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]

In mice, no significant weight loss (<5%) or hepatotoxicity (ALT/AST <1.5×normal) was observed at a dose of 25 mg/kg [1]
In the prenatal exposure model, embryo viability was not affected at a dose of 10 mg/kg (survival rate >95%) [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 currently undergoing clinical trial NCT03620474 (a study on the safety and efficacy of PRI-724 in treating hepatitis C or B virus-related cirrhosis). Foscenvivint is a potent and specific inhibitor of the classic Wnt signaling pathway with potential anti-tumor activity in cancer stem cells. Foscenvivint specifically inhibits the recruitment of β-catenin and its co-activator CBP (the binding protein of cAMP response element-binding protein CREB); β-catenin/CBP, along with other transcription factors, binds to WREs (Wnt response elements), activating the transcription of multiple target genes in the Wnt/β-catenin signaling pathway. This drug may inhibit cancer cell growth by blocking the interaction between CBP and β-catenin, thereby inhibiting the expression of genes of multiple growth-essential proteins. The Wnt/β-catenin signaling pathway regulates cell morphology, motility, and proliferation; abnormal regulation of this pathway can lead to tumor cell proliferation. Emami and colleagues discovered a small molecule, PRI-724 (also known as ICG-001), which downregulates the Wnt/β-catenin signaling pathway by specifically binding to CBP. PRI-724 has been shown to selectively induce apoptosis in colon cancer cells but has no effect on normal colon cells and has shown antitumor activity in a mouse xenograft model of colon cancer. Interestingly, PRI-724 specifically binds to the coactivator CBP but not to its closely related homolog p300. [3] Blocking immune checkpoints with specific antibodies can accelerate antitumor immunity, thereby enabling clinical efficacy in a variety of cancer patients. However, these antibodies can only achieve partial tumor regression. Therefore, various combination therapies based on programmed death-ligand 1 (PD-L1) pathway inhibition are currently being developed to enhance the efficacy of such therapies. This study investigated the effect of the combination therapy of the CBP/β-catenin selective inhibitor PRI-724 with an anti-PD-L1 antibody in a mouse model of colon cancer liver metastases. Liver metastases were induced in mice after inoculation with SL4 colon cancer cells. Combination therapy can inhibit tumor growth, while neither drug alone showed anti-tumor activity. In addition, compared with control mice, the combination of inhibitor and antibody can induce the proliferation of CD8+CD44lowCD62Llow cells in the liver and promote the production of interferon (IFN)-γ by CD8+ T cells. The use of anti-CD8 antibody alone weakened the anti-tumor effect of the combination therapy of PRI-724 and anti-PD-L1 antibody. In summary, targeting CBP/β-catenin combined with PD-1/PD-L1 immune checkpoint blockade therapy is expected to become a new strategy for treating liver metastases of colorectal cancer. [1] Background: Based on the genetically modified mouse model, the Wnt/β-catenin signaling pathway is believed to regulate the proximal-distal differentiation of embryonic lung epithelial cells. The previously discovered and characterized small molecule inhibitor IQ1 can enhance the interaction between β-catenin and its transcriptional coactivator p300 by pharmacological means. Inhibition of the β-catenin/p300 interaction by IQ1 blocked the differentiation of embryonic stem cells and epicardial progenitor cells; however, it remains unclear whether the differential coactivator of β-catenin plays a role in proximal-distal differentiation of the lung epithelium. Methods: We investigated the effects of IQ1 inhibition of the β-catenin/p300 interaction on lung branching morphology in mouse embryos (in utero) and in vitro cultured mouse embryos. 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 distal branching of the mouse lung epithelium, both in utero and in vitro. Quantitative PCR and in situ hybridization results showed that IQ1 proximalized the lung epithelium, leading to decreased expression of distal lung differentiation markers Bmp4 and Fgf10, while increasing expression of proximal genes Sox2 and Scgb1a1 (CC10). In vitro experiments showed that branching interruption was reversible because branching restarted after IQ1 was removed from the culture medium. Conclusion: The results indicate that the β-catenin/p300 interaction plays a key role in the proximal-distal determination of epithelial cells during lung branching morphogenesis in mice, and that β-catenin/p300 inhibitors can induce proximal branching of lung epithelial cells. [2] As previously mentioned, the small molecule PRI-724 blocks the interaction between CBP and β-catenin, thereby inhibiting the Wnt signaling pathway. Preliminary results of the Phase I clinical trial of PRI-724 have been published. A total of 18 patients received continuous infusion of PRI-724 for 7 days. The toxicity of the drug was acceptable, with only one dose-limiting toxicity, namely grade 3 reversible hyperbilirubinemia, occurring. An open-label dose-escalation Phase I/II study of PRI-724 in patients with advanced myeloid malignancies is still underway. In addition, clinical trials of PRI-724 in combination with other therapeutic agents are also underway. For example, a phase I trial has been initiated to treat colorectal cancer patients by administering PRI-724 in combination with a modified FOLFOX6 regimen (mFOLFOX 6). In addition, an ongoing phase I trial is testing the efficacy of PRI-724 in combination with gemcitabine via continuous intravenous administration in patients with advanced or metastatic pancreatic adenocarcinoma. [3] PRI-724 is a small molecule inhibitor of β-catenin/CBP interaction designed to target the Wnt/β-catenin signaling pathway, which is frequently activated in a variety of cancers. By selectively blocking β-catenin/CBP binding, PRI-724 inhibits the transcription of Wnt target genes involved in cell proliferation and survival. [3] PRI-724 upregulates PD-L1 expression in colorectal cancer metastases, suggesting that its 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.)