Her3 (also known as ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be 'undruggable' using ATP-competitive small molecules because it lacks appreciable kinase activity. Here we report what is to our knowledge the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3-dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and 60 other pseudokinases found in human cells.

  • Compound C34H39N9O3


  • Compound C23H22N4O


  • Compound C22H19N3O3


  • Compound C32H36N8O3


  • Compound C50H68N12O8S


  • Compound C42H52N8O3


  • Compound C42H54N8O3


  • Compound C26H27N7O2


  • Compound C32H38N8O3


  • Compound C5H4IN5


  • Compound C11H12IN5O


  • Compound C20H30IN7O2

    tert-Butyl 4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate

  • Compound C17H24IN7O


  • Compound C26H33FN8O4

    tert-Butyl 4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate

  • Compound C32H38N8O5

    tert-Butyl 4-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate

  • Compound C23H27FN8O3


  • Compound C29H32N8O4


  • Compound C42H54N8O9

    tert-Butyl 4-(4-(4-((tert-butoxycarbonyl)amino)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate

  • Compound C42H56N8O7

    tert-Butyl 4-(4-(3-(3-amino-4-phenoxyphenyl)-4-((tert-butoxycarbonyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate

  • Compound C30H34N8O2


  • Compound C39H48N8O8

    tert-Butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate

  • Compound C39H50N8O6

    tert-Butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate

  • Compound C15H21IN6O2

    tert-Butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate

  • Compound C10H13IN6


  • Compound C17H26IN7O2

    tert-Butyl (2-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate

  • Compound C23H29FN8O4

    tert-Butyl (2-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate

  • Compound C29H34N8O5

    tert-Butyl (2-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate

  • Compound C39H48N8O4


  • Compound C49H64N8O8

    tert-Butyl (1-(1-(2-((R)-4-((3R,5R,7R)-adamantan-1-yl)-2-methylbutanamido)ethyl)piperidin-4-yl)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate

  • Compound C49H66N8O6

    tert-Butyl (1-(1-(2-((R)-4-((3R,5R,7R)-adamantan-1-yl)-2-methylbutanamido)ethyl)piperidin-4-yl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate

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  1. 1.

    et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N. Engl. J. Med. 353, 1659–1672 (2005).

  2. 2.

    et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N. Engl. J. Med. 353, 1673–1684 (2005).

  3. 3.

    et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304, 1497–1500 (2004).

  4. 4.

    et al. Gastric cancer—molecular and clinical dimensions. Nat. Rev. Clin. Oncol. 10, 643–655 (2013).

  5. 5.

    , , , & An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137–1149 (2006).

  6. 6.

    , , , & Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3. Proc. Natl. Acad. Sci. USA 106, 21608–21613 (2009).

  7. 7.

    , , , & ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation. Proc. Natl. Acad. Sci. USA 107, 7692–7697 (2010).

  8. 8.

    , , , & Insect cell-expressed P180 (ErbB3) possesses an impaired tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 91, 8132–8136 (1994).

  9. 9.

    , , & Biochemical characterization of the protein tyrosine kinase homology domain of the ErbBB (HER3) receptor protein. Biochem. J. 322, 757–763 (1997).

  10. 10.

    et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007).

  11. 11.

    & Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2, 127–137 (2001).

  12. 12.

    & ERBB receptors and cancer: the complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341–354 (2005).

  13. 13.

    & Novel anticancer targets: revisiting ERBB2 and discovering ERBB3. Nat. Rev. Cancer 9, 463–475 (2009).

  14. 14.

    et al. ErbB3 predicts survival in ovarian cancer. J. Clin. Oncol. 24, 4317–4323 (2006).

  15. 15.

    et al. HER3 is required for HER2-induced preneoplastic changes to the breast epithelium and tumor formation. Cancer Res. 72, 2672–2682 (2012).

  16. 16.

    et al. A central role for HER3 in HER2-amplified breast cancer: implications for targeted therapy. Cancer Res. 68, 5878–5887 (2008).

  17. 17.

    et al. A five-gene signature and clinical outcome in non-small-cell lung cancer. N. Engl. J. Med. 356, 11–20 (2007).

  18. 18.

    et al. Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature 445, 437–441 (2007).

  19. 19.

    et al. Her3 (ErbB3) compensates for inhibition of the Her2 tyrosine kinase. Proc. Natl. Acad. Sci. USA 108, 5021–5026 (2011).

  20. 20.

    et al. An activated ErbB3/NRG1 autocrine loop supports in vivo proliferation in ovarian cancer cells. Cancer Cell 17, 298–310 (2010).

  21. 21.

    et al. A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies. Cancer Cell 20, 472–486 (2011).

  22. 22.

    et al. An ErbB3 antibody, MM-121, is active in cancers with ligand-dependent activation. Cancer Res. 70, 2485–2494 (2010).

  23. 23.

    et al. A first-in-human phase I study of U3–1287 (AMG 888), a HER3 inhibitor, in patients (pts) with advanced solid tumors. J. Clin. Oncol. 29 (Suppl): abstr 3026 (2011).

  24. 24.

    et al. Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N. Engl. J. Med. 366, 109–119 (2012).

  25. 25.

    et al. Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543 (2011).

  26. 26.

    et al. Identification of hydrophobic tags for the degradation of stabilized proteins. ChemBioChem 13, 538–541 (2012).

  27. 27.

    et al. Developing irreversible inhibitors of the protein kinase cysteinome. Chem. Biol. 20, 146–159 (2013).

