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Pharmacological targeting of the pseudokinase Her3

Abstract

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.

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Figure 1: Hit identification and development of the Her3 irreversible inhibitor TX1-85-1.
Figure 2: TX1-85-1 is insufficient to inactivate the Her3-PI3K-AKT pathway, explaining lack of growth inhibition for Her3-dependent cells.
Figure 3: Adamantane-conjugated compounds induce Her3 degradation.
Figure 4: C721S rescue from compound-induced degradation.
Figure 5: Mechanism of TX2-121-1 compound-induced degradation and interferences with Her2-Her3 and c-Met–Her3 interactions.

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Acknowledgements

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.).

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Authors

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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.

Corresponding authors

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

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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.

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Supplementary Results, Supplementary Figures 1–11, Supplementary Tables 1–3 and Supplementary Note. (PDF 2738 kb)

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Xie, T., Lim, S., Westover, K. et al. Pharmacological targeting of the pseudokinase Her3. Nat Chem Biol 10, 1006–1012 (2014). https://doi.org/10.1038/nchembio.1658

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