Monocyclic β-Lactams Are Selective, Mechanism-Based Inhibitors of Rhomboid Intramembrane Proteases

Pierrat, Olivier A.; Střı́šovský, Kvido; Christova, Yonka; Large, Jonathan M.; Ansell, Keith H.; Bouloc, Nathalie; Smiljanic, Ela; Freeman, Matthew

doi:10.1021/cb100314y

Cited by 62 publications

(91 citation statements)

References 33 publications

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“…Drugs that target specifically presenilin activity, an i-CLiP have been obtained (reviewed in [74]) and more recently monocyclic ␤-lactams have been shown to be selective rhomboid inhibitors [75]. In this study, over 57,000 molecules were tested for their ability to inhibit the AarA rhomboid activity of P. stuartii; among others, a monocyclic ␤-lactam compound was a specific, potent inhibitor.…”

Section: Potential As Drug Targetsmentioning

confidence: 99%

New insights into parasite rhomboid proteases

Santos

Graindorge

Soldati‐Favre

2012

Molecular and Biochemical Parasitology

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a b s t r a c tThe rhomboid-like proteins constitute a large family of intramembrane serine proteases that are present in all branches of life. First studied in Drosophila, these enzymes catalyse the release of the active forms of proteins from the membrane and hence trigger signalling events. In protozoan parasites, a limited number of rhomboid-like proteases have been investigated and some of them are associated to pathogenesis. In Apicomplexans, rhomboid-like protease activity is involved in shedding adhesins from the surface of the zoites during motility and host cell entry. Recently, a Toxoplasma gondii rhomboid was also implicated in an intracellular signalling mechanism leading to parasite proliferation. In Entamoeba histolytica, the capacity to adhere to host cells and to phagocytose cells is potentiated by a rhomboid-like protease. Survey of a small number of protozoan parasite genomes has uncovered species-specific rhomboidlike protease genes, many of which are predicted to encode inactive enzymes. Functional investigation of the rhomboid-like proteases in other protozoan parasites will likely uncover novel and unexpected implications for this family of proteases.

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Section: Potential As Drug Targetsmentioning

confidence: 99%

New insights into parasite rhomboid proteases

Santos

Graindorge

Soldati‐Favre

2012

Molecular and Biochemical Parasitology

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“…One other isocoumarin (JLK-6; 20, Table S1) has been reported to inhibit E. coli rhomboid GlpG (23). Sulfonylated β-lactams recently were found to inhibit bacterial rhomboids (24), as well as two fluorophosphonates (25,26).…”

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confidence: 99%

“…However, gel analysis is not optimal for identifying inhibitors because of the low throughput. One FRET-based assay for the rhomboid AarA of the Gram-negative bacterium Providencia stuartii has been reported (24); it made use of a 16-mer FRET peptide, but many rhomboids do not cleave this substrate efficiently. The development of small molecule fluorescent reporters for rhomboids is difficult because the details of their substrate specificities still are not well defined.…”

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confidence: 99%

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Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Vosyka

Vinothkumar

Wolf

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Even when high throughput screening is employed to discover novel compounds, the mechanistic knowledge will be helpful in evaluating early hits and in deciding which chemical classes are worth pursuing further. Both approaches were used in the recent discovery of the ␤-lactam class of rhomboid inhibitors (36).…”

Section: Catalytic Mechanism and Inhibitor Bindingmentioning

confidence: 99%

Structure and Mechanism of Rhomboid Protease

Akiyama

Xue

2013

Journal of Biological Chemistry

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Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

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Monocyclic β-Lactams Are Selective, Mechanism-Based Inhibitors of Rhomboid Intramembrane Proteases

Cited by 62 publications

References 33 publications

New insights into parasite rhomboid proteases

New insights into parasite rhomboid proteases

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Structure and Mechanism of Rhomboid Protease

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