Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

Compounds that form covalent bonds with specific target proteins offer a variety of advantages as chemical probes and therapeutic agents. Most commonly, mildly reactive, electrophilic small molecules are employed to form covalent bonds with select cysteine side chains in specific proteins. Electrophile-first approaches of ligand discovery, whereby a library of electrophilic small molecules are screened against a protein target, have become popular as they avoid the need for time-consuming downstream installation of an electrophilic warhead. Such screening is complicated, however, as electrophilic ligands can exhibit a wide range of different rates of spontaneous reaction with cysteines. Quantitative-irreversible tethering (qIT) offers a fluorescence-based method for hit identification and development that normalizes data for these differences in intrinsic compound reactivity. Rates of reaction of individual compounds with a target protein are determined and compared to compound reactivity with the unstructured tripeptide glutathione (this being a proxy for spontaneous compound reaction), enabling the identification of compounds that preferentially react with the protein of interest. This methodology has been successfully applied to identify selective covalent fragments against several drug targets, including SARS-CoV-2 main protease, cyclin-dependent kinase 2, and RAP27A. Here, we demonstrate the application of qIT to a target protein to generate a quantitative and robust data set, allowing prioritization of hit ligands for future development.