Data Concordance from a Comparison between Filter Binding and Fluorescence Polarization Assay Formats for Identification of ROCK-II Inhibitors

Abstract

The Rho-associated coiled-coil-containing protein serine/threonine kinases ROCK-I and ROCK-II are thought to play a major role in cytoskeletal dynamics by serving as downstream effectors of the Rho/Rac family of cytokine-and growth factoractivated small GTPases. As such, the ROCK family members are attractive intervention targets for a variety of pathologies, including cancer and cardiovascular disease. The authors developed a high-throughput screen to identify ROCK-II inhibitors and report results from a direct comparison of 2 screening campaigns for ROCK-II inhibitors using fluorescence polarization (FP) and filter binding (FB). Screening protocols to identify inhibitors of ROCK-II were developed in FB and FP formats under similar assay and kinetic conditions. A 30,000-member compound library was screened using FB ( 33 P) and FP detection systems, and compounds that were active in either assay were retested in 5-point curve confirmation assays. Analysis of these data showed an approximate 95% agreement of compounds identified as active in both assay formats. Also, compound potency determinations from FB and FP had a high degree of correlation and were considered equivalent. These data suggest that the assay methodology has little impact on the quality and productivity of the screen, provided that the assays are developed to standardize kinetic conditions. (Journal of Biomolecular Screening 2003:399-409)

The Minimum Significant Ratio: A Statistical Parameter to Characterize the Reproducibility of Potency Estimates from Concentration-Response Assays and Estimation by Replicate-Experiment Studies

“…Initially, as in the NPY2 example, a replicate-experiment protocol can be used when the assay is first developed to estimate the MSR, the key outcome of statistical reliability of concentration-response assays. Then, as assays are transferred to new laboratories or undergo 16 and Sun and others 7 used a Bland-Altman analysis to compare 2 assays. In this article, we adapt the analysis to estimate the MSR using 2 runs of a single assay, an approach not used in those articles or in the original Bland-Altman article.…”

Assay Concordance between SPA and TR-FRET in High-Throughput Screening

“…This question has previously been addressed by others. [9][10][11][12][13][14] Sills et al 11 screened a randomly selected subset of 30,000 compounds for tyrosine kinase inhibitors with the alarming result that there was a very limited overlap of hits derived from the scintillation proximity assay (SPA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP) assays and also that there was no significant correlation between IC 50 values obtained with SPA and the fluorescence-based techniques. A similarly alarming result was published by Wu et al 12 for the nuclear receptor FXR.…”

Adopting a Practical Statistical Approach for Evaluating Assay Agreement in Drug Discovery

“…10,11 Hubert et al recently used the Bland and Altman method to analyze the equivalence of 2 assay data sets. 12 A commonly used approach for assessing data sets generated by 2 different assays is correlation analysis. Typically, data sets generated by 2 different assays are visualized using a correlation plot, and the relationship between the 2 data sets is defined by the line of best fit and coefficient of determination (r 2 ).…”