Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages

Abstract

The dual-specificity phosphatase 6 (Dusp6) functions as a feedback regulator of fibroblast growth factor (FGF) signaling to limit the activity of extracellular signal–regulated kinases (ERKs) 1 and 2. We have identified a small-molecule inhibitor of Dusp6—(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI)—using a transgenic zebrafish chemical screen. BCI treatment blocked Dusp6 activity and enhanced FGF target gene expression in zebrafish embryos. Docking simulations predicted an allosteric binding site for BCI within the phosphatase domain. In vitro studies supported a model in which BCI inhibits Dusp6 catalytic activation by ERK2 substrate binding. We used BCI treatment at varying developmental stages to uncover a temporal role for Dusp6 in restricting cardiac progenitors and controlling heart organ size. This study highlights the power of in vivo zebrafish chemical screens to identify new compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded traditional high-throughput in vitro screens.

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Figure 1: Identification of a small molecule that hyperactivates FGF signaling in zebrafish.
Figure 2: BCI structure-activity relationship studies.
Figure 3: BCI activity require FGF ligand, and BCI inhibits ectopic expression of Dusp6.
Figure 4: BCI directly inhibits Dusp6 in both chemical complementation and pERK2 dephosphorylation assays.
Figure 5: Modeling of BCI-Dusp6 interactions and in vitro testing of an allosteric inhibition mechanism.
Figure 6: Dusp6 and FGFs regulate heart size.

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References

  1. 1

    Thisse, B. & Thisse, C. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev. Biol. 287, 390–402 (2005).

  2. 2

    Dailey, L., Ambrosetti, D., Mansukhani, A. & Basilico, C. Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev. 16, 233–247 (2005).

  3. 3

    Tsang, M. & Dawid, I.B. Promotion and attenuation of FGF signaling through the Ras-MAPK pathway. Sci. STKE 2004, pe17 (2004).

  4. 4

    Abraira, V.E. et al. Changes in Sef levels influence auditory brainstem development and function. J. Neurosci. 27, 4273–4282 (2007).

  5. 5

    Li, C., Scott, D.A., Hatch, E., Tian, X. & Mansour, S.L. Dusp6 (Mkp3) is a negative feedback regulator of FGF-stimulated ERK signaling during mouse development. Development 134, 167–176 (2007).

  6. 6

    Maillet, M. et al. DUSP6 (MKP3) null mice show enhanced ERK1/2 phosphorylation at baseline and increased myocyte proliferation in the heart affecting disease susceptibility. J. Biol. Chem. 283, 31246–31255 (2008).

  7. 7

    Vogt, A. et al. Automated image-based phenotypic analysis in zebrafish embryos. Dev. Dyn. 238, 656–663 (2009).

  8. 8

    Zon, L.I. & Peterson, R.T. In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4, 35–44 (2005).

  9. 9

    Peterson, R.T., Link, B.A., Dowling, J.E. & Schreiber, S.L. Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc. Natl. Acad. Sci. USA 97, 12965–12969 (2000).

  10. 10

    Yu, P.B. et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4, 33–41 (2008).

  11. 11

    North, T.E. et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447, 1007–1011 (2007).

  12. 12

    Molina, G.A., Watkins, S.C. & Tsang, M. Generation of FGF reporter transgenic zebrafish and their utility in chemical screens. BMC Dev. Biol. 7, 62 (2007).

  13. 13

    Callahan, J.F. & Chabot-Fletcher, M.C. Inhibitors of transcription factor NF-kB. US patent application WO 99/65495 (1999).

  14. 14

    Latinkic, B.V. et al. The Xenopus Brachyury promoter is activated by FGF and low concentrations of activin and suppressed by high concentrations of activin and by paired-type homeodomain proteins. Genes Dev. 11, 3265–3276 (1997).

  15. 15

    Maves, L., Jackman, W. & Kimmel, C.B. FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain. Development 129, 3825–3837 (2002).

