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Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria

Nature Chemical Biology volume 11, pages 292–298 (2015)Cite this article

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Abstract

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond–forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

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Figure 1: DsbB pathway and screening basis.
Figure 2: In vivo and in vitro inhibition of EcDsbB by compounds 9 and 12.
Figure 3: In vivo inhibition of DsbB enzymes from Gram-negative bacteria expressed in E. coli.

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  • 19 February 2015

    In the version of this article initially published online, the organism Mycobacterium tuberculosis was incorrectly named Mycoplasma tuberculosis. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank the ICCB-Longwood screening facility for access to their compound libraries, equipment and, screening supplies, and the staff, including S. Chiang, K. Lee, S. Rudnicki and D. Flood, for helpful advice and data handling. We thank R. Goldman of the US National Institute of Allergy and Infectious disease for the collection of M. tuberculosis growth inhibitors. We thank R. Tomaino of the Taplin Mass Spectrometry Core Facility at Harvard Medical School for his assistance in sample analysis. This work was supported by US National Institute of General Medical Sciences grants GMO41883 (to J.B. and D.B.), Harvard Catalyst Pilot Grant Harvard #149734 (to J.B.), US National Institutes of Health (NIH) Grant 5-UL1RR02568-01 (to J.B.), Blavatnik Biomedical Accelerator at Harvard University (to J.B.), NIH Grant 3P01-A1AI074805-04S1 Subaward R01638 (to E.J.R.), NIH Grant PO1-HL087203 (to B.F.), NIH Grant RO1-HL092125 (to B.C.F.) and New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE-BEID) Grant U54-AI057159. C.L. was partially supported by a Consejo Nacional de Ciencia y Tecnología (CONACYT) postdoctoral fellowship. J.L.B. was supported by T32-HL07917 Grant (to B.F.). B.M.M. was supported by a Ruth L. Kirschstein National Research Service Award. M.E. was supported by a New England BioLabs Grant. A.M. was supported by a Harvard School of Public Health Yerby postdoctoral fellowship. J.B. is an American Cancer Society Professor.

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Author notes

  1. Holly Arnold, Na Ke & Rachel Dutton

    Present address: Current addresses: New England BioLabs, Ipswich, Massachusetts, USA (H.A.); School of Electrical Engineering and Computer Science, Oregon State University, Corvallis, Oregon, USA (N.K.); FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA (R.D.).,

  2. Cristina Landeta, Jessica L Blazyk and Feras Hatahet: These author contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Cristina Landeta, Jessica L Blazyk, Feras Hatahet, Brian M Meehan, Markus Eser, Holly Arnold, Na Ke, Jon Beckwith, Rachel Dutton & Dana Boyd

  2. Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA

    Alissa Myrick, Shoko Minami & Eric J Rubin

  3. Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Ludmila Bronstain, Barbara C Furie & Bruce Furie

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Contributions

M.E. developed the agar assay. J.L.B., M.E., R.D., H.A., N.K. and D.B. performed the HTS. J.L.B. performed cherry-pick tests. J.L.B. and C.L. performed inhibitor retests. C.L. performed substructure analysis, in vivo DsbA and DsbB inhibition and other Gram-negative bacteria assays. F.H. performed enzyme kinetics and in vitro analysis. B.M.M. performed anaerobic and M. smegmatis growth assays. L.B., B.C.F. and B.F. performed in vitro mice VKOR assays. A.M., S.M. and E.J.R. performed M. tuberculosis growth assays. C.L., J.L.B., F.H., B.M.M., D.B. and J.B. analyzed and discussed the data. C.L. and J.B. wrote the paper.

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Correspondence to Jon Beckwith.

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Landeta, C., Blazyk, J., Hatahet, F. et al. Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria. Nat Chem Biol 11, 292–298 (2015). https://doi.org/10.1038/nchembio.1752

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