Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

1st Faculty of Medicine Charles University Prague Czech Republic

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Department of Natural Sciences University of Applied Sciences Bonn Rhein Sieg Rheinbach Germany

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Pharmaceutical Institute Pharmaceutical and Medicinal Chemistry University of Bonn Germany

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Li H, Xiao Z, Quarles LD, Li W.. Osteoporosis: mechanism, molecular target and current status on drug development. Curr Med Chem 2021;28:1489–507. PubMed PMC

Costa AG, Cusano NE, Silva BC, et al. . Cathepsin K: its skeletal actions and role as a therapeutic target in osteoporosis. Nat Rev Rheumatol 2011;7:447–56. PubMed

Makras P, Delaroudis S, Anastasilakis AD.. Novel therapies for osteoporosis. Metabolism 2015;64:1199–214. PubMed

Kramer L, Turk D, Turk B.. The future of cysteine cathepsins in disease management. Trends Pharmacol Sci 2017;38:873–98. PubMed

Drake MT, Clarke BL, Oursler MJ, Khosla S.. Cathepsin K inhibitors for osteoporosis: biology, potential clinical utility, and lessons learned. Endocr Rev 2017;38:325–50. PubMed PMC

Brömme D, Lecaille F.. Cathepsin K inhibitors for osteoporosis and potential off-target effects. Expert Opin Investig Drugs 2009;18:585–600. PubMed PMC

Lu J, Wang M, Wang Z, et al. . Advances in the discovery of cathepsin K inhibitors on bone resorption. J Enzyme Inhib Med Chem 2018;33:890–904. PubMed PMC

Dejica VM, Mort JS, Laverty S, et al. . Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage. Arthritis Res Ther 2012;14:R113–9. PubMed PMC

Yasuda Y, Kaleta J, Brömme D.. The role of cathepsins in osteoporosis and arthritis: rationale for the design of new therapeutics. Adv Drug Deliv Rev 2005;57:973–93. PubMed

Aguda AH, Panwar P, Du X, et al. . Structural basis of collagen fiber degradation by cathepsin K. Proc Natl Acad Sci USA 2014;111:17474–9. PubMed PMC

Novinec M, Lenarčič B.. Cathepsin K: a unique collagenolytic cysteine peptidase. Biol Chem 2013;394:1163–79. PubMed

Novinec M, Kovačič L, Lenarčič B, Baici A.. Conformational flexibility and allosteric regulation of cathepsin K. Biochem J 2010;429:379–89. PubMed

Cherney MM, Lecaille F, Kienitz M, et al. . Structure-activity analysis of cathepsin K/chondroitin 4-sulfate interactions. J Biol Chem 2011;286:8988–98. PubMed PMC

Verbovšek U, Van Noorden CJ, Lah TT.. Complexity of cancer protease biology: cathepsin K expression and function in cancer progression. Semin Cancer Biol 2015;35:71–84. PubMed

Olson OC, Joyce JA.. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat Rev Cancer 2015;15:712–29. PubMed

Jiang H, Cheng XW, Shi GP, et al. . Cathepsin K-mediated Notch1 activation contributes to neovascularization in response to hypoxia. Nat Commun 2014;5:3838. PubMed

Herroon MK, Rajagurubandara E, Rudy DL, et al. . Macrophage cathepsin K promotes prostate tumor progression in bone. Oncogene 2013;32:1580–93. PubMed PMC

Kamolmatyakul S, Chen W, Yang S, et al. . IL-1alpha stimulates cathepsin K expression in osteoclasts via the tyrosine kinase-NF-kappaB pathway. J Dent Res 2004;83:791–6. PubMed PMC

Asagiri M, Hirai T, Kunigami T, et al. . Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 2008;319:624–7. PubMed

Hira VV, Ploegmakers KJ, Grevers F, et al. . CD133+ and Nestin + glioma stem-like cells reside around CD31+ arterioles in niches that express SDF-1α, CXCR4, osteopontin and cathepsin K. J Histochem Cytochem 2015; 63: 481–93. PubMed

Calio A, Brunelli M, Gobbo S, et al. . Cathepsin K: a novel diagnostic and predictive biomarker for renal tumors. Cancers 2021;13:2441. PubMed PMC

Leusink FK, Koudounarakis E, Frank MH, et al. . Cathepsin K associates with lymph node metastasis and poor prognosis in oral squamous cell carcinoma. BMC Cancer 2018;18:385. PubMed PMC

Kos J, Lah TT.. Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer (review). Oncol Rep 1998;5:1349–410. PubMed

Lecaille F, Brömme D, Lalmanach G.. Biochemical properties and regulation of cathepsin K activity. Biochimie 2008;90:208–26. PubMed

McQueney MS, Amegadzie BY, D'Alessio K, et al. . Autocatalytic activation of human cathepsin K. J Biol Chem 1997;272:13955–60. PubMed

Linnevers CJ, Mcgrath ME, Armstrong A, et al. . Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex. Protein Sci 1997;6:919–21. PubMed PMC

Sivaraman J, Lalumière M, Ménard R, Cygler M.. Crystal structure of wild-type human procathepsin K. Protein Sci 1999;8:283–90. PubMed PMC

LaLonde JM, Zhao B, Janson CA, et al. . The crystal structure of human procathepsin K. Biochemistry 1999;38:862–9. PubMed

Lemaire PA, Huang L, Zhuo Y, et al. . Chondroitin sulfate promotes activation of cathepsin K. J Biol Chem 2014;289:21562–72. PubMed PMC

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

Frizler M, Lohr F, Furtmann N, et al. . Structural optimization of azadipeptide nitriles strongly increases association rates and allows the development of selective cathepsin inhibitors. J Med Chem 2011;54:396–400. PubMed

