Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.
- MeSH
- benzoxaziny chemická syntéza chemie MeSH
- chymotrypsin chemie MeSH
- DNA vazebné proteiny antagonisté a inhibitory chemie genetika MeSH
- Drosophila chemie MeSH
- endopeptidasy chemie genetika MeSH
- enzymatické testy MeSH
- Escherichia coli enzymologie MeSH
- inhibitory serinových proteinas chemická syntéza chemie MeSH
- katalytická doména MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory chemie genetika MeSH
- mutace MeSH
- objevování léků MeSH
- proteiny Drosophily metabolismus MeSH
- proteiny z Escherichia coli antagonisté a inhibitory chemie genetika MeSH
- serin chemie MeSH
- simulace molekulového dockingu MeSH
- skot MeSH
- styreny chemická syntéza chemie MeSH
- transformující růstový faktor alfa metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.
- MeSH
- design s pomocí počítače MeSH
- HEK293 buňky MeSH
- inhibitory serinových proteinas chemie farmakologie MeSH
- lidé MeSH
- membránové proteiny MeSH
- racionální návrh léčiv * MeSH
- sacharin analogy a deriváty farmakologie MeSH
- serinové proteasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
