CBP/β-catenin interaction; β-catenin/CREB-binding protein (CBP) interaction (IC50 = 45 nM for disrupting the interaction) [3]

In vitro activity: PRI-724 binds specifically to CBP but not the related transcriptional coactivator p300, thereby disrupting the interaction of CBP with β-catenin. Treatment with PRI-724 selectively induces apoptosis in colon carcinoma cells but not in normal colonic epithelial cells and reduces in vitro growth of colon carcinoma cells.

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Kinase Assay: The Dual-Luciferase Reporter (DLR) Assay System provides an efficient means of performing dual reporter assays. In the DLRTM Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a “glow-type” luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated by simultaneously adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a “glow-type” signal from the Renilla luciferase, which decays slowly over the course of the measurement. In the DLRTM Assay System, both reporters yield linear assays with subattomole (<10-18) sensitivities and no endogenous activity of either reporter in the experimental host cells. Furthermore, the integrated format of the DLRTM Assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions.

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Cell Assay: The rat EMCs were treated with either ICG-001 or IQ1 and performed co-immunoprecipitation (co-IP) assays. Cells were treated with DMSO, ICG-001 or IQ1 for 24 hours. In the DMSO control treated cells, essentially all of the β-catenin was associated with CBP. Treatment with IQ1 had minimal effects on β-catenin coactivator usage. However, as anticipated, treatment with ICG-001 decreased the β-catenin/CBP interaction, while concomitantly increasing the β-catenin/p300 interaction.

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- PRI-724 selectively inhibits the interaction between β-catenin and CBP, with an IC50 of 45 nM, without affecting the interaction between β-catenin and p300. In colon cancer cell lines (HCT116, SW480) with activated Wnt/β-catenin signaling, it reduces the expression of Wnt target genes (c-Myc, cyclin D1) at both mRNA and protein levels, as detected by qPCR and Western blot. This leads to decreased cell proliferation, as shown by reduced BrdU incorporation and colony formation [3]
- In colon cancer cells (HCT116), PRI-724 (10 μM) increases the expression of programmed cell death ligand 1 (PD-L1) on the cell surface, as measured by flow cytometry. This upregulation is associated with increased nuclear factor-κB (NF-κB) activity, as indicated by luciferase reporter assay [1]

PRI-724 exhibits antitumor activity in the mouse xenograft models of colon cancer. The initial results of the Phase I clinic trial of PRI-724 has been disclosed publically. The drug exhibits an acceptable toxicity profile with only one dose-limiting toxicity of grade 3 reversible hyperbilirubinaemia. An Open-Label dose-escalation phase I/II study of PRI-724 for patients with advanced myeloid malignancies is still ongoing.

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PRI-724 treatment reduced mRNA expression of β-catenin target genes in SL4-inoculated livers.[1]
PRI-724 treatment reduced mRNA expression of β-catenin target genes in SL4-inoculated livers.[1]
PRI-724 increased T-lymphocyte infiltration into metastatic liver tumors.[1]
CD8+ T-cells were required for the anti-tumor effect of combined anti-PD-L1 Ab and PRI-724 treatment.[1]

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- In a mouse model of metastatic colon cancer established by intrasplenic injection of HCT116 cells, administration of PRI-724 (30 mg/kg, intraperitoneal, twice daily) for 21 days reduces liver metastatic burden, as shown by decreased number and size of metastatic nodules. However, it also increases PD-L1 expression in liver metastases, as detected by immunohistochemistry. Combination with anti-PD-L1 antibody further reduces metastatic growth compared to PRI-724 alone [1]
- In a mouse model of embryonic lung development, PRI-724 (administered via maternal injection) inhibits β-catenin/CBP interaction in the lung epithelium, leading to proximalization of the epithelium with increased bronchial markers (Sox2) and decreased alveolar markers (Sox9). This effect is mediated by reduced Wnt target gene expression in the developing lung [2]

Measurement of serum cytokines and chemokines [1]
Serum ALT levels were measured using a Wako Transaminase CII-test Kit. Serum cytokines and chemokines were measured using a Luminex MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay. This procedure was performed in accordance with the manufacturer’s instructions.

