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A Comparative Study of Fluorescence Assays in Screening for BRD4
Pia Hansson1, Helen Boyd1, Ian L Dale2
11 AstraZeneca R&D, Discovery Biology , Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden .
Assay and Drug Development Technologies
|October 12, 2018
Summary
Comparing three fluorescence assay technologies for high-throughput screening revealed differences in hit rates and artifact generation. Overlapping hits across assays were more likely to be confirmed in cellular assays, highlighting the importance of assay selection in drug discovery.
Area of Science:
- Biochemistry
- Drug Discovery
- Assay Development
Background:
- Fluorescence assay technologies are vital for high-throughput screening (HTS) due to their sensitivity and ease of use.
- Assay selection involves balancing performance, cost, and reagent availability, with susceptibility to artifacts being a critical consideration.
- Artifacts can lead to false positives or negatives, wasting resources and hindering drug development.
Purpose of the Study:
- To compare the performance of three distinct fluorescence assay technologies (fluorescence lifetime, fluorescence polarization, homogeneous time-resolved fluorescence) in a screening campaign.
- To evaluate primary hit rates, compound overlap, and hit confirmation rates across the different assay formats.
- To assess the ability of each assay to discriminate true hits from artifact compounds using a known inhibitor set.
Main Methods:
- A diverse set of 7,038 compounds was screened against BRD4(1) using fluorescence lifetime, fluorescence polarization, and homogeneous time-resolved fluorescence assays.
- Confirmed hits were further validated using NanoBRET™ cellular assays.
- A separate screen utilized a well-annotated set of compounds with known undesirable mechanisms of inhibition.
Main Results:
- Significant differences were observed between the fluorescence assay technologies, resulting in distinct hit lists with varying degrees of overlap.
- Hits that overlapped across multiple assay formats showed a higher probability of confirmation in cellular assays.
- The assays demonstrated varying capabilities in identifying false positives and false negatives.
Conclusions:
- The choice of fluorescence assay technology impacts screening outcomes, including hit identification and artifact profiles.
- Assay overlap is a valuable metric for prioritizing hits with higher confidence.
- Careful assay selection and validation are crucial for successful drug discovery campaigns to minimize false results.
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