Activity-based probes composed of a fluorophosphonate reactive group linked to a fluorescent or biotin tag have been developed for serine hydrolases. These probes are used for both selective detection and enrichment of serine hydrolases from proteomes. The activity-based nature of labeling enables simultaneous evaluation of inhibitor selectivity and potency. Moreover, the use of cell permeable probes permit live cell assays to monitor the efficacy and permeability of inhibitors.
Six inhibitors were profiled in multiple proteomes in Xhibit™. One inhibitor demonstrated significant off-target inhibitory activity and was further analysed by XSite™ to determine the target enzymes.
Cells were cultured in 96-well plates and treated with inhibitor prior to probe landing. Automated cell treatment, probe labeling, lysis, SDS-PAGE, and data analysis enables rapid evaluation of cell permeability and inhibitor efficacy.
Animals were treated with an inhibitor at a single dose and were sacrificed at selected time points (n=3) followed by analysis of the target tissue using Xhibit. Time-dependent recovery of the inhibited target is demonstrated.