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Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39

Abstract

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

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Figure 1
Figure 2: CMAP analog 5 inhibited Gli1-stimulated Hh pathway signaling, whereas the Smo antagonist HhAntag had little effect.
Figure 3: The IP3 response in TM3 cells to 3 was exploited to identify candidate CMAP targets.
Figure 4: Modulation of GPR39 levels alters CMAP potency.
Figure 5: Phospho-Erk activation by CMAPs contributes to Hedgehog pathway inhibition.

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Acknowledgements

We thank A. Abrams for assistance with production of a figure for this publication.

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Authors and Affiliations

Authors

Contributions

F.B., J.F.K. III, K.S., R.K.J. and S.J.L. designed experiments. A.C., J.A.D., A.B., A.R., S.J.L. and F.H. performed Hh pathway assays. F.B., B.G., S.V., V.B., B.W., A.S. and A.C. designed and performed GPCR-focused cell-based experiments. X.W. performed the initial screen. L.A.L., T.B.S., P.R. and R.K.J. synthesized compounds. S.P. and L.L. performed cell-free experiments. H.R., P.G., M.S., J.J. and T.B. designed and performed proteomics experiments. T.B., J.A.P., V.M., P.M.F. and J.A.T. provided intellectual input during the target identification campaign. S.J.L., R.K.J. and F.B. wrote the manuscript.

Corresponding authors

Correspondence to Rishi K Jain or Sarah J Luchansky.

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Competing interests

The authors are employees of Novartis and Cellzome Ag. The research was fully funded by Novartis.

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Supplementary Results, Supplementary Figures 1–17, Supplementary Tables 1–3 and Supplementary Note. (PDF 33061 kb)

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Bassilana, F., Carlson, A., DaSilva, J. et al. Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39. Nat Chem Biol 10, 343–349 (2014). https://doi.org/10.1038/nchembio.1481

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