  16. 16

    Fürthauer, M., Reifers, F., Brand, M., Thisse, B. & Thisse, C. sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128, 2175–2186 (2001).

  17. 17

    Tsang, M., Friesel, R., Kudoh, T. & Dawid, I.B. Identification of Sef, a novel modulator of FGF signalling. Nat. Cell Biol. 4, 165–169 (2002).

  18. 18

    Tsang, M. et al. A role for MKP3 in axial patterning of the zebrafish embryo. Development 131, 2769–2779 (2004).

  19. 19

    Reifers, F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 2381–2395 (1998).

  20. 20

    Qian, F. et al. Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes. Dev. Dyn. 233, 1163–1172 (2005).

  21. 21

    Sumanas, S., Jorniak, T. & Lin, S. Identification of novel vascular endothelial-specific genes by the microarray analysis of the zebrafish cloche mutants. Blood 106, 534–541 (2005).

  22. 22

    Mandl, M., Slack, D.N. & Keyse, S.M. Specific inactivation and nuclear anchoring of extracellular signal-regulated kinase 2 by the inducible dual-specificity protein phosphatase DUSP5. Mol. Cell. Biol. 25, 1830–1845 (2005).

  23. 23

    Camps, M. et al. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280, 1262–1265 (1998).

  24. 24

    Chen, P. et al. Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MAP kinase phosphatase-2 and their ability to activate the phosphatase catalytically. J. Biol. Chem. 276, 29440–29449 (2001).

  25. 25

    Slack, D.N., Seternes, O.M., Gabrielsen, M. & Keyse, S.M. Distinct binding determinants for ERK2/p38alpha and JNK map kinases mediate catalytic activation and substrate selectivity of map kinase phosphatase-1. J. Biol. Chem. 276, 16491–16500 (2001).

  26. 26

    Vogt, A. & Lazo, J.S. Chemical complementation: a definitive phenotypic strategy for identifying small molecule inhibitors of elusive cellular targets. Pharmacol. Ther. 107, 212–221 (2005).

  27. 27

    Vogt, A. & Lazo, J.S. Implementation of high-content assay for inhibitors of mitogen-activated protein kinase phosphatases. Methods 42, 268–277 (2007).

  28. 28

    Almo, S.C. et al. Structural genomics of protein phosphatases. J. Struct. Funct. Genomics 8, 121–140 (2007).

  29. 29

    Jeong, D.G. et al. Crystal structure of the catalytic domain of human DUSP5, a dual specificity MAP kinase protein phosphatase. Proteins 66, 253–258 (2007).

  30. 30

    Jeong, D.G. et al. Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase. J. Mol. Biol. 360, 946–955 (2006).

  31. 31

    Stewart, A.E., Dowd, S., Keyse, S.M. & McDonald, N.Q. Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation. Nat. Struct. Biol. 6, 174–181 (1999).

  32. 32

    Morris, G.M. et al. Automated docking using a lamarckian genetic algorithm and an empirical binding free energy functions. J. Comput. Chem. 19, 1639–1662 (1998).

  33. 33

    Jones, G., Willett, P., Glen, R.C., Leach, A.R. & Taylor, R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748 (1997).

  34. 34

    Atilgan, A.R. et al. Anisotropy of fluctuation dynamics of proteins with an elastic network model. Biophys. J. 80, 505–515 (2001).

  35. 35

    Owens, D.M. & Keyse, S.M. Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene 26, 3203–3213 (2007).

  36. 36

    Bahar, I., Chennubhotla, C. & Tobi, D. Intrinsic dynamics of enzymes in the unbound state and relation to allosteric regulation. Curr. Opin. Struct. Biol. 17, 633–640 (2007).

  37. 37

    Brown, J.L. et al. Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification. Proc. Natl. Acad. Sci. USA 105, 12337–12342 (2008).

