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.