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Comparison of Assay Technologies for a Tyrosine Kinase Assay Generates Different Results in High Throughput Screening

Novartis Institute for Biomedical Research, Summit, NJ

Donna Weiss

Novartis Institute for Biomedical Research, Summit, NJ

Quynhchi Pham

Novartis Institute for Biomedical Research, Summit, NJ

Robert Schweitzer

Novartis Institute for Biomedical Research, Summit, NJ

Xiang Wu

Novartis Institute for Biomedical Research, Summit, NJ

Jinzi J. Wu

Immunex Corporation, Seattle, WA

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC₅₀ values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC₅₀ values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.

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