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Small molecules intercept Notch signaling and the early secretory pathway

Abstract

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch–enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.

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Figure 1: Chemical interference of Notch trafficking and processing is amenable to automated microscopy.
Figure 2: Selected compounds from the final hit list show distinct phenotypes.
Figure 3: Selected compounds inhibit endogenous Notch signaling.
Figure 4: FLI-06 affects the recruitment of cargo to ERESs.
Figure 5: FLI-06 induces ER sheet formation.

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Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to C.K. (KA1751/4-1), the Leibniz Gemeinschaft to A.P. and C.K. (PAKT) and the Thüringer Ministerium für Bildung, Wissenschaft und Kultur (TMBWK; 43-5572-321-12040-12) to H.-D.A. We are especially grateful to S. Radetzki, M. Neuenschwander and J. von Kries (Leibniz-Institut für Molekulare Pharmakologie Berlin) for the primary screen of the ChemBioNet library, ChemBioNet for setting up the library and B. Bulic for initial assistance with NMR data interpretation. We thank L. Bally-Cuif (Ecole des Neurosciences), C. Haass (German Center for Neurodegenerative Diseases (DZNE) München), E.L. Snapp (Albert Einstein College of Medicine), M. Kuro-o (University of Texas Southwestern), E. Sztul (University of Alabama, Birmingham) and P. Keller (European Molecular Biology Laboratory Heidelberg) for kind gifts of reagents, C. Hahn for zebrafish stock maintenance and J. Reiling for help in analyzing ER stress.

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C.K. and A.K. conceived the project and designed experiments. A.K. developed and implemented the assay and, together with T.M. and D.R. performed experiments and analyzed the data. B.K. performed in vitro budding experiments. E.R.-M. and C. Englert contributed zebrafish data. C. Enzensperger, O.W., E.T., R.N. and H.-D.A. designed, synthesized and analyzed chemical compounds. H.G. performed X-ray crystal structure analysis. A.P. provided technical support and conceptual advice. C.K., A.K. and H.-D.A. wrote the paper with help of all authors. All authors discussed the results and implications at all stages.

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Correspondence to Christoph Kaether.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–18, Supplementary Tables 1–6 and Supplementary Note (PDF 6030 kb)

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Krämer, A., Mentrup, T., Kleizen, B. et al. Small molecules intercept Notch signaling and the early secretory pathway. Nat Chem Biol 9, 731–738 (2013). https://doi.org/10.1038/nchembio.1356

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