Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology.

A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 10(1) to 10(6) plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT(50) or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT(50) was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies.

Screening and identification of small molecule compounds perturbing mitosis using time-dependent cellular response profiles.

Cellular processes such as cell cycle progression, mitosis, apoptosis, and cell migration are characterized by well-defined events that are modulated as a function of time. Measuring these events in the context of time and its perturbation by small molecule compounds and RNAi can provide mechanistic information about cellular pathways being affected. We have used impedance-based time-dependent cell response profiling (TCRP) to measure and characterize cellular responses to antimitotic compounds or siRNAs. Our findings indicate that small molecule perturbation of mitosis leads to unique TCRP. We have further used this unique TCRP signature to screen 119 595 compound library and identified novel antimitotic compounds based on clustering analysis of the TCRPs. Importantly, 113 of the 117 hit compounds in the TCRP antimitotic cluster were confirmed as antimitotic based on independent assays, thus establishing the robust predictive nature of this profiling approach. In addition, potent and novel agents that induce mitotic arrest either by directly interfering with tubulin polymerization or by other mechanisms were identified. The TCRP approach allows for a practical and unbiased phenotypic profiling and screening tool for small molecule and RNAi perturbation of specific cellular pathways and time resolution of the TCRP approach can serve as a complement for other existing multidimensional profiling approaches.

Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects.

We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This noninvasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested by screening a library containing FDA approved drugs, experimental compounds, and nature compounds. Compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRPs). The compounds were clustered based on TCRP similarity. We identified novel mechanisms for existing drugs, confirmed previously reported calcium modulating activity for COX-2 inhibitor celecoxib, and identified an additional mechanism for the experimental compound monastrol. We also identified and characterized a new antimitotic agent. Our findings indicate that the TCRP approach provides predictive mechanistic information for small molecule compounds.