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Utility of Large-Scale Transiently Transfected Cells for Cell-Based High-Throughput Screens to Identify Transient Receptor Potential Channel A1 (TRPA1) Antagonists

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Marc R. Lake

Protein Biochemistry, Advanced Technology, Abbott Laboratories, Abbott Park, IL

Reza S. Sabet

High Throughput Screening, Target and Lead Discovery, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Wende Niforatos

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Steve D. Pratt

High Throughput Screening, Target and Lead Discovery, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Steven C. Cassar

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Jing Xu

Protein Biochemistry, Advanced Technology, Abbott Laboratories, Abbott Park, IL

Sujatha Gopalakrishnan

High Throughput Screening, Target and Lead Discovery, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Ana Pereda-Lopez

Protein Biochemistry, Advanced Technology, Abbott Laboratories, Abbott Park, IL

Murali Gopalakrishnan

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Thomas F. Holzman

Protein Biochemistry, Advanced Technology, Abbott Laboratories, Abbott Park, IL

Robert B. Moreland

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Karl A. Walter

Protein Biochemistry, Advanced Technology, Abbott Laboratories, Abbott Park, IL

Connie R. Faltynek

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Usha Warrior

High Throughput Screening, Target and Lead Discovery, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Victoria E. Scott

Neuroscience Research, Abbott Laboratories, Abbott Park, IL

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

Key Words: TRPA1 channel • cell-based assay • Ca2+ influx • high-throughput screening • large-scale transient transfection

This version was published on February 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 1, 61-69 (2007)
DOI: 10.1177/1087057106295220


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