Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

Chemical Biology Program Memorial Sloan Kettering Cancer Center 1275 York Avenue Box 428 New York NY 10065 USA

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

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

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo n 2 Prague 166 10 Czech Republic; Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 Prague 128 43 Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo n 2 Prague 166 10 Czech Republic; Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 Prague 128 44 Czech Republic

Leibniz Institute for Analytical Sciences ISAS Otto Hahn Strasse 6b 44227 Dortmund Germany

Leibniz Institute for Analytical Sciences ISAS Otto Hahn Strasse 6b 44227 Dortmund Germany; KU Leuven University of Leuven Herestraat 49 Box 802 3000 Leuven Belgium

Medical Research Council Laboratory of Molecular Biology Cambridge Biomedical Campus Francis Crick Avenue Cambridge CB2 0QH UK

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Cell Chem Biol. 2017 Dec 21;24(12):1431-1433. doi: 10.1016/j.chembiol.2017.12.004. PubMed

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Ahlrichs R., Bär M., Häser M., Horn H., Kölmel C. Electronic structure calculations on workstation computers: the program system turbomole. Chem. Phys. Lett. 1989;162:165–169.

Bachovchin D.A., Koblan L.W., Wu W., Liu Y., Li Y., Zhao P., Woznica I., Shu Y., Lai J.H., Poplawski S.E. A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity. Nat. Chem. Biol. 2014;10:656–663. PubMed PMC

Baker R.P., Wijetilaka R., Urban S. Two Plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria. PLoS Pathog. 2006;2:e113. PubMed PMC

Bastiaans H.M.M., vanderBaan J.L., Ottenheijm H.C.J. Flexible and convergent total synthesis of cyclotheonamide B. J. Org. Chem. 1997;62:3880–3889.

Cao H., Liu H., Domling A. Efficient multicomponent reaction synthesis of the schistosomiasis drug praziquantel. Chemistry. 2010;16:12296–12298. PubMed

Chan E.Y., McQuibban G.A. The mitochondrial rhomboid protease: its rise from obscurity to the pinnacle of disease-relevant genes. Biochim. Biophys. Acta. 2013;1828:2916–2925. PubMed

Chatterjee S., Dunn D., Tao M., Wells G., Gu Z.Q., Bihovsky R., Ator M.A., Siman R., Mallamo J.P. P2-achiral, P'-extended alpha-ketoamide inhibitors of calpain I. Bioorg. Med. Chem. Lett. 1999;9:2371–2374. PubMed

Cho S., Dickey S.W., Urban S. Crystal structures and inhibition kinetics reveal a two-stage catalytic mechanism with drug design implications for rhomboid proteolysis. Mol. Cell. 2016;61:329–340. PubMed PMC

Chu C.T. A pivotal role for PINK1 and autophagy in mitochondrial quality control: implications for Parkinson disease. Hum. Mol. Genet. 2010;19:R28–R37. PubMed PMC

Copeland R.A. John Wiley; 2013. Reversible modes of inhibitor interactions with enzymes. In Evaluation of Enzyme Inhibitors in Drug Discovery; pp. 57–121.

Copeland R.A. John Wiley; 2013. Slow binding inhibitors. In Evaluation of Enzyme Inhibitors in Drug Discovery; pp. 203–244.

Coste J., Frerot E., Jouin P. Coupling N-methylated amino-acids using pybrop and pyclop halogenophosphonium salts - mechanism and fields of application. J. Org. Chem. 1994;59:2437–2446.

D'Andrea, S., and Scola, P.M.. (2008). Inhibitors of hepatitis C virus. Bristol-Myers Squibb Company. US patent US2008107623 (A1), filed October 25, 2007, and published May 8, 2008.

Dickey S.W., Baker R.P., Cho S., Urban S. Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity. Cell. 2013;155:1270–1281. PubMed PMC

Dondoni A., Perrone D. 2-Thiazolyl alpha-amino ketones - a new class of reactive intermediates for the stereocontrolled synthesis of unusual amino-acids. Synthesis (Stuttg) 1993:1162–1176.