Jílková A, Horn M, Fanfrlík J, et al. . Azanitrile inhibitors of the SmCB1 protease target are lethal to Schistosoma mansoni: structural and mechanistic insights into chemotype reactivity. ACS Infect Dis 2021;7:189–201. PubMed PMC

Yang PY, Wang M, Li L, et al. . Design, synthesis and biological evaluation of potent azadipeptide nitrile inhibitors and activity-based probes as promising anti-Trypanosoma brucei agents. Chemistry 2012;18:6528–41. PubMed

Breidenbach J, Lemke C, Pillaiyar T, et al. . Targeting the main protease of SARS-CoV-2: from the establishment of high throughput screening to the design of tailored inhibitors. Angew Chem Int Ed Engl 2021;60:10423–9. PubMed PMC

Chingle R, Proulx C, Lubell WD.. Azapeptide synthesis methods for expanding side-chain diversity for biomedical applications. Acc Chem Res 2017;50:1541–56. PubMed

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

Verhelst SH, Witte MD, Arastu-Kapur S, et al. . Novel aza peptide inhibitors and active-site probes of papain-family cysteine proteases. ChemBioChem 2006;7:943–50. PubMed

Sexton KB, Kato D, Berger AB, et al. . Specificity of aza-peptide electrophile activity-based probes of caspases. Cell Death Differ 2007;14:727–32. 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

Lemke C, Benýšek J, Brajtenbach D, et al. . An activity-based probe for cathepsin K imaging with excellent potency and selectivity. J Med Chem 2021;64:13793–806. PubMed

Jílková A, Horn M, Řezáčová P, et al. . Activation route of the Schistosoma mansoni cathepsin B1 drug target: structural map with a glycosaminoglycan switch. Structure 2014;22:1786–98. PubMed

Horn M, Jílková A, Vondrášek J, et al. . Mapping the pro-peptide of the Schistosoma mansoni cathepsin B1 drug target: modulation of inhibition by heparin and design of mimetic inhibitors. ACS Chem Biol 2011;6:609–17. PubMed

Mueller U, Förster R, Hellmig M, et al. . The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives. Eur Phys J Plus 2015;130:141.

Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr 2010;66:125–32. PubMed PMC

Vagin A, Teplyakov A.. An approach to multi-copy search in molecular replacement. Acta Crystallogr D Biol Crystallogr 2000;56:1622–4. PubMed

Winn MD, Ballard CC, Cowtan KD, et al. . Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 2011;67:235–42. PubMed PMC

Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr 2006;62:1002–11. PubMed

Long F, Nicholls RA, Emsley P, et al. . AceDRG: a stereochemical description generator for ligands. Acta Crystallogr D Struct Biol 2017;73:112–22. PubMed PMC

Davis IW, Leaver-Fay A, Chen VB, et al. . MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 2007;35:W375–83. PubMed PMC

Salentin S, Schreiber S, Haupt VJ, et al. . PLIP: fully automated protein-ligand interaction profiler. Nucleic Acids Res 2015;43:W443–7. PubMed PMC

Case DA, Babin V, Berryman JT, et al. . AMBER 14. San Francisco (USA): University of California San Francisco; 2014.

Stewart JJ. Optimization of parameters for semiempirical methods V: modification of NDDO approximations and application to 70 elements. J Mol Model 2007;13:1173–213. PubMed PMC

Řezáč J, Hobza P.. Advanced corrections of hydrogen bonding and dispersion for semiempirical quantum mechanical methods. J Chem Theory Comput 2012;8:141–51. PubMed

Klamt A, Schuurmann G.. Cosmo - a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J Chem Soc Perk T 2 1993;5:799–805.

Kříž K, Řezáč J.. Reparametrization of the COSMO solvent model for semiempirical methods PM6 and PM7. J Chem Inf Model 2019;59:229–35. PubMed

Řezáč J. Cuby: an integrative framework for computational chemistry. J Comput Chem 2016;37:1230–7. PubMed

Stewart JJP. Optimization of parameters for semiempirical methods IV: extension of MNDO, AM1, and PM3 to more main group elements. J Mol Model 2004;10:155–64. PubMed

Kuzmic P, Sideris S, Cregar LM, et al. . High-throughput screening of enzyme inhibitors: automatic determination of tight-binding inhibition constants. Anal Biochem 2000;281:62–7. PubMed

Horn M, Nussbaumerová M, Šanda M, et al. . Hemoglobin digestion in blood-feeding ticks: mapping a multipeptidase pathway by functional proteomics. Chem Biol 2009;16:1053–63. PubMed PMC

Barrett AJ, Kembhavi AA, Brown MA, et al. . L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J 1982;201:189–98. PubMed PMC

Billington CJ, Mason P, Magny MC, Mort JS.. The slow-binding inhibition of cathepsin K by its propeptide. Biochem Biophys Res Commun 2000;276:924–9. PubMed

Husmann K, Muff R, Bolander ME, et al. . Cathepsins and osteosarcoma: expression analysis identifies cathepsin K as an indicator of metastasis. Mol Carcinog 2008;47:66–73. PubMed

Ottersbach PA, Schnakenburg G, Gütschow M.. Induction of chirality: experimental evidence of atropisomerism in azapeptides. Chem Commun 2012;48:5772–4. PubMed

Ottersbach PA, Schnakenburg G, Gütschow M.. Atropisomerism in azadipeptides: evaluation of N1-methylation and thioamide introduction. Tetrahedron Lett 2015;56:4889–91.

Chia-en AC, Chen W, Gilson MK.. Ligand configurational entropy and protein binding. Proc Natl Acad Sci USA 2007;104:1534–9. PubMed PMC

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans