In addition VRK1 but not VRK2 is sensitive to a non-competitive inhibitor

We therefore reasoned that it would be beneficial to develop an inhibitor assay for rhomboid proteases that does not rely on a substrate at all. A few years ago Cravatt and co-workers developed a highthroughput inhibitor screening method that uses fluorescent activity-based probes. ABPs are small molecules that covalently bind to the active form of an enzyme, but not to an inactivated or zymogen form. ABPs generally consist of a tag, a spacer and an electrophilic group that traps an active site nucleophile. The binding event can be detected by a variety of techniques, such as gel-scanning, biotin blot or fluorescent microscopy, depending on the tagging moiety. When appended to a fluorescent dye, the binding of an ABP can be detected by fluorescence polarization. This so-called fluorescence polarization activity-based protein profiling has been used in inhibitor high-throughput screens for a variety of poorly characterized enzymes. We here report the first FluoPol ABPP screen against a membrane enzyme: the E. coli rhomboid GlpG. Using this method, we have found a novel class of inhibitors for rhomboid proteases: b-lactones. These compounds represent new scaffolds for future rhomboid inhibitor and ABP development. Recently, we and others reported the first fluorescent ABPs for bacterial rhomboids. One ABP is the fluorophosphonate FP-PEG-rhodamine, the other one is based on the 4-chloro-isocoumarin scaffold. Both FP-PEG-R and EK2 have only been used in gel-based applications. In view of previous work of the Cravatt laboratory, we expected that fluorescent rhomboid ABPs would be suitable for the development of a gel-free FluoPol ABPP screening method. Hence, we took EK2 and the commercially available fluorophosphonate FP-rhodamine and verified whether these probes label rhomboid in an activity-based manner. Gratifyingly, both FP-R and EK2 labeled 478-01-3 wild-type GlpG from E. coli, but not the inactive S201A mutant. Labeling was also prevented by pre-inhibition of GlpG WT with the isocoumarin inhibitor S016, which we have identidfied in a previous MALDI-based screen. FP-R gave rise to a more intense labeling, PD1-PDL1 inhibitor 2 probably due to the higher reactivity of the fluorophosphonate electrophile compared to the isocoumarin. We therefor