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.

Chemical Biology Program Memorial Sloan Kettering Cancer Center 1275 York Avenue Box 428 New York New York 10065 United States

Chemical Proteomics Group Leibnitz Institute for Analytical Sciences e 5 Otto Hahn Strasse 6b 44227 Dortmund Germany

Clemens Schoepf Institute for Organic Chemistry and Biochemistry Technical University of Darmstadt Alarich Weiss Strasse 4 8 64287 Darmstadt Germany

Department of Neuropathology Heinrich Heine University Duesseldorf Moorenstrasse 5 40225 Duesseldorf 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

Laboratory of Chemical Biology Department of Cellular and Molecular Medicine University of Leuven Herestraat 49 Box 802 3000 Leuven Belgium

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