These compounds represent new scaffolds for future rhomboid inhibitor and ABP development. In the last decade, small molecule ABPs have substantially impacted protease research, with applications ranging from activity profiling to target discovery and fluorescent imaging. ABPs have also facilitated HTS for ill-characterized enzymes using fluorescent polarization. This HTS has been executed on soluble, but not on membrane enzymes. Recent reports of the first ABPs for intramembrane proteases from the rhomboid family have therefore urged us to investigate FluoPol ABPP for use with membrane enzymes. We have managed this by employing a low concentration of a mild detergent and also found that the surfactant Pluronic F-127 is essential for a good signal-to-noise ratio, probably by facilitating the solubilization of the fluorescent dye. Overall, this resulted in an HTS compatible assay with a high Z-value of 0.9. We are confident that the assay will enable the screening of other poorly characterized membrane-anchored or membrane embedded enzymes. The screening of rhomboids from different organisms is subject of our future research efforts. The special advantage of FluoPol ABPP is that it does not require a substrate, but uses a broad-spectrum ABP. For rhomboids, no small molecule fluorogenic or chromogenic substrates are available as for soluble proteases. One FRET-based polypeptide has been used for screening, but this cannot be used universally. Protein substrates are still the standard assay technique to monitor rhomboid activity. However, the detection of cleavage of these substrates is laborious. Hence, the development and optimization of fluorescent ABPs for rhomboids and other membrane enzymes will likely assist inhibitor discovery for such enzymes. Since the discovery of rhomboids as intramembrane proteases in 2001, inhibitor development has gained momentum slowly. FP-R, for example, reacts with 82% of all mouse metabolic serine hydrolases, which makes it an excellent broad-spectrum ABP. The rhomboid inhibitors based on 4-chloro-isocoumarins have gone through several optimization steps, from the weakly inhibiting DCI, to JLK-6 and S016, which is currently the most potent isocoumarin inhibitor for the E. coli rhomboid GlpG. Still, S016 is more potent against chymotrypsin than against GlpG. The b-lactone scaffold that we have found here, is structurally related to b-lactams. b-lactones are more reactive than b-lactams, and unsurprisingly, b-lactams only act as rhomboid inhibitors when activated with a N-sulfonyl group. The b-lactones 31 and 43 are less potent than the 4chloro-isocoumarin S016, but they have a higher XL-184 potency against GlpG than against trypsin and chymotrypsin. Hence, b-lactones may have the potential to be more selective inhibitors than 4chloro-isocoumarins. Although compounds 31 and 43 also target other serine hydrolases, the b-lactone scaffold can be readily influenced in its selectivity by changing the substituents on the lactone ring. Compound 43 for example, is an acylated form of 44, the natural product vibralactone. Vibralactone is inactive against rhomboid, probably due to the presence of a polar hydroxyl group that may result in unfavourable interactions with the hydrophobic rhomboid TMDs. When this hydroxyl group is blocked as an ester function in compound 43, it yields an active inhibitor. These structures illustrate the possibility to optimize the b-lactone scaffold for usage against rhomboids. We have shown that the b-lactones covalently and irreversibly react with the active site serine of GlpG. This makes them well suitable for use as ‘warheads’ for ABPs. Compounds 31 and 43 contain an alkyne group in their structure, amenable to click chemistry-mediated derivatization. This feature allowed the direct on-gel visualization of the active rhomboid form. Hence, this study adds two new ABPs to the rhomboid chemical toolbox. Since blactones have already been successfully used for ABPP of serine hydrolases in lysates and live bacterial cells, we expect them to be useful tools for the in vivo functional study of bacterial rhomboids. VE-821 Influenza A viruses infect a wide range of avian and mammalian hosts. The worldwide spread of avian flu as well as the subsequent outbreak of the 2009 H1N1 flu has raised public concerns of the global influenza pandemics due to the high morbidity and mortality.