Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effective inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited S. mansoni and I. ricinus legumains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable candidates for further development into therapeutic agents to potentially treat multiple myeloma, neurodegenerative diseases, and parasitic infections.

Center for Discovery and Innovation in Parasitic Diseases Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA USA

Department of Chemistry and Biochemistry The Ohio State University at Newark Newark OH USA

Department of Chemistry and Biochemistry The Ohio State University Columbus OH USA

Division of Medicinal Chemistry and Pharmacognosy College of Pharmacy The Ohio State University Columbus OH USA

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Ceske Budejovice Czech Republic

Sanford Burnham Prebys Medical Discovery Institute La Jolla CA USA

Zobrazit více v PubMed

Proulx C, Sabatino D, Hopewell R, et al. . Azapeptides and their therapeutic potential. Future Med Chem 2011;3:1139–64. PubMed

Aubin S, Martin B, Delcros JG, et al. . Retro hydrazino-azapeptoids as peptidomimetics of proteasome inhibitors. J Med Chem 2005;48:330–4. PubMed

Graybill TL, Ross MJ, Gauvin BR, et al. . Synthesis and evaluation of azapeptide-derived inhibitors of serine and cysteine proteases. Bioorg Med Chem Lett 1992;2:1375–80.

Ekici OD, Götz MG, James KE, et al. . Aza-peptide michael acceptors: a new class of inhibitors specific for caspases and other clan cd cysteine proteases. J Med Chem 2004;47:1889–92. PubMed

Asgian JL, James KE, Li ZZ, et al. . Aza-peptide epoxides: a new class of inhibitors selective for clan cd cysteine proteases. J Med Chem 2002;45:4958–60. PubMed

James KE, Asgian JL, Li ZZ, et al. . Design, synthesis, and evaluation of aza-peptide epoxides as selective and potent inhibitors of caspases-1, -3, -6, and -8. J Med Chem 2004;47:1553–74. PubMed

Götz MG, James KE, Hansell E, et al. . Aza-peptidyl michael acceptors. A new class of potent and selective inhibitors of asparaginyl endopeptidases (legumains) from evolutionarily diverse pathogens. J Med Chem 2008;51:2816–32. PubMed

Löser R, Frizler M, Schilling K, Gütschow M. Azadipeptide nitriles: highly potent and proteolytically stable inhibitors of papain-like cysteine proteases. Angew Chem Int Ed Engl 2008;47:4331–4. PubMed

Schmitz J, Beckmann AM, Dudic A, et al. . 3-Cyano-3-aza-β-amino acid derivatives as inhibitors of human cysteine cathepsins. ACS Med Chem Lett 2014;5:1076–81. PubMed PMC

Bochtler M, Ditzel L, Groll M, et al. . The proteasome. Annu Rev Biophys Biomol Struct 1999; 28:295–317. PubMed

Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell 1994;79:13–21. PubMed

Dahlmann B. Role of proteasomes in disease. BMC Biochem 2007;8(Suppl 1): S3. PubMed PMC

Delcros JG, Floc'h MB, Prigent C, Arlot-Bonnemains Y. Proteasome inhibitors as therapeutic agents: current and future strategies. Curr Med Chem 2003;10:479–503. PubMed

Zhou HJ, Aujay MA, Bennett MK, et al. . Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (pr-047). J Med Chem 2009;52:3028–38. PubMed

Demo SD, Kirk CJ, Aujay MA, et al. . Antitumor activity of pr-171, a novel irreversible inhibitor of the proteasome. Cancer Res 2007;67:6383–91. PubMed

Adams J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 2004;5:417–21. PubMed

Meregalli C. An overview of bortezomib-induced neurotoxicity. Toxics 2015;3:294–303. PubMed PMC

Manasanch EE, Orlowski RZ. Proteasome inhibitors in cancer therapy. Nat Rev Clin Oncol 2017;14:417–33. PubMed PMC

Park JE, Miller Z, Jun Y, et al. . Next-generation proteasome inhibitors for cancer therapy. Transl Res 2018;198:1–16. PubMed PMC

Waxman AJ, Clasen S, Hwang WT, et al. . Carfilzomib-associated cardiovascular adverse events a systematic review and meta-analysis. JAMA Oncol 2018;4:e174519. PubMed PMC

Kumar S, Moreau P, Hari P, et al. . Management of adverse events associated with ixazomib plus lenalidomide/dexamethasone in relapsed/refractory multiple myeloma. Br J Haematol 2017;178:571–82. PubMed PMC

Salvesen GS. Caspases and apoptosis. Essays Biochem 2002;38:9–19. PubMed

Salvesen GS. Caspases: opening the boxes and interpreting the arrows. Cell Death Differ 2002;9:3–5. PubMed

Schulz JB, Weller M, Moskowitz MA. Caspases as treatment targets in stroke and neurodegenerative diseases. Ann Neurol 1999;45:421–9. PubMed

Thornberry NA, Peterson EP, Zhao JJ, et al. . Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry 1994;33:3934–40. PubMed

Thornberry NA, Rano TA, Peterson EP, et al. . A combinatorial approach defines specificities of members of the caspase family and granzyme b. Functional relationships established for key mediators of apoptosis. J Biol Chem 1997;272:17907–11. PubMed

