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

Nature Chemical Biology volume 5, pages 680–687 (2009)Cite this article

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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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.

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Author notes

  1. Gabriela Molina

    Present address: Present address: SRI International, Center for Advanced Drug Research, Harrisonburg, Virginia, USA.,

  2. Gabriela Molina, Andreas Vogt and Ahmet Bakan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Gabriela Molina, Wade Znosko, Thomas E Smithgall & Michael Tsang

  2. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Andreas Vogt, Pierre Queiroz de Oliveira & John S Lazo

  3. Pittsburgh Molecular Libraries Screening Center, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Andreas Vogt, Thomas E Smithgall, Ivet Bahar & John S Lazo

  4. Department of Computational Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Ahmet Bakan & Ivet Bahar

  5. Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Weixiang Dai & Billy W Day

  6. Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Billy W Day

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Contributions

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.

Corresponding author

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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