Drag M., Salvesen G.S. Emerging principles in protease-based drug discovery. Nat. Rev. Drug Discov. 2010;9:690–701. PubMed PMC

Eggert U.S., Ruiz N., Falcone B.V., Branstrom A.A., Goldman R.C., Silhavy T.J., Kahne D. Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin. Science. 2001;294:361–364. PubMed

Emsley P., Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004;60:2126–2132. PubMed

Evans P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. Sect. D Biol. Crystallogr. 2011;67:282–292. PubMed PMC

Fanfrlik J., Holub J., Ruzickova Z., Rezac J., Lane P.D., Wann D.A., Hnyk D., Ruzicka A., Hobza P. Competition between halogen, hydrogen and dihydrogen bonding in brominated carboranes. ChemPhysChem. 2016;17:3373–3376. PubMed

Fleig L., Bergbold N., Sahasrabudhe P., Geiger B., Kaltak L., Lemberg M.K. Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins. Mol. Cell. 2012;47:558–569. PubMed

Gerwig J., Kiley T.B., Gunka K., Stanley-Wall N., Stulke J. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis. Microbiology. 2014;160:682–691. PubMed PMC

Gibson D.G. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol. 2011;498:349–361. PubMed PMC

Grimme S. Semiempirical GGA-type density functional constructed with a long-range dispersion correction. J. Comput. Chem. 2006;27:1787–1799. PubMed

Harper J.W., Powers J.C. Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors. Biochemistry. 1985;24:7200–7213. PubMed

Harper J.W., Hemmi K., Powers J.C. Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor. Biochemistry. 1985;24:1831–1841. PubMed

Hedstrom L. Serine protease mechanism and specificity. Chem. Rev. 2002;102:4501–4524. PubMed

Jensen F. Wiley; 2006. Introduction to Computational Chemistry.

Kabsch W. Xds. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010;66:125–132. PubMed PMC

Klamt A., Schüürmann G. COSMO: a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J. Chem. Soc. Perkin Trans. 1993;2:799–805.

Krissinel E., Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004;60:2256–2268. PubMed

Laskowski R.A., Swindells M.B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 2011;51:2778–2786. PubMed

Lebedev A.A., Young P., Isupov M.N., Moroz O.V., Vagin A.A., Murshudov G.N. JLigand: a graphical tool for the CCP4 template-restraint library. Acta Crystallogr. Sect. D Biol. Crystallogr. 2012;68:431–440. PubMed PMC

Lee C., Kang H.J., Hjelm A., Qureshi A.A., Nji E., Choudhury H., Beis K., de Gier J.W., Drew D. MemStar: a one-shot Escherichia coli-based approach for high-level bacterial membrane protein production. FEBS Lett. 2014;588:3761–3769. PubMed

Lemberg M.K., Menendez J., Misik A., Garcia M., Koth C.M., Freeman M. Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases. EMBO J. 2005;24:464–472. PubMed PMC

Lin J.W., Meireles P., Prudencio M., Engelmann S., Annoura T., Sajid M., Chevalley-Maurel S., Ramesar J., Nahar C., Avramut C.M. Loss-of-function analyses defines vital and redundant functions of the Plasmodium rhomboid protease family. Mol. Microbiol. 2013;88:318–338. PubMed

Liu Y., Stoll V.S., Richardson P.L., Saldivar A., Klaus J.L., Molla A., Kohlbrenner W., Kati W.M. Hepatitis C NS3 protease inhibition by peptidyl-alpha-ketoamide inhibitors: kinetic mechanism and structure. Arch. Biochem. Biophys. 2004;421:207–216. PubMed

McCoy A.J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007;63:32–41. PubMed PMC

McDonald I.K., Thornton J.M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 1994;238:777–793. PubMed

Meissner C., Lorenz H., Hehn B., Lemberg M.K. Intramembrane protease PARL defines a negative regulator of PINK1- and PARK2/Parkin-dependent mitophagy. Autophagy. 2015;11:1484–1498. PubMed PMC

Miroux B., Walker J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 1996;260:289–298. PubMed

Mitchell E.M., Artymiuk P.J., Rice D.W., Willett P. Use of techniques derived from graph-theory to compare secondary structure motifs in proteins. J. Mol. Biol. 1990;212:151–166. PubMed

Morrison J.F. The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions. Trends Biochem. Sci. 1982;7:102–105.