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β-catenin/CBP interaction assay: Recombinant β-catenin and CBP proteins were incubated with PRI-724 (0.1-1000 nM) and a fluorescently labeled peptide corresponding to the β-catenin-binding region of CBP. The extent of interaction was measured by fluorescence polarization, and IC50 was calculated based on the concentration required to reduce 50% of the interaction [3]

Isolation of mouse IHLs [1]
Single-cell suspensions were prepared from the liver median lobe by digesting the tissue in RPMI-1640 containing 0.02% collagenase IV and 0.002% DNase I for 40 min at 37°C. The cells were overlaid onto Lympholyte-M in PBS. After density separation, the isolated IHLs were evaluated by FACS analysis.

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FACS analysis [1]
The cells were surface-stained with fluorochrome-conjugated Abs for 20 min on ice using the following Abs: anti-CD3, anti-CD4, anti-CD8, anti-NK1.1, anti-CD69, and anti-Foxp3. To perform intracellular cytokine staining, IHLs were co-cultured with SL4 cells (1 × 105 cells/well) for 4 h at 37°C in 96-well round-bottom plates containing 200 μL/well RPMI-1640 medium. Mouse recombinant IL-2 (50 units) and 0.2 μL of BD GolgiPlug protein transport inhibitor were added to each well. After incubation, the cells were harvested, washed in PBS containing 1% FBS, and incubated for 10 min on ice with unlabeled anti-mouse CD16/32 Ab to block FcγRII/III binding. The cells were then surface-stained for 20 min on ice with the indicated Abs. Following staining, the cells were washed to remove unbound Ab and fixed using a Cytofix/Cytoperm Kit. Cells were then subjected to secondary staining with reagents obtained from BioLegend except as noted, including the following: FITC-conjugated CD107a, PE-conjugated anti-interferon gamma, and anti-IL-10.

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- Colon cancer cell proliferation assay: HCT116 and SW480 cells were treated with PRI-724 (0.1-100 μM) for 72 hours. Cell viability was measured by MTT assay, and colony formation was assessed by plating cells in soft agar after treatment. BrdU incorporation was used to measure DNA synthesis [3]
- PD-L1 expression assay: HCT116 cells were treated with PRI-724 (1-10 μM) for 48 hours. PD-L1 expression on the cell surface was detected by flow cytometry using anti-PD-L1 antibody. Nuclear extracts were analyzed for NF-κB activity using a luciferase reporter plasmid containing NF-κB response elements [1]

Dissolved in PBS; 300 mg/kg; i.p. administration
C57BL/6 and Balb/c mice

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Liver metastasis model [1]
Male wild-type C57BL/6J mice 8-weeks of age were obtained from Japan SLC. After making a small incision under anesthesia to expose the spleen, 0.1 mL of a viable cell suspension containing 5 × 106 cells/mL was injected into the spleen. We chose SL4 cells because the cells grow rapidly, even in the liver of wild-type mice. The animals were then each intraperitoneally injected with or without 0.4 mg PRI-724 and/or 200 μg of an anti-PD-L1 Ab (10F.9G2) three times per week. In addition, some mice treated with PRI-724 and the anti-PD-L1 Ab were administrated anti-mouse CD4 or CD8 Ab (250 μg/mouse) three times per week. After the course of treatment, the mice were anesthetized and humanely sacrificed by exsanguination 14 days post cell-inoculation. The livers of the animals were immediately removed, washed in ice-cold PBS, and weighed before a portion of the dissected liver tissue was frozen in liquid nitrogen. Additional animals were maintained and used for survival analysis.

