Group Competition Strategy for Covalent Ligand Discovery

As a powerful chemoproteomic tool, activity-based protein profiling (ABPP) has been extensively used for covalent ligand discovery. However, the current ABPP-based approaches are inherently based on indirect probe labeling competed by covalent ligands, and cannot directly compare the preferences of different ligands head-to-head. Herein, we report a group competition-based ABPP strategy (GC-ABPP) to allow the direct comparison of multiple ligands’ binding ability on a proteome-wide scale. By dividing a library of fully functionalized probes (FFPs) into different subgroups and labeling the proteome simultaneously, the direct competition enables comparison of the labeling ability of different probes in drawing a global protein–ligand affinity metric. When it is applied to an expanded probe library, this strategy can be used iteratively to select the highest-affinity ligand toward a certain target protein in a multiple-round process. As a proof of concept, we synthesized 65 FFPs and employed the GC-ABPP to screen the ligand–protein reactivity for >6000 cysteine sites. After three rounds of screening, we identified high-affinity ligands targeting BCAT2 and UGDH. Our “multiple ligands versus multiple proteins” screening paradigm demonstrates great potential for applications in covalent ligand/drug discovery.