Analysing Properties of Proteasome Inhibitors Using Kinetic and X-Ray Crystallographic Studies

Part of the Methods in Molecular Biology book series (MIMB, volume 832)


The combination of X-ray crystallography and kinetic studies of proteasome:ligand complexes has proven to be an important tool in inhibitor analysis of this crucial protein degradation machinery. Here, we describe in detail the purification protocols, proteolytic activity assays, crystallisation methods, and structure determination for the yeast 20S proteasome (CP) in complex with its inhibitors. The fusion of these advanced techniques offers the opportunity to further optimise drugs which are already tested in different clinical phase studies, as well as to design new promising proteasome lead structures which might be suitable for their application in medicine, plant protection, and antibiotics.

Key words

Proteasome Crystallography Kinetic studies Inhibitors Drug design Cancer 


  1. 1.
    Gallastegui N, Groll M (2010) The 26S proteasome: assembly and function of a destructive machine. Trends Biochem Sci. 35:634–642.PubMedCrossRefGoogle Scholar
  2. 2.
    Borissenko L, Groll M (2007) 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem Rev 107, 687–717.PubMedCrossRefGoogle Scholar
  3. 3.
    Groll M, Huber R, Moroder L (2009) The persisting challenge of selective and specific proteasome inhibition. J Pept Sci 15, 58–66.PubMedCrossRefGoogle Scholar
  4. 4.
    Kisselev AF, Goldberg AL (2001) Proteasome inhibitors: from research tools to drug candidates. Chem. Biol. 8, 739–758.PubMedCrossRefGoogle Scholar
  5. 5.
    Groll M, Ditzel L, Lowe J, et al (1997) Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386, 463–471.PubMedCrossRefGoogle Scholar
  6. 6.
    Löwe J, Stock D, Jap B, et al (1995) Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539.PubMedCrossRefGoogle Scholar
  7. 7.
    Adams J, Palombella VJ, Elliott PJ (2000) Proteasome inhibition: a new strategy in cancer treatment. Invest New Drugs 18, 109–121.PubMedCrossRefGoogle Scholar
  8. 8.
    Richardson PG, Barlogie B, Berenson J, et al (2003) A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 348, 2609–2617.PubMedCrossRefGoogle Scholar
  9. 9.
    Mikhael JR, Belch AR, Prince HM, et al (2009) High response rate to bortezomib with or without dexamethasone in patients with relapsed or refractory multiple myeloma: results of a global phase 3b expanded access program. Br J Haematol 144, 169–175.PubMedCrossRefGoogle Scholar
  10. 10.
    Argyriou AA, Iconomou G, Kalofonos HP (2008) Bortezomib-induced peripheral neuropathy in multiple myeloma: a comprehensive review of the literature. Blood 112, 1593–1599.PubMedCrossRefGoogle Scholar
  11. 11.
    Groll M, Berkers CR, Ploegh HL, Ovaa H (2006) Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure 14, 451–456.PubMedCrossRefGoogle Scholar
  12. 12.
    Feling RH, Buchanan GO, Mincer TJ, et al (2003) Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus salinospora. Angew Chem Int Ed Engl 42, 355–357.PubMedCrossRefGoogle Scholar
  13. 13.
    Groll M, Huber R, Potts BC (2006) Crystal Structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in Complex with the 20S Proteasome Reveal Important Consequences of beta-Lactone Ring Opening and a Mechanism for Irreversible Binding. J Am Chem Soc 128, 5136–5141.PubMedCrossRefGoogle Scholar
  14. 14.
    Parlati F, Lee SJ, Aujay M, et al (2009) Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome. Blood 114, 3439–3447.PubMedCrossRefGoogle Scholar
  15. 15.
    de Bettignies G, Coux O (2010) Proteasome inhibitors: Dozens of molecules and still counting. Biochimie 92, 1530–1545.PubMedCrossRefGoogle Scholar
  16. 16.
    Groll M, Gotz M, Kaiser M, et al (2006) TMC-95-based inhibitor design provides evidence for the catalytic versatility of the proteasome. Chem Biol 13, 607–614.PubMedCrossRefGoogle Scholar
  17. 17.
    Groll M, Schellenberg B, Bachmann AS, et al (2008) A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature 452, 755–758.PubMedCrossRefGoogle Scholar
  18. 18.
    Groll M, Koguchi Y, Huber R, Kohno J (2001) Crystal structure of the 20S proteasome:TMC-95A complex: a non-covalent proteasome inhibitor. J Mol Biol 311, 543–548.PubMedCrossRefGoogle Scholar
  19. 19.
    Groll M, Gallastegui N, Maréchal X, et al (2010) 20S Proteasome Inhibition: Designing Non-Covalent Linear Peptide Mimics of the Natural Product TMC-95A. ChemMedChem 5:1701–1705.PubMedCrossRefGoogle Scholar
  20. 20.
    Dahlmann B, Kopp F, Kuehn L, et al (1986) Studies on the multicatalytic proteinase from rat skeletal muscle. Biomed Biochim Acta 45, 1493–1501.PubMedGoogle Scholar
  21. 21.
    Groll M, Bajorek M, Kohler A, et al (2000) A gated channel into the proteasome core particle.” Nature Structural Biology. Nature Structural Biology 7, 1062–1067.PubMedCrossRefGoogle Scholar
  22. 22.
    Lesslie AG (1994) Mosfilm user guide, mosfilm version 5.2. MRC Laboratory of Molecular Biology, Cambrige, UK.Google Scholar
  23. 23.
    Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Cryst 26, 795–800.CrossRefGoogle Scholar
  24. 24.
    Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr. 59, 1131–1137.PubMedCrossRefGoogle Scholar
  25. 25.
    Brünger A, Adams P, Clore G, et al (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1, 905–921.CrossRefGoogle Scholar
  26. 26.
    Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240–255.PubMedCrossRefGoogle Scholar
  27. 27.
    Turk D (1992) Improvement of a programm for molecular graphics and manipulation of electron densities and its application for protein structure determination. Thesis, Technische Universitaet Muenchen.Google Scholar
  28. 28.
    Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486–501.PubMedCrossRefGoogle Scholar
  29. 29.
    Groll M, Huber R (2004) Inhibitors of the eukaryotic 20S proteasome core particle: a structural approach. Biochim Biophys Acta 1695, 33–44.PubMedCrossRefGoogle Scholar
  30. 30.
    Borissenko L, and Groll M (2007) Diversity of proteasomal missions: fine tuning of the immune response. Biol Chem 388, 947–955.PubMedCrossRefGoogle Scholar
  31. 31.
    Brünger AT (1992) Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of BiochemistryTechnische Universität MünchenGarchingGermany

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