Ligand-Directed Modification of Active Matrix Metalloproteases: Activity-based Probes with no Photolabile Group

Activity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S-3 ' region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.

Automated summary

Activity-based probes can discriminate active enzymes from inactive counterparts, targeting metalloproteases through reversible inhibitors with photo-crosslinkers. Novel probes were developed to covalently modify active matrix metalloproteases without external trigger, allowing for nanogram-scale reactions with recombinant MMPs in complex proteomes. This ligand-directed chemistry successfully labeled active MMP-12 secreted by eukaryote cells, with potential applications for other metalloproteases in unresolved proteomic profiling in vivo.