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Identification of small molecule inhibitors of anthrax lethal factor

Abstract

The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have Ki values in the 0.5–5 μM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

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Figure 1: A two-stage assay for screening and validating small molecule inhibitors of anthrax lethal factor.
Figure 2: General pharmacophore model of the LF inhibitors.
Figure 3: X-ray crystal structure of the LF–NSC 12155–Zn complex.
Figure 4: Efficacy of LF inhibitors in a cell-based toxicity assay.

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References

  1. Bradley, K.A., Mogridge, J., Mourez, M., Collier, R.J. & Young, J.A. Identification of the cellular receptor for anthrax toxin. Nature 414, 225–229 (2001).

    Article  CAS  Google Scholar 

  2. Scobie, H.M., Rainey, G.J., Bradley, K.A. & Young, J.A. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc. Natl. Acad. Sci. USA 100, 5170–5174 (2003).

    Article  CAS  Google Scholar 

  3. Klimpel, K.R., Molloy, S.S., Thomas, G. & Leppla, S.H. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc. Natl. Acad. Sci. USA 89, 10277–10281 (1992).

    Article  CAS  Google Scholar 

  4. Molloy, S.S., Bresnahan, P.A., Leppla, S.H., Klimpel, K.R. & Thomas, G. Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J. Biol. Chem. 267, 16396–16402 (1992).

    CAS  PubMed  Google Scholar 

  5. Leppla, S.H. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc. Natl. Acad. Sci. USA 79, 3162–3166 (1982).

    Article  CAS  Google Scholar 

  6. Vitale, G. et al. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem. Biophys. Res. Commun. 248, 706–711 (1998).

    Article  CAS  Google Scholar 

  7. Duesbery, N.S. et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734–737 (1998).

    Article  CAS  Google Scholar 

  8. Duesbery, N.S. et al. Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways. Proc. Natl. Acad. Sci. USA 98, 4089–4094 (2001).

    Article  CAS  Google Scholar 

  9. Park, J.M., Greten, F.R., Li, Z.W. & Karin, M. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048–2051 (2002).

    Article  CAS  Google Scholar 

  10. Sellman, B.R., Mourez, M. & Collier, R.J. Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax. Science 292, 695–697 (2001).

    Article  CAS  Google Scholar 

  11. Singh, Y., Khanna, H., Chopra, A.P. & Mehra, V. A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo. J. Biol. Chem. 276, 22090–22094 (2001).

    Article  CAS  Google Scholar 

  12. Mourez, M. et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 19, 958–961 (2001).

    Article  CAS  Google Scholar 

  13. Hammond, S.E. & Hanna, P.C. Lethal factor active-site mutations affect catalytic activity in vitro. Infect. Immun. 66, 2374–2378 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Tonello, F., Seveso, M., Marin, O., Mock, M. & Montecucco, C. Screening inhibitors of anthrax lethal factor. Nature 418, 386 (2002).

    Article  CAS  Google Scholar 

  15. Turk, B.E. et al. The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor. Nat. Struct. Mol. Biol. 11, 60–66 (2004).

    Article  CAS  Google Scholar 

  16. Pannifer, A.D. et al. Crystal structure of the anthrax lethal factor. Nature 414, 229–233 (2001).

    Article  CAS  Google Scholar 

  17. Segel, I.H. Enzyme Kinetics (Wiley, New York, 1975).

    Google Scholar 

  18. Vosko, S.J., Wilk, L. & Nusair, M. Accurate spin-dependent electron liquid correlation energies for local spin density calculations. Can. J. Phys. 58, 1200–1211 (1980).

    Article  CAS  Google Scholar 

  19. Godbout, N., Salahub, D., Andzelm, J. & Wimmer, E. Optimization of Gaussian-type basis-sets for local spin-density functional calculations. 1. Boron through neon, optimization technique and validation. Can. J. Chem. 70, 560–571 (1992).

    Article  CAS  Google Scholar 

  20. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J.Chem. Phys. 98, 5648–5652 (1993).

    Article  CAS  Google Scholar 

  21. Lee, C., Yang, W. & Parr, R.G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B Condens. Matter 37, 785–789 (1988).

    Article  CAS  Google Scholar 

  22. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  25. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D. 50, 760–763 (1994).

Download references

Acknowledgements

This research was sponsored by the Medical Biological Defense Research Program, US Army Medical Research and Materiel Command, project no. 0242C012. We acknowledge the US National Cancer Institute for the allocation of computing time and staff support at the Advanced Biomedical Computing Center of the Frederick Cancer Research and Development Center. We thank the staff of the Stanford Synchrotron Radiation Laboratory (SSRL) for assistance during data collection. We also thank S. Leppla (US National Institutes of Health (NIH)) and D. Hsu for the LF protein preparation used in the crystal structure. Portions of this research were carried out at the SSRL, a national user facility operated by Stanford University on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences (BES). The SSRL Structural Molecular Biology Program is supported by the DOE, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences.

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Correspondence to Rekha G Panchal or Sina Bavari.

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Panchal, R., Hermone, A., Nguyen, T. et al. Identification of small molecule inhibitors of anthrax lethal factor. Nat Struct Mol Biol 11, 67–72 (2004). https://doi.org/10.1038/nsmb711

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