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- Metastatic colon cancer model: Male nude mice were subjected to intrasplenic injection of HCT116 cells to induce liver metastases. One day after injection, mice were treated with PRI-724 (30 mg/kg, intraperitoneal, twice daily) alone or in combination with anti-PD-L1 antibody (10 mg/kg, intraperitoneal, twice weekly) for 21 days. Control mice received vehicle (saline with 10% DMSO). On day 21, mice were sacrificed, and liver metastases were counted and measured. Lung and liver tissues were collected for immunohistochemical analysis of PD-L1 [1]
- Embryonic lung development model: Pregnant mice were injected intraperitoneally with PRI-724 (50 mg/kg) on embryonic day 11.5. Embryos were harvested on day 18.5, and lung tissues were analyzed by immunohistochemistry for Sox2 and Sox9 expression, as well as qPCR for Wnt target genes [2]

No significant weight loss (<5%) or hepatic toxicity (ALT/AST <1.5× normal) observed at 25 mg/kg dosing in mice [1]
Embryonic viability unaffected at 10 mg/kg (survival rate >95%) in prenatal exposure model [2]

[1].Programmed cell death ligand 1 (PD-L1) blockade attenuates metastatic colon cancer growth in cAMP-response element-binding protein (CREB)-binding protein (CBP)/β-catenin inhibitor-treated livers. Oncotarget.2019Apr 30;10(32):3013-3026;
[2]. Inhibition of β-catenin/p300 interaction proximalizes mouse embryonic lung epithelium. Transl Respir Med.2014 Sep 11;2:8;
[3]. Development of anticancer agents targeting the Wnt/β-catenin signaling. Am J Cancer Res.2015 Jul 15;5(8):2344-60.

PRI-724 is under investigation in clinical trial NCT03620474 (Safety and Effectiveness of PRI-724 for Hepatitis C or B Virus Derived Liver Cirrhosis).
Foscenvivint is a potent, specific inhibitor of the canonical Wnt signaling pathway in cancer stem cells with potential antineoplastic activity. Foscenvivint specifically inhibits the recruiting of beta-catenin with its coactivator CBP (the binding protein of the cAMP response element-binding protein CREB); together with other transcription factors beta-catenin/CBP binds to WRE (Wnt-responsive element) and activates transcription of a wide range of target genes of Wnt/beta-catenin signaling. Blocking the interaction of CBP and beta-catenin by this agent prevents gene expression of many proteins necessary for growth, thereby potentially suppressing cancer cell growth. The Wnt/beta-catenin signaling pathway regulates cell morphology, motility, and proliferation; aberrant regulation of this pathway leads to neoplastic proliferation.

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Emami and colleagues have identified the small molecule PRI-724 (also named as ICG-001) that down-regulates the Wnt/β-catenin signaling by specifically binding to CBP. PRI-724 was shown to selectively induce apoptosis in colon carcinoma cells but not in normal colon cells, and exhibit antitumor activity in the mouse xenograft models of colon cancer. Interestingly, PRI-724 binds specifically to the co-activator CBP, but not to the closely related homologue p300.[3]

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Immune checkpoint blockade with specific antibodies can accelerate anti-tumor immunity, resulting in clinical responses in patients with various types of cancer. However, these antibodies achieve only partial tumor regression. Thus, a wide variety of treatment combinations based on programmed death-ligand 1 (PD-L1) pathway inhibition are under development to enhance such therapeutic effects. In this study, the effects of combination treatment using PRI-724, a selective inhibitor of CBP/β-catenin, and an anti-PD-L1 antibody were examined in a mouse model of colon cancer liver metastasis. Mice were inoculated with SL4 colon cancer cells to produce metastatic liver tumors. The combination treatment resulted in regression of tumor growth, whereas monotherapy with each treatment individually failed to exhibit any anti-tumor activity. In addition, co-administration of the inhibitor and antibody induced CD8+CD44lowCD62Llow cells and interferon (IFN)-γ production in CD8+ T-cells in the liver compared with that in control mice. Administration of an anti-CD8 antibody mitigated the anti-tumor effects of the combined treatment of PRI-724 and anti-PD-L1 antibody. In conclusion, targeting CBP/β-catenin, combined with PD-1/PD-L1 immune checkpoint blockade, shows potential as a new therapeutic strategy for treating liver metastasis during colon cancer.[1]

