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Type IIA topoisomerase inhibition by a new class of antibacterial agents

Nature volume 466, pages 935–940 (2010)Cite this article

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Abstract

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 Å crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor ‘bridges’ the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

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Figure 1
Figure 2: The GyrB27–A56 fusion protein.
Figure 3: The 2.1 Å GyrB27–A56 complex with GSK299423 and DNA.
Figure 4: Comparison of topoisomerase active sites.
Figure 5: Metal-binding site associated with cleavage of DNA by type IIA topoisomerases.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported structures have been deposited in the Protein Data Bank (PDB) under accession codes 2XCO, 2XCQ, 2XCR, 2XCS and 2XCT.

Change history

  • 19 August 2010

    Author Andrew J. Theobald's middle initial was corrected for the print issue.

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Acknowledgements

We thank H. Hiasa, A. Maxwell, J. C. Wang and D. B. Wigley for discussions. We thank S. Erskine, A. West and S. Brooks for experimental work that helped initiate this project. We thank P. Rowland for help with data collection and processing, the antimicrobial profiling group at GlaxoSmithKline (GSK) for antibacterial data, and K. Smith and D. Payne and members of the project team for discussions. A.W. was supported by the Wellcome Trust Seeding Drug Discovery Initiative and contract HDTRA1-07-9-0002 with the US Department of Defense (DoD) Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD), and the Defense Threat Reduction Agency (DTRA) Transformational Medical Technologies (TMT). The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the DoD or the US Government.

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Author notes

  1. Drake S. Eggleston, Fabrice Gorrec, Earl W. May & Alexandre Wohlkonig

    Present address: Present addresses: Innovalyst, 1000 Centre Green Way, Suite 200, Cary, North Carolina 27513, USA (D.S.E.); MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK (F.G.); OSI Pharmaceuticals, 1 Bioscience Park Drive, Farmingdale, New York 11735, USA (E.W.M.); Vrije Universiteit Brussel, VIB Department of Molecular and Cellular Interactions, Pleinlaan 2, 1050 Brussels, Belgium (A.W.).,

Authors and Affiliations

  1. Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK,

    Benjamin D. Bax, Michael M. Hann, Martin R. Saunders, Onkar Singh & Claus E. Spitzfaden

  2. Antibacterial Discovery Performance Unit, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline, 1250 Collegeville Road, Collegeville, 19426, Pennsylvania, USA

    Pan F. Chan, Daniel R. Gentry, Jianzhong Huang, Earl W. May, Carol Shen, Neil D. Pearson & Michael N. Gwynn

  3. Molecular Discovery Research, GlaxoSmithKline, Third Ave, Harlow, Essex, CM19 5AW, UK,

    Drake S. Eggleston, Andrew Fosberry, Fabrice Gorrec, Martin Hibbs, Emma Jones, Jo Jones, Ceri J. Lewis, Anthony Shillings & Andrew J. Theobald

  4. Antibacterial Discovery Performance Unit, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK,

    Ilaria Giordano, Alan Hennessy & Alexandre Wohlkonig

  5. Molecular Discovery Research, GlaxoSmithKline, 1250 Collegeville Rd., Collegeville, 19426, Pennsylvania, USA

    Kristin Koretke Brown

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Contributions

M.N.G. and D.R.G. defined the relevant domains for crystallography by mutational analysis. K.K.B. performed gene sequence analysis and designed B27–A56 translation fusion. J.H. and E.W.M. made the initial fusion constructs, and tested them for activity. J.H., C.S. and P.F.C. performed studies with GSK299423 on target potency and the inhibition mechanism and demonstrated the lack of cross-resistance with quinolones. B.D.B. and M.R.S. designed the Greek key deletion. A.F. made the Greek-key-deletion and catalytic Tyr-to-Phe constructs. J.J. optimized expression in fermentors and grew cells for purification. M.H., E.J., A.S. and A.F.T. purified apo protein and complexes of various constructs for crystallography and assay. I.G. and A.H. designed and synthesized the compound. C.E.S. performed analytical size exclusion chromatography experiments that were used to find a stable particle that could be crystallized. F.G. crystallized apo and GSK299423 complex in fluidigm chips. O.S. and A.W. grew quinolone crystals by microbatch. B.D.B. grew apo and GSK299423 complex crystals by vapour diffusion, solved and refined structures. C.J.L. lead biochemistry. N.D.P. led chemistry and provided compound. D.S.E., M.N.G. and N.D.P. initiated and led the project. B.D.B. and M.N.G. wrote the manuscript with the assistance of M.M.H. and the other authors.

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Correspondence to Benjamin D. Bax or Michael N. Gwynn.

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Bax, B., Chan, P., Eggleston, D. et al. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 466, 935–940 (2010). https://doi.org/10.1038/nature09197

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