Proteases are important targets for the treatment of human disease. Several protease inhibitors have failed in clinical trials due to a lack of in vivo specificity, indicating the need for studies of protease function and inhibition in complex, disease-related models. The tight post-translational regulation of protease activity complicates protease analysis by traditional proteomics methods. Activity-based protein profiling is a powerful technique that can resolve this issue. It uses small-molecule tools-activity-based probes-to label and analyze active enzymes in lysates, cells, and whole animals. Over the last twelve years, a wide variety of protease activity-based probes have been developed. These synthetic efforts have enabled techniques ranging from real-time in vivo imaging of protease activity to high-throughput screening of uncharacterized proteases. This Review introduces the general principles of activity-based protein profiling and describes the recent advancements in probe design and analysis techniques, which have increased the knowledge of protease biology and will aid future protease drug discovery.

Activity-based probes (ABPs) are powerful tools for the analysis of active enzyme species in whole proteomes, cells or animals. Quenched fluorescent ABPs (qABPs) can be applied for real time imaging, allowing the visualization of dynamic enzyme activation by fluorescent microscopy. Unfortunately, qABPs are only available for a few enzymes. We here describe the design and synthesis of qABPs for serine proteases based on a phosphonate ester scaffold.

Activity-based probes (ABPs) are powerful tools for functional proteomics studies. Their selectivity can be influenced by modification of a recognition element that interacts with pockets near the active site. For serine proteases there are a limited number of simple and efficient synthetic procedures for the development of selective probes. Here we describe a new synthetic route combining solid and solution phase chemistries to generate a small library of diphenyl phosphonate probes. Building blocks carrying a P1 recognition element and an electrophilic phosphonate warhead were prepared in solution and 'clicked' on-resin onto a tripeptide. We show the ability to modulate the activity and selectivity of diphenyl phosphonate ABPs and demonstrate activity-dependent labeling of endogenous proteases within a tissue proteome. The herein described synthetic approach therefore serves as a valuable method for rapid diversification of serine protease ABPs.