Murshudov G.N., Skubak P., Lebedev A.A., Pannu N.S., Steiner R.A., Nicholls R.A., Winn M.D., Long F., Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. Sect. D Biol. Crystallogr. 2011;67:355–367. PubMed PMC

Nguyen M.T., Van Kersavond T., Verhelst S.H. Chemical tools for the study of intramembrane proteases. ACS Chem. Biol. 2015;10:2423–2434. PubMed

Njoroge F.G., Chen K.X., Shih N.Y., Piwinski J.J. Challenges in modern drug discovery: a case study of boceprevir, an HCV protease inhibitor for the treatment of hepatitis C virus infection. Acc. Chem. Res. 2008;41:50–59. PubMed

O'Donnell R.A., Hackett F., Howell S.A., Treeck M., Struck N., Krnajski Z., Withers-Martinez C., Gilberger T.W., Blackman M.J. Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite. J. Cell Biol. 2006;174:1023–1033. PubMed PMC

O'Donoghue A.J., Eroy-Reveles A.A., Knudsen G.M., Ingram J., Zhou M., Statnekov J.B., Greninger A.L., Hostetter D.R., Qu G., Maltby D.A. Global identification of peptidase specificity by multiplex substrate profiling. Nat. Methods. 2012;9:1095–1100. PubMed PMC

Pierrat O.A., Strisovsky K., Christova Y., Large J., Ansell K., Bouloc N., Smiljanic E., Freeman M. Monocyclic beta-lactams are selective, mechanism-based inhibitors of rhomboid intramembrane proteases. ACS Chem. Biol. 2011;6:325–335. PubMed PMC

Powers J.C., Kam C.M., Narasimhan L., Oleksyszyn J., Hernandez M.A., Ueda T. Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design. J. Cell. Biochem. 1989;39:33–46. PubMed

Powers J.C., Asgian J.L., Ekici O.D., James K.E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 2002;102:4639–4750. PubMed

Purich D.L. Elsevier; 2010. Kinetic behavior of enzyme inhibitors. In Enzyme Kinetics: Catalysis & Control; pp. 485–574.

Ruiz N., Falcone B., Kahne D., Silhavy T.J. Chemical conditionality. Cell. 2005;121:307–317. PubMed

Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967;27:157–162. PubMed

Schneider C.A., Rasband W.S., Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012;9:671–675. PubMed PMC

Schrodinger . Schrödinger LLC; 2012. The PyMOL Molecular Graphics System, Version 1.5.0.4.

Semple J.E., Owens T.D., Nguyen K., Levy O.E. New synthetic technology for efficient construction of alpha-hydroxy-beta-amino amides via the Passerini reaction. Org. Lett. 2000;2:2769–2772. PubMed

Serim S., Haedke U., Verhelst S.H. Activity-based probes for the study of proteases: recent advances and developments. ChemMedChem. 2012;7:1146–1159. PubMed

Singh J., Petter R.C., Baillie T.A., Whitty A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 2011;10:307–317. PubMed

Song W., Liu W., Zhao H., Li S., Guan X., Ying J., Zhang Y., Miao F., Zhang M., Ren X. Rhomboid domain containing 1 promotes colorectal cancer growth through activation of the EGFR signalling pathway. Nat. Commun. 2015;6:8022. PubMed PMC

Souček M., Urban J. An efficient method for preparation of optically active N-protected α-amino aldehydes from N-protected α-amino alcohols. Collect. Czechoslov. Chem. Commun. 1995;60:693–696.

