Summary We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This non-invasive 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 (TCRP). 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 anti-mitotic agent. Our findings indicate TCRP approach provides predictive mechanistic information for small molecule compounds.

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Cell-based assays have become an integral part of the preclinical drug development process. Recently, noninvasive label-free cell-based assay technologies have taken center stage, offering important and distinct advantages over and in addition to traditional label-based endpoint assays. Dynamic monitoring of live cells, the preclusion of label, and kinetics are some of the fundamental features of cell-based label-free technologies. In this article we will discuss the real-time cell electronic sensing (RT-CES, ACEA Biosciences Inc., San Diego, CA) system and some of its key applications for cell-based assays such as cell proliferation and cytotoxicity, functional assays for receptor-ligand analysis, cell adhesion and spreading assays, dynamic monitoring of endothelial barrier function, and dynamic monitoring of cell migration and invasion. Also, where appropriate we will briefly discuss other label-free technologies in an application-specific manner.

In previous work, we identified genetic correlations between cAMP accumulation in the cerebellum and sensitivity to the incoordinating effects of ethanol. A genetic correlation suggests that common genes underlie the phenotypes investigated. One method for provisionally identifying genes involved in a given phenotypic measure is quantitative trait locus (QTL) analysis. Using a panel of 30 BXD recombinant inbred strains of mice and the progenitors (DBA/2J and C57BL/6J), and the dowel test for ataxia, we measured the blood ethanol concentrations at the time an animal first fell from the dowel and acute functional tolerance (AFT), and investigated cAMP signaling in the cerebellum. Cyclic AMP accumulation was measured in whole-cell preparations of cerebellar minces from individual mice under basal or stimulated conditions. We conducted a genome-wide QTL analysis of the behavioral and biochemical measures with Ͼ2000 genetic markers to identify significant associations. Western blot and comparative sequencing analysis were used to compare cAMP response element binding protein (CREB) levels and protein-coding sequence, respectively. QTL analyses correlating strain means with allelic status at genetic markers identified several significant associations (p Ͻ 0.01). Analysis of variance revealed an effect of strain on behavioral and biochemical measures. There was a significant genetic correlation between initial sensitivity and basal cAMP accumulation in the cerebellum. We identified 6 provisional QTLs for initial sensitivity on four chromosomes, 6 provisional QTLs for AFT on four chromosomes, and 11 provisional QTLs for cAMP signaling on nine chromosomes. Two loci were found to overlap for measures of initial sensitivity and for cAMP signaling. Given the genetic correlation between initial sensitivity and basal cAMP accumulation, we investigated candidate genes in a QTL on chromosome 1. Comparative sequence analysis was performed, and protein levels were compared between C57 and DBA mice for Creb1. No significant differences were detected in coding sequence or protein levels for CREB. These results suggest that although ethanol sensitivity and cAMP signaling are determined by multiple genes, they may share certain genetic codetermination.

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