Key result
Computational score penalizing spectral distance improves hit follow-up confirmation rates ~10-fold vs FRET ratio alone.
Why the study?
In untargeted screening campaigns using FRET proximity assays, analyzing output solely by the acceptor to donor fluorescence ratio leads to high rates of artefact hits and wasted resources.
Effect estimate: over 10-fold improvement
A novel computational score for FRET assays improves hit confirmation rates by over 10-fold by reducing interference artefacts.
Förster resonance energy transfer (FRET) proximity assays are commonly used in hit discovery for investigating protein-protein interactions, protein modification, molecular glues and small analytes like cyclicAMP or GDP. While they are thought to be robust to artefacts, analyzing their output solely using the ratio of acceptor to donor channel fluorescence intensity hides information about interference. We have observed that in untargeted screening campaigns, with true hit rates around 0.1-0.5 %, up to half of the actives based on ratio can be made up of artefacts. Therefore, using the FRET ratio identifies the wrong equity leading to wasted resources and time in follow-up. We propose a simple computational score to prioritize compounds displaying expected spectral behavior without requiring any extra reads or experimental resources. The score penalizes compounds based on their distance from the theoretical spectral path connecting the medians of an untreated (neutral) control and a full-response control. It is easily tuned based on tool compounds and orthogonal assays and can improve confirmation rate in hit follow-up by over 10-fold.
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Turberville et al. (2026) studied this question. Computational score for FRET prioritization vs. FRET ratio alone was evaluated on Confirmation rate in hit follow-up (over 10-fold improvement). A simple computational score penalizing compounds based on their distance from the theoretical spectral path can improve confirmation rate in hit follow-up by over 10-fold compared to FRET ratio alone.
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