A FRET-Based Assay for Assessing Covalent Warhead Reactivity

High Resolution Image Download MS PowerPoint Slide Covalent modalities have become increasingly important in drug discovery, yet reliable methods for evaluating the intrinsic reactivity of electrophilic warheads toward different nucleophiles remain a challenge. While Förster resonance energy transfer (FRET)-based fluorescent sensors are widely exploited in chemical biology for the detection of diverse analytes, their potential for probing covalent warhead reactivity has not been explored. Herein, we report the design and application of a FRET-based fluorescent platform for assessing covalent warhead reactivity toward biological nucleophiles. The system incorporates a sulfone-based nucleophilic aromatic substitution (S N Ar) warhead bridging a coumarin fluorescent donor and a dinitrophenyl dark quencher, forming a FRET dyad. The dyad operates as a reaction-based fluorescent sensor, in which S N Ar reaction disrupts FRET and allows for fluorescence readout, quantifying covalent warhead reactivity and demonstrating strong selectivity for thiols. The assay is performed in a 96-well plate format, which enables high-throughput screening of nucleophiles and reaction conditions. This versatile approach provides a robust tool for the evaluation of covalent warhead reactivity, facilitating mechanistic studies in chemical biology and drug discovery.