Stevenson L.G., Strisovsky K., Clemmer K.M., Bhatt S., Freeman M., Rather P.N. Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase. Proc. Natl. Acad. Sci. USA. 2007;104:1003–1008. PubMed PMC

Strisovsky K. Structural and mechanistic principles of intramembrane proteolysis - lessons from rhomboids. FEBS J. 2013;280:1579–1603. PubMed

Strisovsky K. Rhomboid protease inhibitors: emerging tools and future therapeutics. Semin. Cell Dev. Biol. 2016;60:52–62. PubMed

Strisovsky K. Why cells need intramembrane proteases - a mechanistic perspective. FEBS J. 2016;283:1837–1845. PubMed

Strisovsky K., Sharpe H.J., Freeman M. Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Mol. Cell. 2009;36:1048–1059. PubMed PMC

Ticha A., Stanchev S., Skerle J., Began J., Ingr M., Svehlova K., Polovinkin L., Ruzicka M., Bednarova L., Hadravova R. Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases. J. Biol. Chem. 2017;292:2703–2713. PubMed PMC

Tulla-Puche J., Torres A., Calvo P., Royo M., Albericio F. N,N,N',N'-Tetramethylchloroformamidinium hexafluorophosphate (TCFH), a powerful coupling reagent for bioconjugation. Bioconj. Chem. 2008;19:1968–1971. PubMed

Venkatraman S., Bogen S.L., Arasappan A., Bennett F., Chen K., Jao E., Liu Y.T., Lovey R., Hendrata S., Huang Y. Discovery of (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]- 3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a selective, potent, orally bioavailable hepatitis C virus NS3 protease inhibitor: a potential therapeutic agent for the treatment of hepatitis C infection. J. Med. Chem. 2006;49:6074–6086. PubMed

Vinothkumar K.R., Strisovsky K., Andreeva A., Christova Y., Verhelst S., Freeman M. The structural basis for catalysis and substrate specificity of a rhomboid protease. EMBO J. 2010;29:3797–3809. PubMed PMC

Vinothkumar K.R., Pierrat O.A., Large J.M., Freeman M. Structure of rhomboid protease in complex with beta-lactam inhibitors defines the S2' cavity. Structure. 2013;21:1051–1058. PubMed PMC

Vosyka O., Vinothkumar K.R., Wolf E.V., Brouwer A.J., Liskamp R.M., Verhelst S.H. Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proc. Natl. Acad. Sci. USA. 2013;110:2472–2477. PubMed PMC

Walker B., Lynas J.F. Strategies for the inhibition of serine proteases. Cell. Mol. Life Sci. 2001;58:596–624. PubMed PMC

Wang Y., Ha Y. Open-cap conformation of intramembrane protease GlpG. Proc. Natl. Acad. Sci. USA. 2007;104:2098–2102. PubMed PMC

Wang Y., Zhang Y., Ha Y. Crystal structure of a rhomboid family intramembrane protease. Nature. 2006;444:179–180. PubMed

Wolf E.V., Zeissler A., Vosyka O., Zeiler E., Sieber S., Verhelst S.H. A new class of rhomboid protease inhibitors discovered by activity-based fluorescence polarization. PLoS One. 2013;8:e72307. PubMed PMC

Wolf E.V., Zeissler A., Verhelst S.H. Inhibitor fingerprinting of rhomboid proteases by activity-based protein profiling reveals inhibitor selectivity and rhomboid autoprocessing. ACS Chem. Biol. 2015;10:2325–2333. PubMed

Xue Y., Chowdhury S., Liu X., Akiyama Y., Ellman J., Ha Y. Conformational change in rhomboid protease GlpG induced by inhibitor binding to its S' subsites. Biochemistry. 2012;51:3723–3731. PubMed PMC

Yin J., Gallis C.E., Chisholm J.D. Tandem oxidation/halogenation of aryl allylic alcohols under Moffatt-Swern conditions. J. Org. Chem. 2007;72:7054–7057. PubMed

Zoll S., Stanchev S., Began J., Skerle J., Lepsik M., Peclinovska L., Majer P., Strisovsky K. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 2014;33:2408–2421. PubMed PMC

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