Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S′ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.MALDI screening | covalent inhibition | regulated intramembrane proteolysis P roteolysis controls many important biological processes, such as apoptosis, antigen presentation, and blood coagulation. Selective digestion of protein substrates is possible by a combination of tight posttranslational control of protease activity (1) and the protease's substrate specificity, which generally is governed by the primary sequence around the scissile bond (2). The use of inhibitors and activity-based probes (ABPs) has led to a tremendous gain in understanding the roles of proteases within physiological and pathological processes (3). ABPs are small molecules that bind only to active enzymes, but not to zymogen or inhibitor-bound forms (4). ABPs generally consist of a detection tag, a spacer, and a "warhead." The warhead covalently binds to the target enzyme(s) and often is derived from a mechanism-based inhibitor. In the past, ABPs were used to study the activation, localization, and function of soluble proteases in a variety of organisms and disease models (5).Most proteases are soluble and surrounded by an aqueous environment. However, several families of intramembrane proteases exist (6-8): the metalloprotease family M50 (site-2 protease), the aspartic protease family A22 (signal peptide peptidase and γ-secretase), and the serine protease family S54 [rhomboid; numbering according to the MEROPS database (9)]. Rhomboid was discovered in 2001 as a protease in the EGF receptor signaling pathway in the fruitfly Drosophila melanogaster (10). Interestingly, rhomboid genes occur in all kingdoms of nature and are found in most sequenced organisms (11,12). Rhomboids appear to have a wide range of physiological functions, including bacterial protein export (13) and invasion by apicomplexan parasites (14,15), but the roles of many rhomboids remain to be discovered.Rhomboids catalyze peptide bond hydrolysis using a catalytic dyad formed by a serine residue in transmembrane domain...

Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.

Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) and a few crystal structures of the Escherichia coli rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of rhomboids from other species, probably because of a lack of general methods to measure inhibition against different rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen rhomboids for their activity and inhibition. Using ABPP, we compare the inhibitory capacity of 50 small molecules against 13 different rhomboids. We find one new pan rhomboid inhibitor and several inhibitors that display selectivity. We also demonstrate that inhibition profile and sequence similarity of rhomboids are not related, which suggests that related rhomboids may be selectively inhibited. Finally, by making use of the here discovered inhibitors, we were able to show that two bacterial rhomboids autoprocess themselves in their N-terminal part.

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Although activity-based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity-based imaging of rhomboid proteases in membrane environments.

Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes. Several assays have been developed that are able to monitor rhomboid activity. With the aid of these assays, different types of inhibitors have been found, all based on electrophiles that covalently react with the active site machinery. Although the currently available inhibitors have limited selectivity and moderate potency, they can function as research tools and as starting point for the development of activity-based probes, which are reagents that can specifically detect active rhomboid species. Structural studies on complexes of inhibitors with the Escherichia coli rhomboid GlpG have provided insight into how substrate recognition may occur. Future synthetic efforts, aided by high-throughput screening or structure-based design, may lead to more potent and selective inhibitors for this interesting family of proteases.

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