Overcoming Compound Interference in Fluorescence Polarization-Based Kinase Assays Using Far-Red Tracers

Abstract

Kinase-mediated phosphorylation of proteins is critical to the regulation of many biological processes, including cell growth, apoptosis, and differentiation. Because of the central role that kinases play in processes that can lead to disease states, the targeting of kinases with small-molecule inhibitors is a validated strategy for therapeutic intervention. Classic methods for assaying kinases include nonhomogenous enzyme-linked immunosorbent assays or scintillation-based formats using [gamma-(32)P]ATP. However, homogenous fluorescence-based assays have gained in popularity in recent years due to decreased costs in reagent usage through miniaturization, increased throughput, and avoidance of regulatory costs associated with the use of radiation. Whereas the readout signal from a nonhomogenous or radioactive assay is largely impervious to interferences from matrix components (such as library compounds), all homogenous fluorescent assay formats are subject to such interferences. Interference from intrinsically fluorescent compounds or from scattered light due to precipitated compounds can interfere with assays that depend on a fluorescence intensity (or fluorescence quenching), fluorescence resonance energy transfer, or fluorescence polarization-based readout. Because these interfering factors show a greater effect at lower wavelengths, one strategy to overcome such interferences is to develop fluorescent assays using longer wavelength (red-shifted) fluorescent probes. In this article, we describe the PanVera PolarScreen far-red fluorescence polarization assay format, which mitigates assay interference from autofluorescent compounds or scattered light through the use of a far-red tracer. The tracer shows substantially less interference from light scatter or autofluorescent library compounds than do fluorescein-based tracers, and gives rise to a larger assay window than the popular far-red fluorophore Cy5.

Development of a Transcreener™ Kinase Assay for Protein Kinase A and Demonstration of Concordance of Data with a Filter-Binding Assay Format

“…Clearly, the use of the longer wavelength Alexa Fluor-633 probe (647 nm emission peak) enables this resistance to low-wavelength fluorescent interference from small-molecule libraries, as the use of red-shifted fluorescent probes to avoid compound fluorescent interference in FP assays is well documented in the literature. [11][12][13][14][15][16] We speculate (based on our experience and on BellBrook Labs data) that large-molecularweight compounds, especially natural product molecules that possess many conjugated bonds, may cause fluorescent interference at the wavelengths used in the assay. 17 It is possible that the reason we did not observe any fluorescent interference from compounds is because of limited diversity of our compound libraries (and the focus on lower molecular weight compounds).…”

Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes

“…Furthermore, the use of a far-red-shifted fluorophore (Alexa Fluor633) provides the benefit of lower assay interference from compound fluorescence and light scatter. 28,29 The combined properties of the detection reagents are reflected in the Z ′ values observed in standard curves mimicking enzyme reactions ( Fig. 5 ).…”

A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy

“…The two-color ratiometric approach results in precise, quantitative analysis of cell signaling with good reproducibility. In addition, the use of NIR detection should avoid the problem of compound interference, which has been reported for other methods of kinase analysis [15][16][17]. The value of using a cell-based approach for evaluation of kinase inhibitors was demonstrated by the different observed effects of the EGFR inhibitor PD168393 on phosphorylation of EGFR, Stat3, and ERK.…”