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.

  • Compound C23H35N7O2S

    N-(((1r,4r)-4-(((4-(Methylamino)-6-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)cyclohexyl)methyl)piperidine-1-sulfonamide

  • Compound C24H29FN6O2S

    4-Fluoro-N-(((1r,4r)-4-(((4-(methylamino)-6-(pyridin-3-yl)pyrimidin-2-yl)amino)methyl)cyclohexyl)methyl)benzenesulfonamide

  • Compound C26H33FN6O3S

    4-Fluoro-N-(((1r,4r)-4-(((4-((2-methoxyethyl)amino)-6-(pyridin-2-yl)pyrimidin-2-yl)amino)methyl)cyclohexyl)methyl)benzenesulfonamide

  • Compound C24H28FN5O3S

    3-Fluoro-N-(((1r,4r)-4-(((4-methoxy-6-(pyridin-2-yl)pyrimidin-2-yl)amino)methyl)cyclohexyl)methyl)benzenesulfonamide

  • Compound C25H29N7O2S

    3-Cyano-N-(((1r,4r)-4-(((4-(methylamino)-6-(pyridin-2-yl)pyrimidin-2-yl)amino)methyl)cyclohexyl)methyl)benzenesulfonamide

  • Compound C29H38N8O2S

    N-(((1r,4r)-4-(((4-((5-Aminopentyl)amino)-6-(pyridin-2-yl)pyrimidin-2-yl)amino)methyl)cyclohexyl)methyl)-3-cyanobenzenesulfonamide

  • Compound C31H40N8O3S

    N-(5-((2-((((1r,4r)-4-((3-Cyanophenylsulfonamido)methyl)cyclohexyl)methyl)amino)-6-(pyridin-2-yl)pyrimidin-4-yl)amino)pentyl)acetamide

  • Compound C62H77N10O9S3+

    2-((1E,3E,5E)-5-(1-(6-((5-((2-((((1r,4r)-4-((3-Cyanophenylsulfonamido)methyl)cyclohexyl)methyl)amino)-6-(pyridin-2-yl)pyrimidin-4-yl)amino)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indol-1-ium

  • Compound C35H44N8O7S2

    2-((2-((5-((2-((((1r,4r)-4-((3-Cyanophenylsulfonamido)methyl)cyclohexyl)methyl)amino)-6-(pyridin-2-yl)pyrimidin-4-yl)amino)pentyl)amino)-2-oxoethyl)thio)succinic acid

  • Compound C19H26ClN5O2

    tert-Butyl (5-((2-chloro-6-(pyridin-2-yl)pyrimidin-4-yl)amino)pentyl)carbamate

  • Compound C15H21N3O2S

    N-(((1r,4r)-4-(Aminomethyl)cyclohexyl)methyl)-3-cyanobenzenesulfonamide

  • Compound C10H9ClN4

    2-Chloro-N-methyl-6-(pyridin-2-yl)pyrimidin-4-amine

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Acknowledgements

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

Author information

Author notes

    • Xu Wu
    • , Heinz Ruffner
    • , Markus Schirle
    • , Tewis Bouwmeester
    •  & Peter M Finan

    Present addresses: Novartis Institutes for Biomedical Research, Basel, Switzerland (T.B. and H.R.); Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA (M.S.); Massachusetts General Hospital, Charlestown, Massachusetts, USA. (X.W.); Novartis Institutes for Biomedical Research, Horsham, West Sussex, UK (P.M.F.).

Affiliations

  1. Novartis Institutes for Biomedical Research, Basel, Switzerland.

    • Frederic Bassilana
    • , Bianka Grosshans
    • , Solange Vidal
    • , Valerie Beck
    • , Barbara Wilmeringwetter
    • , Pascal Rigollier
    • , Andreas W Sailer
    •  & Klaus Seuwen
  2. Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

    • Adam Carlson
    • , Jennifer A DaSilva
    • , Luis A Llamas
    • , Todd B Showalter
    • , Aaron Bourret
    • , Arun Ramamurthy
    • , Fred Harbinski
    • , Samantha Plonsky
    • , Lac Lee
    • , Jeremy Jenkins
    • , Jeffrey A Porter
    • , Vic Myer
    • , Peter M Finan
    • , John A Tallarico
    • , Joseph F Kelleher III
    • , Rishi K Jain
    •  & Sarah J Luchansky
  3. Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.

    • Xu Wu
  4. Cellzome AG, Heidelberg, Germany.

    • Heinz Ruffner
    • , Paola Grandi
    • , Markus Schirle
    •  & Tewis Bouwmeester

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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.

Competing interests

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

Corresponding authors

Correspondence to Rishi K Jain or Sarah J Luchansky.

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    Supplementary Results, Supplementary Figures 1–17, Supplementary Tables 1–3 and Supplementary Note.

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DOI

https://doi.org/10.1038/nchembio.1481

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