The Notch inhibitor cowanin accelerates nicastrin degradation

Aberrant activation of Notch signaling contributes to the pathogenesis of several different types of cancer, and Notch pathway inhibitors may have significant therapeutic potential. Using a unique cell-based assay system, we isolated twelve compounds, including one new natural product from Garcinia speciosa, that inhibit the Notch signaling pathway. HES1 and HES5 are target genes of the Notch cascade, and compound 2, referred to as cowanin, decreased the protein levels of HES1 and HES5 in assay cells. Furthermore, cowanin (2) showed potent cytotoxicity against human leukemic HPB-ALL cells. The Notch signaling inhibitory activity of cowanin (2) is linked to the increased degradation of nicastrin, which is one of the components of the γ-secretase complex. To the best of our knowledge, this is the first example of a compound with Notch pathway inhibitory activity mediated by nicastrin degradation.

Notch signaling plays a pivotal role in the regulation of many fundamental cellular processes, including proliferation and stem cell maintenance. It also influences cell differentiation decisions at various stages during embryonic and adult development 1,2 . It is apparent that an important direct output of Notch activity is the upregulation of basic helix-loop-helix (bHLH) transcriptional repressors such as the hairy and enhancer of split 1 (HES1), HES5 and HES related (HESR/HEY) family genes. Notch signaling has an important role in the regulation of neural stem cell differentiation 3,4 , and the differentiation of neural stem cells into neurons can be inhibited by Notch signaling. In direct contrast, the Notch pathway also promotes astrocyte differentiation from glial progenitors derived from neural stem cells. Aberrant upregulation of Notch signaling is oncogenic in multiple hematologic and solid malignancies [5][6][7] . For example, activating mutations of NOTCH1, a transmembrane receptor that regulates normal T cell development, drives the development of human T-cell acute lymphoblastic leukemia (T-ALL) 8 . Notch has been proven to be an oncoprotein in melanocytes 9 , prostate 10 , and breast 11 cancer cells, providing significant impetus for the development of clinically effective Notch pathway inhibitors 12 .
Signal transduction from Notch receptors is shown in Fig. 1. Notch signaling is activated by interaction between the ligand-expressing cell and the signal-receiving cell. The binding of Jagged or Delta ligand proteins to the Notch receptor induces a conformational change, exposing the S2 cleavage site in the Notch receptor to the metalloproteinase containing protein, ADAM/TACE. Following S2 cleavage, the subsequent S3 cleavage is mediated by γ-secretase, which is a multi-subunit complex composed of presenilin 1 (PS1), nicastrin, presenilin enhancer 2 (PEN-2) and anterior pharynx-defective 1 (APH-1). The S3 cleavage results in the release of the Notch intracellular domain (NICD), which translocates to the nucleus and forms a heterotrimer with mastermind-like protein (MAML) and DNA binding protein recombination signal binding protein for immunoglobulin kappa J region (RBP-J) to initiate transcription of target genes.
Various Notch inhibitors have been reported including γ-secretase inhibitors, DAPT (N-[N-(3,5-difluorop henacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) 13 , RO4929097 14 , MK-0752 15 and PF0384014 16 . SHAM1, a MAML1-derived peptide that inhibits the binding of full-length MAML1 to NICD1-RBP-J, was also reported as a potent Notch signaling inhibitor 17 . The natural products, curcumin 18 and genistein 19 , downregulate Notch1. Butein was also reported as a potent inhibitor 20 . A peptide-based protein knockdown system was also developed for degradation of Notch1 21 . Efforts have been taken to develop Notch signaling inhibitors as agents with clinically used chemotherapeutics to treat cancer 12 . However, the development of Notch inhibitors from natural products has been limited despite the increasing realization that natural products are a valuable repository of Figure 1. The Notch signaling pathway. Interaction between Notch and Delta or Jagged that exist on the surface of signal-sending cells triggers the cleavage of Notch protein by ADAM/TACE metalloproteases and γ-secretase to produce NICD. NICD moves to the nucleus to forms a heterotrimer with RBP-J and MAML to enhance transcription of target genes, such as HES1 and HES5. NICD: Notch intracellular domain, MAML: mastermindlike protein (co-activator), HES1: hairy and enhancer of split 1, HES5: hairy and enhancer of split 5.
