Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipidic cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesized here containing a branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2.

ELI Beamlines Centre ELI ERIC Za Radnicí 835 252 41 Dolní Břežany Czech Republic

Institute of Biological Information Processing 7 IBI 7 Forschungszentrum Jülich 52428 Jülich Germany

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 160 00 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 160 00 Czech Republic; 1st Faculty of Medicine Charles University Kateřinská 32 Prague 121 08 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 160 00 Czech Republic; Department of Molecular Genetics Faculty of Science Charles University Viničná 5 Prague 128 44 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science Flemingovo n 2 Prague 160 00 Czech Republic; University of Chemistry and Technology Technická 5 Prague 166 28 Czech Republic

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