  28. 28.

    et al. Development and applications of a broad-coverage, TR-FRET–based kinase binding assay platform. J. Biomol. Screen. 14, 924–935 (2009).

  29. 29.

    et al. Discovery of A-770041, a src-family selective orally active lck inhibitor that prevents organ allograft rejection. Bioorg. Med. Chem. Lett. 16, 118–122 (2006).

  30. 30.

    et al. Pyrrolo[2,3-d]pyrimidines containing an extended 5-substituent as potent and selective inhibitors of lck I. Bioorg. Med. Chem. Lett. 10, 2167–2170 (2000).

  31. 31.

    et al. Pyrrolo[2,3-d]pyrimidines containing an extended 5-substituent as potent and selective inhibitors of lck II. Bioorg. Med. Chem. Lett. 10, 2171–2174 (2000).

  32. 32.

    et al. 2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3 -thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor. J. Med. Chem. 49, 6819–6832 (2006).

  33. 33.

    et al. Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity. J. Med. Chem. 44, 3965–3977 (2001).

  34. 34.

    et al. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46, 350–358 (2007).

  35. 35.

    et al. In situ kinase profiling reveals functionally relevant properties of native kinases. Chem. Biol. 18, 699–710 (2011).

  36. 36.

    , & Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

  37. 37.

    et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell 17, 77–88 (2010).

  38. 38.

    , & Inhibitor mediated protein degradation. Chem. Biol. 19, 629–637 (2012).

  39. 39.

    & Greasy tags for protein removal. Nature 487, 308–309 (2012).

  40. 40.

    et al. ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system. Nat. Chem. Biol. 9, 307–312 (2013).

  41. 41.

    et al. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat. Chem. Biol. 4, 691–699 (2008).

  42. 42.

    et al. Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function. Nat. Chem. Biol. 6, 359–368 (2010).

  43. 43.

    et al. Characterization of a selective inhibitor of the Parkinsons disease kinase LRRK2. Nat. Chem. Biol. 7, 203–205 (2011).

  44. 44.

    et al. An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels. Proc. Natl. Acad. Sci. USA 99, 2866 (2002).

  45. 45.

    & Structure-based mutagenesis of the substrate-recognition domain of Nrdp1/FLRF identifies the binding site for the receptor tyrosine kinase ErbB3. Protein Sci. 16, 654 (2007).

  46. 46.

    E3 ubiquitin ligases in ErbB receptor quantity control Semin. Cell Dev. Biol. 21, 936 (2010).

  47. 47.

    et al. The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation. J. Biol. Chem. 287, 15996 (2012).

  48. 48.

    et al. Structures of lung cancer–derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11, 217–227 (2007).

  49. 49.

    & A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J. Am. Soc. Mass Spectrom. 9, 225–233 (1998).

  50. 50.

    et al. Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells. Anal. Chem. 81, 3440–3447 (2009).

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We wish to thank staff at The Institute of Chemistry and Cell Biology for the guidance of screening equipment and assay development discussion. We thank J. Minna and M. Peyton of UT Southwestern Medical Center for providing the HCC2935 cell line. This work is supported by the Dana Farber Cancer Institute Lander Fellowship (T.X.), Claudia Adams Barr Program Award (N.S.G.), US National Institutes of Health (NIH) grant AI 084140-03 (C.M.C.), Cancer Prevention Research Institute of Texas grant R1207 (K.D.W.), creative/challenging research program of National Research Foundation of Korea NRF-2011-0028676 (T.S.) and NIH grant P01 CA154303 (P.A.J. and N.S.G.).

Author information


  1. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Ting Xie
    • , Sang Min Lim
    •  & Nathanael S Gray
  2. Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Ting Xie
    • , Sang Min Lim
    • , Scott B Ficarro
    • , Jarrod A Marto
    •  & Nathanael S Gray
  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Ting Xie
    •  & Sang Min Lim
  4. Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

    • Kenneth D Westover
    • , Durga Udayakumar
    •  & Deepak Gurbani
  5. Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Michael E Dodge
    • , Dalia Ercan
    •  & Pasi A Jänne
  6. Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Scott B Ficarro
    •  & Jarrod A Marto
  7. Department of Molecular, Cellular and Development Biology, Yale University, New Haven, Connecticut, USA.

    • Hyun Seop Tae
    •  & Craig M Crews
  8. Primary and Stem Cell Systems, Life Technologies Corporation, Madison, Wisconsin, USA.

    • Steven M Riddle
  9. Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul, Korea.

    • Taebo Sim
  10. KU-KIST Graduate School of Converging Science and Technology, Seoul, Korea.

    • Taebo Sim


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N.S.G. oversaw all aspects of the experiments and manuscript preparation. T.X. and S.M.L. performed the chemical synthesis and structure-activity relationship analysis. H.S.T. and C.M.C. provided assistance and reagents for synthesis of adamantane derivatives. T.X. performed hits/leads valuation by protein- and cell-based assays with assistance from D.E., P.A.J., K.D.W., D.U. and M.E.D. K.D.W., D.G. and T.X. expressed and purified Her3 protein. T.X. and S.M.R. optimized the FRET-based binding assay. T.S. performed molecular docking studies. S.B.F., J.A.M., K.D.W., D.G. and T.X. conducted MS labeling experiments and analyses. T.X. and N.S.G. wrote the manuscript, and all coauthors participated in editing this manuscript.

Competing interests

C.M.C. is founder and an equity shareholder in Arvinas, Inc., which is developing small molecule -induced protein degradation as a therapeutic methodology.

Corresponding authors

Correspondence to Pasi A Jänne or Craig M Crews or Nathanael S Gray.

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