Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166 10

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166 10 the 1st Faculty of Medicine Charles University Kateřinská 32 Prague 121 08 and

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166 10 the Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 Prague 128

From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 166 10 the Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 Prague 128

the Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 Prague 128 43 the Department of Physics and Materials Engineering Tomas Bata University in Zlín Faculty of Technology nám T G Masaryka 5555 76001 Zlín Czech Republic

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