A new screening assay for allosteric inhibitors of cSrc


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.

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Figure 1: Schematic diagram of the fluorescent-labeled kinase assay for detecting allosteric inhibitors.
Figure 2: Screening of a compound library reveals pyrazoloureas binding to cSrc and p38α.
Figure 3: Crystal structures of cSrc in complex with type III inhibitors.

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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.

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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.

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Correspondence to Daniel Rauh.

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Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 874 kb)

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Simard, J., Klüter, S., Grütter, C. et al. A new screening assay for allosteric inhibitors of cSrc. Nat Chem Biol 5, 394–396 (2009). https://doi.org/10.1038/nchembio.162

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