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.

From Fly Wings to Targeted Cancer Therapies: A Centennial for Notch Signaling

“…Based on these findings, H3K27me3 demethylation inhibitors might be an attractive therapy option in NOTCH1-induced T-ALL (or CLL). Finally, recent evidence suggests that it may be possible to inhibit Notch signaling by interfering with its trafficking in cancer cell secretory pathways (Ilagan and Kopan, 2013; Kramer et al, 2013). …”

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel

“…As distinct from BFA, which blocks protein transport from the ER to the Golgi and disrupts the trans-Golgi network (33), FLI-06 is a small molecule that blocks protein trafficking from the ER without disrupting the trans-Golgi network (32). As shown in Fig.…”

“…To further determine the role of recycling in the recovery of hERG channels, we used the ER export inhibitor, FLI-06 (32). As distinct from BFA, which blocks protein transport from the ER to the Golgi and disrupts the trans-Golgi network (33), FLI-06 is a small molecule that blocks protein trafficking from the ER without disrupting the trans-Golgi network (32).…”

New Approaches to Target T-ALL

“…Kramer and colleagues (43) developed a high-content screening assay to identify new regulatory proteins involved in Notch trafficking and processing in human cells. This assay relied on the generation of constitutively active, ligand-independent, NotchΔE-eGF stable cell lines in which nuclear eGFP staining was the surrogate measure of Notch activation.…”

“…The authors identified four γ-secretase modulators and the dihydropyridine FLI-06. They demonstrated that FLI-06 inhibits Notch trafficking early in the protein secretary pathway (43). Although the precise protein target of FLI-06 is yet to be determined, this finding is particularly important because new candidate targets/molecules regulating Notch trafficking are rapidly emerging in studies of Notch in other model organisms (44).…”