Recommendations for the Reduction of Compound Artifacts in Time-Resolved Fluorescence Resonance Energy Transfer Assays

Abstract

Time-resolved (TR) fluorescence resonance energy transfer (FRET) is a widely accepted technology for high throughput screening (HTS), being able to detect and quantify the interactions of specific biomolecules in a homogeneous format. TR-FRET has several advantages for HTS applications that reduce assay artifacts such as compound interference. However, in some cases artifacts due to compound autofluorescence, color quenching, or signal stability are still observed. This report presents strategies addressing these issues by several means. One recommendation is the recording and visualization of differences in the donor/acceptor fluorescence, which allows the identification of compound artifacts. Another suggestion is to adjust the time delay, between excitation and recording of the fluorescence, in order to reduce compound interference. Furthermore, configuring the assay to allow the TR-FRET measurement to be taken at different time points, creating a reaction time course, allows background correction for each sample. Finally, the optimization of the FRET pair, to ensure assay signal stability under screening conditions, can improve the assay quality. This report presents examples of how these simple steps can be applied to enhance the quality of TR-FRET screening campaigns.

Apparent activity in high-throughput screening: origins of compound-dependent assay interference

“…Both of these phenomena occur with greater incidence at shorter wavelengths of light [14,15,18,19]. Obtaining fluorescence intensity data at a range of compound concentrations can help identify if the activity seen is spurious or due to compound fluorescence [14,20]. Compound fluorescence is reproducible and concentration dependent, with the degree of fluorescence detected proportional to compound concentration (Fig.…”

Cellular Ser/Thr-Kinase Assays Using Generic Peptide Substrates

“…Wherever high-quality phospho-specific antibodies are available, protein kinase substrates will be incorporated into our panel as well. However, due to the very limited availability of such reagents it is expected that a rapid expansion and a significant coverage of the Ser/Thr-kinome will be initially achieved through TR-FRET assays relying on more broadly applicable peptide substrates like cSTKpep-1, 2 and -3 in conjunction with the corresponding universal p-STK antibody [17, 21]. In conclusion, the successful translation of the biochemical KinEASE assay platform into a cellular assay format will aid the exploration of biological Ser/Thr-kinase space through automated high-throughput screening and profiling.…”

“…In vitro, a minimal reagent set of three peptide substrates in conjunction with one phospho-specific antibody [17] is sufficient for the development of >100 biochemical Ser/Thr-kinase assays (KinEASE TM -platform, Cisbio International). We have translated this biochemical platform into a cellular format while maintaining the original homogeneous addition-only protocol as well as the HTS-friendly TR-FRET readout [18-21]. In the case of B-Raf(V600E) targeted TR-FRET assays we found that a set of selected reference compounds returned comparable activity profiles for both protein and peptide substrate based cellular assays.…”

Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases

“…Until further studies are done on this compound and purified analogues, one should not exclude the possibility that this compound inhibits Rtt109 activity in vitro via a therapeutically uninteresting mechanism (Baell, 2010). For instance, an impurity such as a degradation product or a remnant from its chemical synthesis could disrupt enzymatic activity, though isolating and characterizing such impurities can be notoriously difficult (Beasley et al , 2003; Imbert et al , 2007). …”