Rhomboid proteases play a variety of physiological roles, but rhomboid protease inhibitors have been mostly developed for the E. coli model rhomboid GlpG. In this work, we screened different electrophilic scaffolds against the human mitochondrial rhomboid PARL and found 4-oxo-β-lactams as submicromolar inhibitors. Multifaceted computations suggest explanations for the activity at the molecular scale and provide models of covalently bound complexes. Together with the straightforward synthesis of the 4-oxo-β-lactam scaffold, this may pave the way toward selective, nonpeptidic PARL inhibitors.

Department of Pharmaceutical Sciences and Medicines Research Institute for Medicines Faculdade de Farmácia Universidade de Lisboa 1600 277 Lisbon Portugal

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Praha 6 160 00 Praha Czech Republic

Laboratory of Chemical Biology Department of Cellular and Molecular Medicine KU Leuven University of Leuven Herestraat 49 box 901b 3000 Leuven Belgium

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