of the DMR response from cell stimulation was used to identify compounds that functioned as agonists or antagonists and to evaluate the associated efficacy and potency. DMR results were shown to have good agreement with data obtained from cyclic AMP and calcium flux assays for compounds evaluated. A further analysis was performed and successfully identified the signaling pathways that the two GPCRs activated. In addition, the DMR measurement was able to detect responses from an endogenous receptor in these cells. The Epic DMR technology provides a generic platform amenable to pharmacological evaluation of cellular responses to GPCR activation in a label-free live cell assay format.

The most widely used assay format for characterizing plasma membrane transporter activity measures accumulation of radiolabeled substrates in tissues or cells expressing the transporters. This assay format had limitations and disadvantages; therefore, there was an unmet need for development of a homogeneous, nonradioactive assay for membrane transporter proteins. In this report, the authors describe the development of a label-free homogeneous assay for the sodium-dependent phosphate transporter NaPi-IIb using the Epic system. The addition of phosphate stimulated a dynamic mass redistribution (DMR) profile unique to cells expressing NaPi-IIb but not on parental cells. This DMR profile was phosphate specific because sulfate or buffer alone did not elicit the same response. Furthermore, the DMR response observed was phosphate and sodium dependent, with Km values in the micromolar and millimolar range, respectively. A known NaPi-IIb noncompetitive inhibitor was shown to completely inhibit the phosphate-stimulated DMR response, suggesting that this observed DMR response is an NaPi-IIb-mediated cellular event. The results demonstrate that a novel label-free assay was developed for studying transporter-mediated cellular activity, and this DMR assay platform could be applicable to other membrane transporter proteins.

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.

Synaptic transmission is characterized by exocytotic events which mediate the release of chemical transmitters to facilitate neuronal communication. Gi/Go coupled GPCRs play an important role in controlling exocytosis. In addition to direct interaction with voltage‐gated calcium channels to inhibit calcium influx, Gβγ subunits bind directly to SNARE proteins in the regulation of fusion events. The current study is aimed at developing small, druglike molecules which target the βγ /SNARE interaction to elucidate details involved in this novel mode of neuromodulation.Compound libraries were screened using a novel, label free detection method, Corning Epic®, which identifies protein‐protein interactions as changes in the local index of refraction due to binding events. Using these signals, we evaluated the ability of small molecules to modulate the Gβγ /SNARE interaction. Initial screening identified five lead compounds. By further application of an iterative analog library synthesis approach, we will develop analogs with improved physiochemical properties to serve as tools to dissect the therapeutic relevance of the Gβγ /SNARE interaction. This work was supported by the National Institutes of Health (EY010291).

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