Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening

Abstract

Small molecules that react to form covalent bonds with proteins are widely used as biological tools and therapeutic agents. Screening cysteine-reactive fragments against a protein target is an efficient way to identify chemical starting points for covalent probe development. Mass spectrometry is often used to identify the site and degree of covalent fragment binding. However, robust hit identification requires characterization of the kinetics of covalent binding that can be readily achieved using quantitative irreversible tethering. This screening platform uses a non-specific cysteine-reactive fluorogenic probe to monitor the rate of reaction between covalent fragments and cysteine containing biomolecules. Fragment libraries are simultaneously screened against the target protein and glutathione, which functions as a control, to identify hit fragments with kinetic selectivity for covalent modification of the target. Screening by quantitative irreversible tethering accounts for variations in the intrinsic reactivity of individual fragments enabling robust hit identification and ranking.

Keywords: Covalent fragments; Cysteine targeting; Fragment-based drug discovery; Irreversible inhibition; Quantitative irreversible tethering.