  38. 38

    Kudoh, T. et al. A gene expression screen in zebrafish embryogenesis. Genome Res. 11, 1979–1987 (2001).

  39. 39

    Keegan, B.R., Meyer, D. & Yelon, D. Organization of cardiac chamber progenitors in the zebrafish blastula. Development 131, 3081–3091 (2004).

  40. 40

    Yelon, D. Cardiac patterning and morphogenesis in zebrafish. Dev. Dyn. 222, 552–563 (2001).

  41. 41

    Chen, J.N. & Fishman, M.C. Genetics of heart development. Trends Genet. 16, 383–388 (2000).

  42. 42

    Marques, S.R., Lee, Y., Poss, K.D. & Yelon, D. Reiterative roles for FGF signaling in the establishment of size and proportion of the zebrafish heart. Dev. Biol. 321, 397–406 (2008).

  43. 43

    Reifers, F., Walsh, E.C., Leger, S., Stainier, D.Y. & Brand, M. Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar). Development 127, 225–235 (2000).

  44. 44

    Schoenebeck, J.J., Keegan, B.R. & Yelon, D. Vessel and blood specification override cardiac potential in anterior mesoderm. Dev. Cell 13, 254–267 (2007).

  45. 45

    Ducruet, A.P., Vogt, A., Wipf, P. & Lazo, J.S. Dual specificity protein phosphatases: therapeutic targets for cancer and Alzheimer's disease. Annu. Rev. Pharmacol. Toxicol. 45, 725–750 (2005).

  46. 46

    Bakan, A., Lazo, J.S., Wipf, P., Brummond, K.M. & Bahar, I. Toward a molecular understanding of the interaction of dual specificity phosphatases with substrates: insights from structure-based modeling and high throughput screening. Curr. Med. Chem. 15, 2536–2544 (2008).

  47. 47

    Lazo, J.S. et al. Novel benzofuran inhibitors of human mitogen-activated protein kinase phosphatase-1. Bioorg. Med. Chem. 14, 5643–5650 (2006).

  48. 48

    Gurtner, G.C., Werner, S., Barrandon, Y. & Longaker, M.T. Wound repair and regeneration. Nature 453, 314–321 (2008).

  49. 49

    Lepilina, A. et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127, 607–619 (2006).

  50. 50

    Dowd, S., Sneddon, A.A. & Keyse, S.M. Isolation of the human genes encoding the Pyst1 and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases. J. Cell Sci. 111, 3389–3399 (1998).

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Acknowledgements

We thank N. Hukriede, M. Rebagliati and I. Dawid for critical reading of the manuscript. We thank M.S. Poslusney for assistance in the syntheses. We thank R. Schultz (Developmental Therapeutics Program, US National Cancer Institute) for providing the National Cancer Institute diversity set and samples of individual compounds. The project described was supported in part by award number R01HL088016 to M.T. from the US National Heart, Lung, and Blood Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health (NIH). This work was also supported by NIH grants HD053287, CA52995, MH074411 and CA78039, and by the Fiske Drug Discovery Fund.

Author information

G.M., A.V., A.B., P.Q.O., W.D., W.Z. and M.T. performed experiments. G.M., A.V., A.B., T.E.S., J.S.L., I.B., B.W.D. and M.T. designed experiments and analyzed data. M.T. wrote the paper with help from A.V., A.B., T.E.S., J.S.L., B.W.D. and I.B.

Correspondence to Michael Tsang.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Methods (PDF 16097 kb)

Supplementary Movie 1

Dusp6 intrinsic flexibility of the general acid loop was generated using 3rd, 4th, and 5th slow modes. (MOV 156 kb)

Supplementary Movie 2

Catalytic activation of Dusp6 was generated using 5% of the entire spectrum of modes in the low frequency regime. (MOV 582 kb)

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Molina, G., Vogt, A., Bakan, A. et al. Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages. Nat Chem Biol 5, 680–687 (2009). https://doi.org/10.1038/nchembio.190

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