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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References
Kopan, R. & Ilagan, M.X. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216–233 (2009).
Le Borgne, R. Regulation of Notch signalling by endocytosis and endosomal sorting. Curr. Opin. Cell Biol. 18, 213–222 (2006).
Real, P.J. & Ferrando, A.A. NOTCH inhibition and glucocorticoid therapy in T-cell acute lymphoblastic leukemia. Leukemia 23, 1374–1377 (2009).
Koch, U. & Radtke, F. Notch in T-ALL: new players in a complex disease. Trends Immunol. 32, 434–442 (2011).
von Kleist, L. & Haucke, V. At the crossroads of chemistry and cell biology: inhibiting membrane traffic by small molecules. Traffic 13, 495–504 (2012).
Wolfe, M.S. γ-secretase inhibitors and modulators for Alzheimer's disease. J. Neurochem. 120 (suppl. 1), 89–98 (2012).
Zanella, F., Lorens, J.B. & Link, W. High content screening: seeing is believing. Trends Biotechnol. 28, 237–245 (2010).
Huenniger, K. et al. Notch1 signaling is mediated by importins α3, 4, and 7. Cell. Mol. Life Sci. 67, 3187–3196 (2010).
Lisurek, M. et al. Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept. Mol. Divers. 14, 401–408 (2010).
McCarthy, J.V., Twomey, C. & Wujek, P. Presenilin-dependent regulated intramembrane proteolysis and γ-secretase activity. Cell. Mol. Life Sci. 66, 1534–1555 (2009).
Bloch, L. et al. Klotho is a substrate for α-, β- and γ-secretase. FEBS Lett. 583, 3221–3224 (2009).
Kopan, R., Nye, J.S. & Weintraub, H. The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development 120, 2385–2396 (1994).
Geling, A., Steiner, H., Willem, M., Bally-Cuif, L. & Haass, C.A. γ-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 3, 688–694 (2002).
Kitzmann, M. et al. Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells. J. Cell. Physiol. 208, 538–548 (2006).
Blader, P. & Strahle, U. Zebrafish developmental genetics and central nervous system development. Hum. Mol. Genet. 9, 945–951 (2000).
Blader, P., Fischer, N., Gradwohl, G., Guillemot, F. & Strahle, U. The activity of neurogenin1 is controlled by local cues in the zebrafish embryo. Development 124, 4557–4569 (1997).
Pavelka, M. & Ellinger, A. Effect of colchicine on the Golgi complex of rat pancreatic acinar cells. J. Cell Biol. 97, 737–748 (1983).
Lippincott-Schwartz, J., Yuan, L.C., Bonifacino, J.S. & Klausner, R.D. Rapid redistribution of Golgi proteins into the ER in cells treated with Brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56, 801–813 (1989).
Sáenz, J.B. et al. Golgicide A reveals essential roles for GBF1 in Golgi assembly and function. Nat. Chem. Biol. 5, 157–165 (2009).
Rothman, J.E. Mechanisms of intracellular protein transport. Nature 372, 55–63 (1994).
Donaldson, J.G., Cassel, D., Kahn, R.A. & Klausner, R.D. ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein β-COP to Golgi membranes. Proc. Natl. Acad. Sci. USA 89, 6408–6412 (1992).
Helms, J.B. & Rothman, J.E. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature 360, 352–354 (1992).
Szul, T. et al. Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1. Traffic 6, 374–385 (2005).
Wang, Y. et al. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J. Biol. Chem. 275, 27013–27020 (2000).
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891 (2001).
Harding, H.P., Zhang, Y. & Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum–resident kinase. Nature 397, 271–274 (1999).
Toomre, D., Keller, P., White, J., Olivo, J.C. & Simons, K. Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells. J. Cell Sci. 112, 21–33 (1999).
Presley, J.F. et al. ER-to-Golgi transport visualized in living cells. Nature 389, 81–85 (1997).
Dukhovny, A., Papadopulos, A. & Hirschberg, K. Quantitative live-cell analysis of microtubule-uncoupled cargo-protein sorting in the ER. J. Cell Sci. 121, 865–876 (2008).
