© 2013 Nature America, Inc. All rights reserved. NATURE CHEMICAL BIOLOGY | ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology 1 ARTICLE PUBLISHED ONLINE: 29 SEPTEMBER 2013 | DOI: 10.1038/NCHEMBIO.1356 N otch is an important regulator of gene transcription and is involved in cellular differentiation, lateral inhibition and tissue homeostasis (reviewed in ref. 1). After interaction with its ligand from the Delta and Serrate (also called Jagged) family, it is subsequently processed by ADAM10 or ADAM17 and by γ-secretase to generate the Notch intracellular domain (NICD). The NICD translocates to the nucleus, where it activates a transcription factor of the CSL family (named for CBF1 (also known as RBP-J) in vertebrates, Su(H) in Drosophila and Lag-1 in Caenorhabditis elegans). One characteristic and important hallmark of the Notch signaling pathway is its independence from second messengers. Notch signaling can be fine tuned at several steps ranging from glycosylation at the ectodomain, interaction with inhibitors such as Numb, ubiquitinylation and phosphorylation of NICD to slight variations at the γ-secretase cleavage site (reviewed in ref. 1). Fine tuning can regulate trafficking of the receptor, ligand binding affin- ity, the endocytosis rate of the receptor and the stability of the NICD 1,2 . Despite the wealth of information about Notch signaling, its trafficking and regulation of signaling are not fully understood. Currently, therapeutic interventions for aberrant Notch signaling, for example, in T cell–lineage acute lymphoblastic leukemia, are aimed and tested at the level of ligand binding and γ-secretase cleav- age, but new strategies are highly desirable 3,4 . Natural products such as brefeldin A (BFA), shiga toxin or cholera toxin have been invaluable tools in dissecting the molecular details of membrane trafficking at various steps in the exocytic or endo- cytic pathway 5 . Similarly, γ-secretase research could not have come this far without the identification of γ-secretase inhibitors (GSIs). GSIs have been important not only in inhibiting γ-secretase but also in purifying it, elucidating structure-function relations within the γ-secretase complex and identifying substrate-docking sites 6 . High-content screening (HCS) describes the process of auto- mated image acquisition of phenotypes of cells or organisms and the subsequent automated analysis of these phenotypes by image analysis algorithms without, or with only little, user intervention, allowing for high-throughput applications such as genome-wide RNA interference or screening large chemical-compound libraries (reviewed in ref. 7). Here we describe the development and success- ful application of a HCS microscope-based assay to identify new small molecules that are involved in the intracellular trafficking and processing of the Notch signaling pathway. Molecular charac- terization of selected compounds identified four new inhibitors of γ-secretase and a new inhibitor of ER export. The latter compound has the unique property to inhibit cargo recruitment to ER exit sites (ERESs). RESULTS Establishment of a microscope-based HCS To identify new regulatory factors that are involved in Notch traffick- ing and processing, we set up an image-based HCS. To this end, we employed a HeLa Kyoto cell line stably expressing an eGFP-tagged, transcriptionally inactive and ligand-independent Notch1 construct (NotchΔE-eGFP 8 ; Fig. 1a). NotchΔE-eGFP is a direct substrate for γ-secretase at the plasma membrane (PM) and under physiological conditions is proteolytically processed to NICD-eGFP (Fig. 1a), resulting in strong nuclear eGFP staining in the steady state 8 (Supplementary Results, Supplementary Fig. 1a). After inhibition of γ-secretase with the GSI DAPT, the reporter accumulated at the PM, and nuclear fluorescence decreased (Supplementary Fig. 1a). Notably, changes in the subcellular localization of the reporter are amenable to quantification by automated microscopy 8 . Accordingly, we determined reporter fluorescence in the nucleus (nuc) and in a ring around the nucleus (enuc), and we evaluated the enuc-to-nuc fluorescence ratio (Fig. 1b). DAPT-induced accumulation of the reporter outside the nucleus was concentration dependent, with an effector concentration for a half-maximum response (EC 50 ) of 0.68 ± 0.2 μM (mean ± s.d.) (18 h incubation; Supplementary Fig. 1b). NICD-eGFP fluorescence in the nucleus decreased with a t 1/2 of 1 Leibniz Institut für Altersforschung–Fritz Lipmann Institut, Jena, Germany. 2 Department of Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands. 3 Institut für Pharmazie, Friedrich-Schiller Universität, Jena, Germany. 4 Institut für Organische und Makromolekulare Chemie, Friedrich-Schiller Universität, Jena, Germany. 5 Institut für Anorganische und Analytische Chemie, Friedrich-Schiller Universität, Jena, Germany. 6 Present address: Alere Technologies, Jena, Germany. *e-mail: ckaether@fli-leibniz.de Small molecules intercept Notch signaling and the early secretory pathway Andreas Krämer 1 , Torben Mentrup 1 , Bertrand Kleizen 2 , Eric Rivera-Milla 1,6 , Daniela Reichenbach 1 , Christoph Enzensperger 3 , Richard Nohl 4 , Eric Täuscher 4 , Helmar Görls 5 , Aspasia Ploubidou 1 , Christoph Englert 1 , Oliver Werz 3 , Hans-Dieter Arndt 4 & Christoph Kaether 1 * 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 g-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.