FRET prioritization score to choose high-quality hit equity and reduce the impact of interference by quenching and...

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.