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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.

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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.

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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.

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Correspondence to Gang Lin or Huilin Li or Carl Nathan.

Additional information

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

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Supplementary Information

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

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

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