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Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles

Nature Chemical Biology volume 8, pages 366–374 (2012)Cite this article

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Abstract

Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently identified, in a DNA-templated macrocycle library, inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated 'gatekeeper' mutant. We solved two cocrystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide–competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bisubstrate-competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance.

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Figure 1: Chemical structures of macrocycles described in this work.
Figure 2: Potency of macrocycle compounds against Src kinase domain constructs.
Figure 3: Macrocycle compounds are ATP- and peptide-competitive inhibitors.
Figure 4: The three-dimensional structure of Src kinase domain bound to macrocyclic inhibitors.
Figure 5: Determinants of macrocycle specificity.
Figure 6: Activity of the macrocycles against the T338I gatekeeper mutant of Src kinase.

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Acknowledgements

3T3 (Src−/−) cells transfected with a plasmid encoding c-SrcY529F or null vector were a gift from J. Cooper (Fred Hutchinson Cancer Research Center). We thank the beamline staff at X29A at the National Synchrotron Light Source at Brookhaven National Laboratory, the use of which was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences contract DE-AC02-98CH10886. G.G. and M.A.S. thank L. Malone for her assistance. This research was supported by the Howard Hughes Medical Institute (D.R.L.) and US National Institutes of Health (NIH)–National Institute of General Medical Sciences grants GM065865 (D.R.L.) and GM080097 (M.A.S.). R.E.K. acknowledges NIH training grant support to the Harvard University Training Program in Molecular, Cellular and Chemical Biology. G.G. acknowledges NIH training grant support to the Graduate Program in Molecular and Cellular Pharmacology at Stony Brook University.

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Author notes

  1. George Georghiou and Ralph E Kleiner: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York, USA

    George Georghiou, Michael Pulkoski-Gross & Markus A Seeliger

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Ralph E Kleiner & David R Liu

  3. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Ralph E Kleiner & David R Liu

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  1. George Georghiou
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Contributions

D.R.L. and R.E.K. developed and synthesized the compounds, performed Z'-LYTE assays and tested activity in NIH 3T3 cells. G.G., M.P.-G. and M.A.S. prepared proteins and performed the structural studies and biochemical assays. All authors analyzed the data and wrote the manuscript.

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Correspondence to David R Liu or Markus A Seeliger.

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Competing interests

D.R.L. is a consultant for Ensemble Therapeutics, a company that uses DNA-templated synthesis in drug discovery and development.

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Georghiou, G., Kleiner, R., Pulkoski-Gross, M. et al. Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles. Nat Chem Biol 8, 366–374 (2012). https://doi.org/10.1038/nchembio.792

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