Kim, J. et al. Biogenesis of γ-secretase early in the secretory pathway. J. Cell Biol. 179, 951–963 (2007).
Lee, T.H. & Linstedt, A.D. Potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-Golgi transport revealed by protein kinase inhibitor H89. Mol. Biol. Cell 11, 2577–2590 (2000).
Snapp, E.L., Sharma, A., Lippincott-Schwartz, J. & Hegde, R.S. Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells. Proc. Natl. Acad. Sci. USA 103, 6536–6541 (2006).
Imbimbo, B.P. & Giardina, G.A. γ-secretase inhibitors and modulators for the treatment of Alzheimer's disease: disappointments and hopes. Curr. Top. Med. Chem. 11, 1555–1570 (2011).
Groth, C. & Fortini, M.E. Therapeutic approaches to modulating Notch signaling: current challenges and future prospects. Semin. Cell Dev. Biol. 23, 465–472 (2012).
Goldmann, S. & Stoltefuss, J. 1,4-dihydropyridines: effects of chirality and conformation on the calcium antagonist and calcium agonist activities. Angew. Chem. Int. Edn Engl. 30, 1559–1578 (1991).
Edraki, N., Mehdipour, A.R., Khoshneviszadeh, M. & Miri, R. Dihydropyridines: evaluation of their current and future pharmacological applications. Drug Discov. Today 14, 1058–1066 (2009).
Meredith, P.A. & Elliott, H.L. Dihydropyridine calcium channel blockers: basic pharmacological similarities but fundamental therapeutic differences. J. Hypertens. 22, 1641–1648 (2004).
Zanetti, G., Pahuja, K.B., Studer, S., Shim, S. & Schekman, R. COPII and the regulation of protein sorting in mammals. Nat. Cell Biol. 14, 20–28 (2011); erratum 14, 221 (2012).
Klopfenstein, D.R., Kappeler, F. & Hauri, H.P. A novel direct interaction of endoplasmic reticulum with microtubules. EMBO J. 17, 6168–6177 (1998).
Shibata, Y., Hu, J., Kozlov, M.M. & Rapoport, T.A. Mechanisms shaping the membranes of cellular organelles. Annu. Rev. Cell Dev. Biol. 25, 329–354 (2009).
Rogalski, A.A., Bergman, J.E. & Singer, S.J. Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane. J. Cell Biol. 99, 1101–1109 (1984).
Cole, N.B., Sciaky, N., Marotta, A., Song, J. & Lippincott-Schwartz, J. Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol. Biol. Cell 7, 631–650 (1996).
Kirk, S.J. & Ward, T.H. COPII under the microscope. Semin. Cell Dev. Biol. 18, 435–447 (2007).
Malo, N., Hanley, J.A., Cerquozzi, S., Pelletier, J. & Nadon, R. Statistical practice in high-throughput screening data analysis. Nat. Biotechnol. 24, 167–175 (2006).
Zhang, J.H., Chung, T.D. & Oldenburg, K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).
Ritz, C. & Streibig, J.C. Bioassay analysis using R. J. Stat. Softw. 12 (2005).
Sastre, M. et al. Presenilin-dependent γ-secretase processing of β-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch. EMBO Rep. 2, 835–841 (2001).
Steiner, H. et al. Glycine 384 is required for presenilin-1 function and is conserved in polytopic bacterial aspartyl proteases. Nat. Cell Biol. 2, 848–851 (2000).
Steiner, H. et al. PEN-2 is an integral component of the γ-secretase complex required for coordinated expression of presenilin and nicastrin. J. Biol. Chem. 277, 39062–39065 (2002).
Wacker, I. et al. Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein. J. Cell Sci. 110, 1453–1463 (1997).
Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. & Schilling, T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995).
Thisse, C. & Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69 (2008).
Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method. Methods 25, 402–408 (2001).
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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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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DOI: https://doi.org/10.1038/nchembio.1356
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