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.

Clemens Schoepf Institute for Organic Chemistry and Biochemistry Technische Universitaet Darmstadt Alarich Weiss Strasse 4 8 64287 Darmstadt Germany

Department of Neuropathology Heinrich Heine University Duesseldorf Moorenstrasse 5 40225 Duesseldorf Germany

Department of Neuropathology Heinrich Heine University Duesseldorf Moorenstrasse 5 40225 Duesseldorf Germany; Clemens Schoepf Institute for Organic Chemistry and Biochemistry Technische Universitaet Darmstadt Alarich Weiss Strasse 4 8 64287 Darmstadt Germany

Institute for Pathobiochemistry University Medical Center of the Johannes Gutenberg University Mainz Duesbergweg 6 55128 Mainz Germany

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo n 2 166 10 Praha 6 Czech Republic

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