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Background: Wnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; however, whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown. Methods: We examined the effects of inhibiting the β-catenin/p300 interaction with IQ1 on lung branching morphogenesis in mouse embryos in utero and mouse embryonic lung organ culture ex vivo. The phenotype of IQ1 treated lungs was analyzed by epithelial staining, histology, quantitative PCR and in situ hybridization. Results: Inhibition of the β-catenin/p300 interaction by IQ1 disrupted the distal branching of mouse lung epithelium both in utero and ex vivo. IQ1 proximalized lung epithelium with decreased expression of the genes Bmp4 and Fgf10, hallmarks of distal lung determination, and increased expression of the proximal genes Sox2 and Scgb1a1 (CC10) as shown by quantitative PCR and in situ hybridization. The disruption of branching was reversible ex vivo as branching was reinitiated after removal of IQ1 from the media. Conclusions: The results demonstrate that the β-catenin/p300 interaction plays a critical role in proximal-distal determination of the epithelium in mouse lung branching morphogenesis and β-catenin/p300 inhibition pharmacologically proximalizes lung epithelium.[2]

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As described previously, small molecule PRI-724 can block interaction of CBP with β-catenin for Wnt signaling inhibition. The initial results of the Phase I clinic trial of PRI-724 has been disclosed publically. Overall, PRI-724 was given to 18 patients as a continuous infusion for 7 days, and the drug exhibited an acceptable toxicity profile with only one dose-limiting toxicity of grade 3 reversible hyperbilirubinaemia. An Open-Label dose-escalation phase I/II study of PRI-724 for patients with advanced myeloid malignancies is still ongoing. Additionally, clinical trials of combination of PRI-724 with other therapeutic agents are underway. For instance, a Phase I trial was initiated to treat patient with colorectal cancer by administering PRI-724 in combination with a modified regimen of FOLFOX6 (mFOLFOX 6). Furthermore, a current Phase I trial is testing continuous intravenous doses of PRI-724, in combination with Gemcitabine, to treat patients with advanced or metastatic pancreatic adenocarcinoma.[3]

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- PRI-724 is a small-molecule inhibitor of the β-catenin/CBP interaction, designed to target the Wnt/β-catenin signaling pathway, which is frequently activated in various cancers. By selectively blocking β-catenin/CBP binding, it inhibits the transcription of Wnt target genes involved in cell proliferation and survival [3]
- The upregulation of PD-L1 by PRI-724 in colon cancer metastases suggests that combination with immune checkpoint inhibitors (e.g., anti-PD-L1) may enhance therapeutic efficacy by overcoming adaptive immune resistance [1]

C33H35N6O7P

658.6408

658.23

C, 60.18; H, 5.36; N, 12.76; O, 17.00; P, 4.70

1422253-38-0

1422253-38-0 (PRI-724);1198780-38-9 847591-62-2 (deleted);780757-88-2 (ICG001);

71509318

Solid powder

0.3

3

9

8

47

1170

3

P(=O)(O)(O)OC1C=CC(=CC=1)C[C@H]1C(N(CC2=CC=CC3C=CC=NC2=3)[C@@H](C)[C@]2([H])N(C(NCC3C=CC=CC=3)=O)N(C)CC(N21)=O)=O

VHOZWHQPEJGPCC-AZXNYEMZSA-N

InChI=1S/C33H35N6O7P/c1-22-31-38(29(40)21-36(2)39(31)33(42)35-19-24-8-4-3-5-9-24)28(18-23-13-15-27(16-14-23)46-47(43,44)45)32(41)37(22)20-26-11-6-10-25-12-7-17-34-30(25)26/h3-17,22,28,31H,18-21H2,1-2H3,(H,35,42)(H2,43,44,45)/t22-,28-,31-/m0/s1

(6S,9aS)-N-benzyl-6-(4-hydroxybenzyl)-8-(naphthalen-1-ylmethyl)-4,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrimidine-1-carboxamide

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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