Size-Dependent Target Engagement of Covalent Probes

Labeling proteins with covalent ligands is finding increasing use in proteomics applications, including identifying nucleophilic residues amenable for labeling and in the development of targeted covalent inhibitors (TCIs). Labeling efficiency is measured by the covalent occupancy of the target or by biochemical activity. Here, we investigate how these observed quantities relate to the intrinsic parameters of complex formation, namely, noncovalent affinity and covalent reactivity, and to experimental conditions, including incubation time and ligand concentration. It is shown that target engagement is beneficially driven by noncovalent recognition for lead-like compounds, which are appropriate starting points for targeted covalent inhibitors owing to their easily detectable occupancy and fixed binding mode, facilitating optimization. In contrast, labeling by fragment-sized compounds is inevitably reactivity-driven as their small size limits noncovalent affinity. They are well-suited for exploring ligandable nucleophilic residues, while small fragments are less appropriate starting points for TCI development.