Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Inhibitors selective for mycobacterial versus human proteasomes


Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M. tuberculosis and act as selective suicide-substrate inhibitors of the M. tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Oxathiazol-2-one compounds inhibit mycobacterial proteasomes and kill non-replicating M. tuberculosis.
Figure 2: Kinetic analysis of inactivation of M. tuberculosis 20SOG and human proteasomes (Hu20S) by oxathiazol-2-one compounds.
Figure 3: LC–MS/MS identification of the modified N terminus of the M. tuberculosis proteasome treated with oxathiazol-2-one compounds.
Figure 4: Crystal structure of the full-length M. tuberculosis 20S proteasome after exposure to HT1171 shows cyclocarbonylation of active site Thr 1 and conformational changes in the β-subunit.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank


  1. Baumeister, W., Walz, J., Zuhl, F. & Seemuller, E. The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380 (1998)

    Article  CAS  Google Scholar 

  2. Kropff, M. et al. Bortezomib in combination with intermediate-dose dexamethasone and continuous low-dose oral cyclophosphamide for relapsed multiple myeloma. Br. J. Haematol. 138, 330–337 (2007)

    Article  CAS  Google Scholar 

  3. Glenn, R. J. et al. Trypanocidal effect of α′, β′-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity. Int. J. Antimicrob. Agents 24, 286–289 (2004)

    Article  CAS  Google Scholar 

  4. Darwin, K. H. et al. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966 (2003)

    Article  ADS  CAS  Google Scholar 

  5. Raviglione, M. C. & Smith, I. M. XDR tuberculosis–implications for global public health. N. Engl. J. Med. 356, 656–659 (2007)

    Article  CAS  Google Scholar 

  6. Corbett, E. L. et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163, 1009–1021 (2003)

    Article  Google Scholar 

  7. Restrepo, B. I. Convergence of the tuberculosis and diabetes epidemics: renewal of old acquaintances. Clin. Infect. Dis. 45, 436–438 (2007)

    Article  Google Scholar 

  8. Pearce, M. J. et al. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis . Science 322, 1104–1107 (2008)

    Article  ADS  CAS  Google Scholar 

  9. Burns, K. E. et al. Proteasomal protein degradation in mycobacteria is dependent upon a prokaryotic ubiquitin-like protein. J. Biol. Chem. 284, 3069–3075 (2009)

    Article  CAS  Google Scholar 

  10. Gandotra, S. et al. In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice. Nature Med. 13, 1515–1520 (2007)

    Article  CAS  Google Scholar 

  11. Borissenko, L. & Groll, M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem. Rev. 107, 687–717 (2007)

    Article  CAS  Google Scholar 

  12. Lin, G. et al. Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates. J. Biol. Chem. 283, 34423–34431 (2008)

    Article  CAS  Google Scholar 

  13. Kisselev, A. F. & Goldberg, A. L. Proteasome inhibitors: from research tools to drug candidates. Chem. Biol. 8, 739–758 (2001)

    Article  CAS  Google Scholar 

  14. Hu, G. et al. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate. Mol. Microbiol. 59, 1417–1428 (2006)

    Article  CAS  Google Scholar 

  15. Lin, G. et al. Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity. Mol. Microbiol. 59, 1405–1416 (2006)

    Article  CAS  Google Scholar 

  16. Bryk, R. et al. Selective killing of nonreplicating mycobacteria. Cell Host Microbe 3, 137–145 (2008)

    Article  CAS  Google Scholar 

  17. Huth, J. R. et al. Toxicological evaluation of thiol-reactive compounds identified using a La assay to detect reactive molecules by nuclear magnetic resonance. Chem. Res. Toxicol. 20, 1752–1759 (2007)

    Article  CAS  Google Scholar 

  18. Copp, L. J. in Enzyme Kinetics: A Modern Approach (ed. Marangoni, A. G.) Ch. 13 158–173 (Wiley, 2003)

    Google Scholar 

  19. Russo, A., Chandramouli, N., Zhang, L. & Deng, H. T. Reductive glutaraldehydation of amine groups for identification of protein N-termini. J. Proteome Res. 7, 4178–4182 (2008)

    Article  CAS  Google Scholar 

  20. Lu, L. Q. et al. A new entry to cascade organocatalysis: reactions of stable sulfur ylides and nitroolefins sequentially catalyzed by thiourea and DMAP. J. Am. Chem. Soc. 130, 6946–6948 (2008)

    Article  CAS  Google Scholar 

  21. Rajca, A., Grobelny, D., Witek, S. & Zbirovsky, M. 5-Aryl-2-oxo-1,2,4-oxathiazoles as cyclocarbonylating agents for 2-aminoalcohols and 1,2-diamines. Synthesis 12, 1032–1033 (1983)

    Article  Google Scholar 

  22. MacMicking, J. D. et al. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc. Natl Acad. Sci. USA 94, 5243–5248 (1997)

    Article  ADS  CAS  Google Scholar 

  23. Walsh, C. Where will new antibiotics come from? Nature Rev. Microbiol. 1, 65–70 (2003)

    Article  CAS  Google Scholar 

  24. Hu, Y. M. et al. Protein synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock. FEMS Microbiol. Lett. 158, 139–145 (1998)

    Article  CAS  Google Scholar 

  25. Copeland, R. A. in Enzymes: a Practical Introduction to Structure, Mechanism, and Data Analysis Chs 9–10 305–349 (Wiley, 2000)

    Book  Google Scholar 

  26. Otwinoswki, Z. & Minor, W. in Methods in Enzymology (eds Carter, C. W. Jr & Sweet, R. M.) 307–326 (Academic, 1997)

    Google Scholar 

  27. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  28. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  29. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  30. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

Download references


S. Eswaramoorthy helped with crystallography software, C. Lipinski, C. Walsh and M. Fischbach proposed reaction mechanisms, and C. Karan assisted with screening. S. Ehrt and S. Gandotra performed some bactericidal assays, C. Tsu and L. Dick donated a fluorimeter and J. Blanchard provided BlaC. Supported by NIH PO1-AI056293, NIH R01AI070285 and the Milstein Program in Chemical Biology of Infectious Diseases. X-ray diffraction data were collected at beamline X6A, X25 and X29 in the National Synchrotron Light Source, a facility supported by the US DOE and NIH. The Department of Microbiology and Immunology is supported by the William Randolph Hearst Foundation.

Author Contributions G.L. purified recombinant proteasome, conducted the screen, designed new oxathiazol-2-one compounds and performed most of the assays. D.L. and H.L. purified and crystallized recombinant proteasomes and solved their structures. L.P.S.C. helped analyse kinetics. H.D. performed mass spectrometry. H.T. synthesized oxathiazol-2-one compounds under the supervision of J.D.W.; G.V. studied human macrophages. K.W. conducted studies with viable M. tuberculosis. J.S. and T.C. performed kinetic, bactericidal and cytotoxicity experiments. C.N. organized the effort and helped design and interpret experiments. C.N., G.L. and H.L. wrote the paper.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Gang Lin, Huilin Li or Carl Nathan.

Additional information

Coordinates have been deposited in the Protein Data Bank under ID codes 3H6F, 3H6I, 3HFA and 3HF9.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Tables S1-S5 and Supplementary Figures S1- S9 with Legends. (PDF 3274 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lin, G., Li, D., de Carvalho, L. et al. Inhibitors selective for mycobacterial versus human proteasomes. Nature 461, 621–626 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing