β-catenin/TCF mediated transcription; β-catenin/CBP interaction; CBP (IC50 = 3 μM)

In MCF7 cells, leptin-induced EMT, invasion, and tumor sphere formation are inhibited by ICG-001 (5 μM) [1]. In presenilin-1 mutant cells, ICG-001 can phenotypically rescue normal nerve growth factor (NGF)-induced neuronal differentiation and neuronal outgrowth, highlighting the significance of neuronal differentiation and the function of the TCF/β-catenin signaling pathway in neurite outgrowth [2]. In SW480 cells, the steady-state levels of survivin and cyclin D1 RNA and protein were decreased by ICG-001 (25 μM) treatment; these two proteins are increased by β-catenin. ICG-001 inhibits the development of colon cancer cells in vitro and specifically triggers apoptosis in transformed cells but not in normal colon cells [3].

In mice, ICG-001 (5 mg/kg daily) strongly suppresses β-catenin signaling while preserving epithelial cells [2]. The development of polyps in the colon and small intestine was reduced by 42% after 9 weeks of administration of a water-soluble version of ICG-001. This impact was comparable to that of the nonsteroidal anti-inflammatory medication MK-231, which has repeatedly demonstrated efficacy in this model. In the SW620 nude mouse tumor regression xenograft model, ICG-001 (150 mg/kg, i.v.) caused a notable reduction in tumor volume over the course of 19 days of therapy without causing death or weight loss [3].

Coimmunoprecipitations [4]
For β-catenin-CBP/P300/E-cadherin/N-cadherin coimmunoprecipitations, MGC-803 cells were incubated 48 h with or without ICG-001 and 100 μg protein extract was diluted to 1 mL in coimmunoprecipitation (Co-IP) buffer. 2 μg of β-catenin antibody was added to the protein samples and the mix were incubated overnight at 4 °C with rotation. 20 μL of 50% protein A/G-agarose bead slurry (equilibrated in Co-IP buffer) were added, and after 2 h–incubation at 4 °C, the beads were washed 4 times with Co-IP buffer (1 ml per wash) and diluted with 1× loading buffer. Western blotting was performed, and CBP, P300, E-cadherin, N-cadherin were detected.

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Affinity Purification.[3]
Cells were lysed in protein-binding buffer [PBB, 20 mM Hepes, pH 7.9/100 mM NaCl/0.5 mM EDTA/0.5% Nonidet P-40/6 mM MgCl2/5 mM 2-mercaptoethanol/one tablet of Complete protease inhibitor mixture]. Biotinylated ICG-002 was bound overnight at room temperature to a 50% slurry of streptavidin-agarose beads in buffer containing 50% DMSO and 50% PBB. Beads were washed to remove unbound ICG-002 and then incubated with whole-cell lysates. Proteins eluted, either specifically with 100 μM ICG-001 or by boiling in SDS, were immunoblotted and silver stained.

RLE-6TN Cell qPCR Studies. [3]
To evaluate effects of ICG-001 on α-SMA and collagen type 1 expression, RLE-6TN cells were treated with TGF-β1 (0.25 ng/mL) in the presence or absence of ICG-001 (5.0 μM). After 24 h, cells were harvested and mRNA isolated for analysis by qPCR. RNA was reverse-transcribed using SuperScript reverse transcriptase. Quantitative PCR was performed with SYBR-Green PCR using Real-Time PCR System HT7900. The amplification protocol was set as follows: 95 °C denaturation for 10 min followed by 40 cycles of 15-s denaturation at 95 °C, 1 min of annealing/extension, and data collection at 60 °C. Primer pairs used are as following: α-SMA forward 5′-ATGGCTCCGGGCTCTGTAA-3′ and reverse 5′-ACAGCCCTGGGAGCATCA-3′; collagen 1α forward 5′-TTGACCCTAACCAAGGATGC-3′ and reverse 5′-CACCCCTTCTGCGTTGTATT-3′. IPF Lung Fibroblasts. [3]
Primary fibroblast cultures were derived from lung tissue from a patient with IPF undergoing transplant surgery, following informed consent and ethics approval from relevant institutions, as previously described. One cell line (CCL-134) established from a patient with IPF was obtained from ATCC. The IPF fibroblasts were treated with ICG-001(5 μM) or DMSO control for 48 h, after which mRNA was isolated for analysis by qPCR.

Bleomycin-Induced Pulmonary Fibrosis in Mice.[2]
All animal use procedures were approved by the University of Washington Animal Care Committee. In prevention of pulmonary fibrosis studies, C57BL/6J mice and BAT-gal transgenic mice that express the Escherichia coli lacZ gene encoding β-galactosidase under the control of β-catenin/TCF responsive elements produced from B6D2F1 mice had s.c. placement of miniosmotic pumps (200 μL Alzet Model 2001 osmotic pumps, 1.0 ± 0.15 μL/h delivery rate; Durect Corporation) containing ICG-001 (5 mg/kg per day in saline), dexamethasone-water soluble (1 mg/kg per day in saline), or saline control on day 0. Twenty-four hours later (day 1), 0.08 units bleomycin in 50 μL of saline was administered intranasally. Control groups received 50 μL of saline intranasally on day 1. Mice were killed on day 10. In intervention studies during the inflammatory and early fibrogenic response period, C57BL/6J mice received bleomycin (0.08 units) or saline intranasally on day 0 followed on day 5 by twice daily oral treatment with pirfenidone [5-methyl-1-phenyl-2-(IH)-pyridone, 400 mg/kg/d in 0.5% carboxymethylcellulose], or miniosmotic pump infusion of ICG-001 (5 mg/kg per day) or saline control for a 10-d treatment period. In long-term reversal of established fibrosis studies, groups of mice administered bleomycin (0.08 units intranasally) on day 0, had implantation on day 21 of miniosmotic pumps containing either ICG-001 (5 mg/kg per day) or saline, and killed on day 42. Lungs were obtained for histopathology, qPCR, and collagen assay.

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Min Mouse Model. [3]
Seven-week-old male C57BL/6J-ApcMin/+ and WT C57BL/6J mice were treated orally for 9 weeks with ICG-001 (300 mg/kg per day) or vehicle (1% carboxymethylcellulose), once daily, six times per week. Sulindac was administered in drinking water (160 ppm, dissolved in 8 mM Na2PO4 buffer, pH 7.6). At 16 weeks, the polyp number was counted manually by using a dissecting microscope.

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In vivo xenograft and treatment experiments [4]
For in vivo studies, 4–6 week old male nude mice were used. MGC-803 cells (2 × 106 cells in 200 μl PBS) were subcutaneously injected into right flank of the nude mice to establish tumors. After 7 days, 50 mg/kg/day ICG-001 was delivered into the tumors. PBS was used as the control. Treatment continued for a total of 4 weeks. The tumor size was measured using digital caliper and tumor volume was calculated with the following formula: volume = 0.5 × width2 × length.

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Dissolved in water; 300 mg/kg; oral administration

Seven-week-old male C57BL/6J-Apc Min/+

[1]. Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires β-catenin activation via Akt/GSK3- and MTA1/Wnt1 protein-dependent pathways. J Biol Chem, 2012, 287(11), 8598-8612.

[2]. Henderson WR Jr, et al, Inhibition of Wnt/beta-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis. Proc Natl Acad Sci USA, 2010, 107(32), 14309-14314.

[3]. A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected]. Proc Natl Acad Sci USA, 2004, 101(34), 12682-12687.

[4]. ICG-001 suppresses growth of gastric cancer cells and reduces chemoresistance of cancer stem cell-like population. J Exp Clin Cancer Res. 2017 Sep 11;36(1):125.

(6S,9aS)-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-3,6,9,9a-tetrahydro-2H-pyrazino[1,2-a]pyrimidine-1-carboxamide is a peptide.

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Perturbations in the adipocytokine profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1), and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with the small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion, and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of the GSK3β-LKB1-Axin complex that leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of the Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation, and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors shows increased expression of Wnt1, pGSK3β, and vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized cross-talk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.[2]

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Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a ravaging condition of progressive lung scarring and destruction. Anti-inflammatory therapies including corticosteroids have limited efficacy in this ultimately fatal disorder. An important unmet need is to identify new agents that interact with key molecular pathways involved in the pathogenesis of pulmonary fibrosis to prevent progression or reverse fibrosis in these patients. Because aberrant activation of the Wnt/beta-catenin signaling cascade occurs in lungs of patients with IPF, we have targeted this pathway for intervention in pulmonary fibrosis using ICG-001, a small molecule that specifically inhibits T-cell factor/beta-catenin transcription in a cyclic AMP response-element binding protein binding protein (CBP)-dependent fashion. ICG-001 selectively blocks the beta-catenin/CBP interaction without interfering with the beta-catenin/p300 interaction. We report here that ICG-001 (5 mg/kg per day) significantly inhibits beta-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently preserving the epithelium. Administration of ICG-001 concurrent with bleomycin prevents fibrosis, and late administration is able to reverse established fibrosis and significantly improve survival. Because no effective treatment for IPF exists, selective inhibition of Wnt/beta-catenin-dependent transcription suggests a potential unique therapeutic approach for pulmonary fibrosis.[2]

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Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.[3]

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Background: ICG-001, a small molecule, binds CREB-binding protein (CBP) to disrupt its interaction with β-catenin and inhibits CBP function as a co-activator of Wnt/β-catenin-mediated transcription. Given its ability to inhibit Wnt/β-catenin signaling pathway, ICG-001 has been used in some tumor types to exert its anticarcinogenic effect. Here, we examined ICG-001 and its potential role as a therapeutic in gastric cancer (GC).
Methods: The gastric cancer cell lines SGC-7901, MGC-803, BGC-823 and MKN-45 were used in vitro and in vivo. The abilities of cell proliferation, tumor sphere formation, metastasis, tumorgenesis and chemoresistance to chemotherapy drugs in vitro were evaluated by MTT assay, colony formation assay, flow cytometry, migration and invasion assay, and tumor spheres culture. The in vivo experiments were performed using a subcutaneous transplantation tumor model in athymic nude mice. Alterations at RNA and protein levels were followed by qRT-PCR, western blot, coimmunoprecipitations and immunofluorescence assay.
Results: In this study, we showed that ICG-001 significantly inhibited growth and metastasis of multiple GC cell lines, induced cell apoptosis, and augmented in vitro tumor spheres suppression when used in combination with chemotherapy drugs probably through robustly blocking association of β-catenin with CBP and N-cadherin, but promoting association of β-catenin with P300 and E-cadherin, instead of altering the distribution and expression of β-catenin.
Conclusions: Our findings suggest that ICG-001 suppresses GC cell line growth, metastasis and reduces its stem cell-like properties and chemoresistance, indicating that ICG-001 is a potentially useful small molecule therapeutic for GC.
Keywords: Gastric cancer; Growth; ICG-001; Stem cell-like; Wnt/β-catenin signaling pathway.[4]

C33H32N4O4

548.63

548.242

C, 72.24; H, 5.88; N, 10.21; O, 11.66

780757-88-2

1422253-38-0 (PRI-724);847591-62-2 (deleted);780757-88-2 (ICG001);

11238147

White to off-white solid powder

1.4±0.1 g/cm3

895.6±65.0 °C at 760 mmHg

133-134ºC

495.4±34.3 °C

0.0±0.3 mmHg at 25°C

1.722

4.01

2

4

6

41

930

2

C1CN([C@H]2CN(C(=O)[C@@H](N2C1=O)CC3=CC=C(C=C3)O)CC4=CC=CC5=CC=CC=C54)C(=O)NCC6=CC=CC=C6

HQWTUOLCGKIECB-IHLOFXLRSA-N

InChI=1S/C33H32N4O4/c38-27-15-13-23(14-16-27)19-29-32(40)35(21-26-11-6-10-25-9-4-5-12-28(25)26)22-30-36(18-17-31(39)37(29)30)33(41)34-20-24-7-2-1-3-8-24/h1-16,29-30,38H,17-22H2,(H,34,41)/t29-,30+/m1/s1

rel-(6R,9aR)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide

ICG-001; ICG 001; ICG-001; 847591-62-2; 780757-88-2; (S,S)-ICG 001; (6S,9aS)-N-benzyl-6-(4-hydroxybenzyl)-8-(naphthalen-1-ylmethyl)-4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxamide; (6S,9aS)-6-(4-hydroxybenzyl)-N-benzyl-8-(naphthalen-1-ylmethyl)-4,7-dioxo-hexahydro-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide; ICG001

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.56 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1.67 mg/mL (3.04 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 1.67 mg/mL (3.04 mM) in 17% Polyethylene glycol 12-hydroxystearate in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)