Activity-based probes to detect MMP activity.

Elucidating the precise biological functions of MMPs remains challenging because conventional analytical methods generally measure total protein abundance rather than enzymatic activity. Since only a fraction of the enzyme pool exists in an active form and proteolytic activity is the primary determinant of MMP function, approaches capable of selectively monitoring active MMPs provide a more accurate representation of their physiological and pathological roles. Recent advances in chemical biology have led to the development of molecular probes that directly interrogate MMP activity in complex biological environments. This chapter reviews two complementary classes of probes: substrate-based activatable probes and affinity-based probes. Activatable probes exploit MMP-mediated cleavage to generate detectable signals and enable sensitive monitoring of proteolytic activity in cells, tissues, biological fluids, and living organisms. In contrast, affinity-based probes selectively bind active MMPs and form covalent adducts that enable their direct visualization, enrichment, and unambiguous identification by proteomic methods. Particular emphasis is placed on probe design, substrate selectivity, analytical performance, and applications ranging from in vivo imaging to activity-based biomarker discovery. Finally, current limitations and emerging opportunities are discussed, including machine learning-guided substrate design, synthetic biomarker technologies, and next-generation affinity-based profiling strategies, which collectively hold promise for advancing both fundamental MMP biology and translational diagnostic applications.