Brief Communication | Published:

A new screening assay for allosteric inhibitors of cSrc

Nature Chemical Biology volume 5, pages 394396 (2009) | Download Citation

Abstract

Targeting kinases outside the highly conserved ATP pocket is thought to be a promising strategy for overcoming bottlenecks in kinase inhibitor research, such as limited selectivity and drug resistance. Here we report the development and application of a direct binding assay to detect small molecules that stabilize the inactive conformation of the tyrosine kinase cSrc. Protein X-ray crystallography validated the assay results and confirmed an exclusively allosteric binding mode.

  • Compound

    1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-(4-chlorophenyl)urea

  • Compound

    1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-phenylurea

  • Compound

    1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-(4-chlorophenyl)urea

  • Compound

    1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-(naphthalen-1-yl)urea

  • Compound

    1-(3-tert-Butyl-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-1H-pyrazol-5-yl)-3-(4-chlorophenyl)urea

  • Compound

    Adenosine 5′-triphosphate

  • Compound

    Staurosporine

  • Compound

    Dasatinib

  • Compound

    Imatinib

  • Compound

    GNF2

  • Compound

    BIRB-796

  • Compound

    3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-amine

  • Compound

    1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-phenylurea

  • Compound

    1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-(4-chlorophenyl)urea

  • Compound

    3-tert-Butyl-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-1H-pyrazol-5-amine

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Acknowledgements

We thank the Dortmund Protein Facility for cloning, expressing and purifying the chicken cSrc and human p38α mutant variants for labeling. We thank M. Weyand, W. Blanckenfeldt, E. Hofmann, I. Vetter and beamline scientists at the Swiss Light Source of the Paul Scherrer Institute for expert assistance during data collection. We thank P. Janning for her assistance with the HPLC and MS analysis of the fluorescent-labeled and unlabeled cSrc, and we thank H. Waldmann and K. Shokat for helpful discussions. J.R.S. was funded by the Alexander von Humboldt Foundation. This work was supported by the German Federal Ministry for Education and Research through the German National Genome Research Network-Plus (NGFN-Plus) (grant number BMBF 01GS08102). Schering Plough, Bayer-Schering Pharma, Merck-Serono and Bayer CropScience are thanked for financial support.

Author information

Affiliations

  1. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany.

    • Jeffrey R Simard
    • , Sabine Klüter
    • , Christian Grütter
    • , Matthäus Getlik
    • , Matthias Rabiller
    • , Haridas B Rode
    •  & Daniel Rauh

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Contributions

J.R.S. designed the p38α and cSrc constructs for labeling, developed the fluorescent-labeled kinase assay system and determined compound Kd, kon and koff values. S.K. overexpressed, purified and crystallized cSrc. C.G. determined the cocrystal structures. M.R. determined IC50 values. M.G. synthesized the compounds with assistance from S.K. and H.B.R. The manuscript was prepared by J.R.S. with assistance by all co-authors. D.R. designed the experiments.

Corresponding author

Correspondence to Daniel Rauh.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods

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DOI

https://doi.org/10.1038/nchembio.162

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