Thornberry NA. The caspase family of cysteine proteases. Br Med Bull 1997;53:478–90. PubMed

Thornberry NA. Caspases: key mediators of apoptosis. Chem Biol 1998;5:R97–103. PubMed

Graham RK, Ehrnhoefer DE, Hayden MR. Caspase-6 and neurodegeneration. Trends Neurosci 2011;34:646–56. PubMed

Ishii S. Legumain: asparaginyl endopeptidase. Meth. Enzymol 1994;244:604–15. PubMed

Abdul Alim M, Tsuji N, Miyoshi T, et al. . Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick haemaphysalis longicornis. Insect Biochem Mol Biol 2007;37:911–22. PubMed

Liu C, Sun C, Huang H, et al. . Overexpression of legumain in tumors is significant for invasion/metastasis and a candidate enzymatic target for prodrug therapy. Cancer Res 2003;63:2957–64. PubMed

Chen JM, Dando PM, Rawlings ND, et al. . Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase. J Biol Chem 1997;272:8090–8. PubMed

Hara-Nishimura I, Inoue K, Nishimura M. A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett 1991;294:89–93. PubMed

Davis AH, Nanduri J, Watson DC. Cloning and gene expression of schistosoma mansoni protease. J Biol Chem 1987;262:12851–5. PubMed

Sojka D, Hajdusek O, Dvorak J, et al. . Irae: an asparaginyl endopeptidase (legumain) in the gut of the hard tick ixodes ricinus. Int J Parasitol 2007;37:713–24. PubMed PMC

Delcroix M, Sajid M, Caffrey CR, et al. . A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem 2006;281:39316–29. PubMed

Sajid M, McKerrow JH, Hansell E, et al. . Functional expression and characterization of schistosoma mansoni cathepsin b and its trans-activation by an endogenous asparaginyl endopeptidase. Mol Biochem Parasitol 2003;131:65–75. PubMed

Kilpatrick AM, Dobson ADM, Levi T, et al. . Lyme disease ecology in a changing world: consensus, uncertainty and critical gaps for improving control. Philos Trans R Soc Lond B Biol Sci 1722;2017:372. PubMed PMC

Steinmann P, Keiser J, Bos R, et al. . Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006;6:411–25. PubMed

Kaiser M, Milbradt AG, Siciliano C, et al. . Tmc-95a analogues with endocyclic biphenyl ether group as proteasome inhibitors. Chem Biodivers 2004;1:161–73. PubMed

Caffrey CR, Mathieu MA, Gaffney AM, et al. . Identification of a cdna encoding an active asparaginyl endopeptidase of schistosoma mansoni and its expression in pichia pastoris. FEBS Lett 2000;466:244–8. PubMed

Fang B, Boross PI, Tozser J, Weber IT. Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition. J Mol Biol 2006;360:654–66. PubMed

Stennicke HR, Salvesen GS. Biochemical characteristics of caspases-3, -6, -7, and -8. J Biol Chem 1997;272:25719–23. PubMed

Liebschner D, Afonine PV, Baker ML, Bunkoczi G, et al. . Macromolecular structure determination using x-rays, neutrons and electrons: recent developments in phenix. Acta Crystallogr D Struct Biol 2019;75:861–77. PubMed PMC

Moriarty NW, Grosse-Kunstleve RW, Adams PD. Electronic ligand builder and optimization workbench (elbow): a tool for ligand coordinate and restraint generation. Acta Crystallogr D Biol Crystallogr 2009;65:1074–80. PubMed PMC

Moriarty NW, Draizen EJ, Adams PD. An editor for the generation and customization of geometry restraints. Acta Crystallogr D Struct Biol 2017;73:123–30. PubMed PMC

Afonine PV, Grosse-Kunstleve RW, Echols N, et al. . Towards automated crystallographic structure refinement with phenix.Refine. Acta Crystallogr D Biol Crystallogr 2012;68:352–67. PubMed PMC

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of coot. Acta Crystallogr D Biol Crystallogr 2010;66:486–501. PubMed PMC

Mathieu MA, Bogyo M, Caffrey CR, et al. . Substrate specificity of schistosome versus human legumain determined by p1-p3 peptide libraries. Mol Biochem Parasitol 2002;121:99–105. PubMed

Bogyo M, Shin S, McMaster JS, Ploegh HL. Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes. Chem Biol 1998;5:307–20. PubMed

Kisselev AF, Goldberg AL. Proteasome inhibitors: from research tools to drug candidates. Chem Biol 2001;8:739–58. PubMed

Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 1998;8:397–403. PubMed

Rock KL, Gramm C, Rothstein L, et al. . Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on mhc class i molecules. Cell 1994;78:761–71. PubMed

Rotonda J, Nicholson DW, Fazil KM, et al. . The three-dimensional structure of apopain/cpp32, a key mediator of apoptosis. Nat Struct Biol 1996;3:619–25. PubMed

Mittl PR, Di Marco S, Krebs JF, et al. . Structure of recombinant human cpp32 in complex with the tetrapeptide acetyl-asp-val-ala-asp fluoromethyl ketone. J Biol Chem 1997;272:6539–47. PubMed