Next, we investigated the Notch signal inhibitory activity of the twelve compounds (Fig. 5). Compounds 1, 2, 3 and 7 showed potent inhibitory activity with IC 50 values of less than 10 μM (8.0, 2.4, 7.3 and 7.5 μM, respectively). Compounds 4, 9, 10 and 11 had moderate activity with IC 50 values of 10.5, 19.6, 15.0 and 11.7 μM, respectively. The remaining compounds, 5, 6, 8 and 12, had weak activity or were inactive. The Structure-Activity Relationship (SAR) of isolated Notch inhibitors indicated that two prenyl moieties at C-2 and C-8 strengthened activity. From the comparison of the activities of compounds 1 and 10, the OH group at the C-6 position was preferred over the methoxy group. The methoxy group at the C-7 position seemed to be preferred for activity. In addition, the ring portions at C-2 and C-3 weakened activity based on comparisons of 1 with 9 or 12, and 2 with 11.  We focused on compound 2, referred to as cowanin, which had the strongest inhibitory activity. The expression of the Notch signaling target proteins, HES1 and HES5, was reduced by the addition of cowanin (2) to the assay cells ( Fig. 6A,C,D). NICD was reduced dose dependently ( Fig. 6B,F), while the expression of Notch1(1704-2531) was increased (Fig. 6B,E). This result indicated that cowanin (2) might affect the activity of the γ-secretase complex.
To investigate the effect of cowanin (2) on cancer cells, the cytotoxicity of the compound on the T-ALL cell line, HPB-ALL, was evaluated ( Fig. 7A). Cowanin (2) reduced the viability of HPB-ALL cells at 10 μM, while the viability of normal human embryonic kidney 293 cells was not affected, although cytotoxicity was detected at 20 μM. Cowanin (2) showed stronger cytotoxicity than that of the known γ-secretase inhibitor DAPT (Fig. 7A). Although IC 50 of Notch signal inhibitory activity of DAPT in our luciferase assay system was 0.12 μM, cowanin (2) showed high efficient cytotoxicity against cancer cells HPB-ALL than that of DAPT (IC 50 >20 μM) (Fig. 7A). The IC 50 values of cowanin (2) were 7.5 μM (HPB-ALL cells) and 15.1 μM (293 cells) (Fig. 7B). This difference of cytotoxicity between cancer cells and normal cells is also attractive. The expression of NICD and HES1 was reduced in HPB-ALL cells at 10 μM significantly ( Fig. 7C-E).
The effects of cowanin (2) on the expression of components of the γ-secretase complex (Fig. 8A), nicastrin (Fig. 8B), APH-1, PS1 and PEN-2 ( Fig. 8C) were evaluated to address the mechanisms by which cowanin (2) inhibits γ-secretase activity. It was known that the activity site exists in PS1 but the complexation of these four proteins is important for activity 33 . Although cowanin (2) didn't affect the expression of APH-1, PS1 and PEN-2, nicastrin levels were reduced (Fig. 8B). To address the possibility that cowanin (2) accelerates the degradation of nicastrin, a proteasome inhibitor, MG132, was co-incubated with cowanin (2) (Fig. 8D-F). We confirmed that the cell viability was 98% under 12 h treatment of cowanin (2) (15 μM). When MG132 (5 μM) was added to HPB-ALL cells with cowanin (2) (15 μM), the decrease in nicastrin expression was not so marked. These data indicated that the mechanism of Notch inhibition by cowanin (2) was acceleration of nicastrin degradation. As Fig. 8F showed, cowanin (2) decreased immature nicastrin stronger than mature nicastrin. Nicastrin was known to be matured by glycosylation of a large extracellular domain. And PS1 interacts preferentially with mature nicastrin 34 . Therefore, hydrophobic compound, cowanin (2), might prefer to bind immature extracellular domain without glycans. By cowanin (2) binding, PS1 binding to nicastrin might be reduced. That might cause to increase the amount of unstable free nicastrin, then degradation of nicastrin might be accelerated.

Conclusion
In conclusion, we report the isolation of twelve compounds, including one new natural product, using our cell-based assay for Notch signal inhibitors. Cowanin (2) was shown to inhibit γ-secretase activity by reducing nicastrin levels, which was the result of the acceleration of nicastrin degradation. To the best of our knowledge, this is the first example of a compound that accelerates nicastrin degradation and shows Notch inhibitory activity.

General Experimental Procedures.
Optical rotations were recorded on a JASCO P-1020 polarimeter.
HPB-ALL cells were incubated in a 6 cm dish in 5 mL of RPMI-1640 medium with 10% FBS, Streptomycin (100 μg/mL) and Penicillin (100 unit/mL) at 5 × 10 5 cells per dish (1 × 10 6 cells per dish, when treatment with MG132). Media also contained different concentrations of compound. Cells were incubated at 37 °C for 72 h (12 h, when treatment with MG132). After incubation, cells were centrifuged and collected, and then washed with 500 μL of PBS twice. To prepare cell lysate and confirm protein expression levels, the same method as described above was used.