HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime de-tection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Automated summary

High-throughput screening (HTS) is crucial for the direct discovery of allosterically active ligands. We developed a high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET) technology to detect allosteric modulators by monitoring protein structure changes. We tested this approach at an industrial scale by adapting an allosteric FRET sensor for cardiac myosin to HTS and screened 1.6 million compounds. The results identified allosteric activators and inhibitors of cardiac myosin with high potential for FLT-based drug discovery.