A ligand selection strategy to customize small molecule probes for activity-based protein profiling (LS-ABPP).

Activity-based probes (ABPs) are the key components of activity-based protein profiling (ABPP). However, designing a probe that shows target-specific as well as site-selective binding can be a challenging and time-consuming task, often requiring complex synthetic procedures to provide a selection of probes from which to choose the ideal one. In this chapter, we present a ligand selection (LS) approach that allows us to rapidly diversify probe molecules in order to meet the steric and electronic demands of the binding site of any target enzyme. The central element of this method is a trifunctional LS probe synthesized from tyrosine in five steps, consisting of a highly reactive pentafluorophenyl (PFP) ester in addition to an electrophilic chloroacetamide warhead, and a bioorthogonal alkyne reporter group. By reacting a variety of primary amine ligands with the PFP ester, a probe library is created and screened for optimal binding characteristics to the target enzyme. With the optimized probe in hand, a compound library is subsequently screened by competitive profiling to identify potential enzyme inhibitors. Conveniently, this protocol is highly adaptable to a large variety of target proteins, representing a valuable tool for enzyme characterization and the discovery of enzyme inhibitors. Here, we apply this method exemplarily to the cysteine protease 3CLpro of the coronavirus SARS-CoV-2.