I-CBP112 targets the bromodomains of CBP/p300 (histone acetyltransferases); cellular IC50 for displacing isolated CBP bromodomain is 600 ± 50 nM (NanoBRET assay) [2]
I-CBP112 shows in vitro IC50 for CBP bromodomain-H3K56ac peptide interaction (AlphaScreen assay) [2]
I-CBP112 has no significant binding to BRD4(1)/BRD4(2) bromodomains (ITC assay) [2]

I-CBP112 significantly enhanced the acetylation of p300 at histone H3K18 and H3K23 sites. H3K18ac is stimulated by I-CBP112 about three times, and these same sites also experience increased CBP and H4K5 acetylation. With an EC50 of less than 2 μM, I-CBP112 activates p300 and CBP-mediated H3K18 acetylation [1]. I-CBP112 treatment of human and murine leukemia cell lines significantly reduced colony formation and induced cell differentiation without appearing to be harmful. BioMAP primary cell sets exhibit distinct reactions to cytokine and marker protein production upon exposure to I-CBP112 [2].

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1. I-CBP112 stimulated p300/CBP-mediated nucleosome acetylation up to 3-fold, while CBP30 had no such effect; this activation was not observed with isolated histone H3 substrate and required nucleosome substrate, with EC50 for p300-mediated nucleosome acetylation determined from standard binding isotherm (duplicate experiments, average ± span) [1]
2. I-CBP112 (40 μM) altered the time course of p300-catalyzed nucleosome acetylation, with linear initial 5 min of acetylation showing significant difference (p = 0.005) compared to control; it also changed the concentration-dependent nucleosome acetylation by p300, with fitted curves significantly different (p < 0.0001) from control [1]
3. I-CBP112 did not affect nucleosome acetylation by the isolated p300 HAT domain (one-way ANOVA, p = 0.8535, n=3); CBP30 could neutralize the activation effect of I-CBP112 on p300/CBP [1]
4. Mass spectrometry and Western blot showed I-CBP112 specifically stimulated acetylation of histone H3 Lys18 (H3K18) in nucleosomes (p < 0.001), an established in vivo acetylation site of p300/CBP; Western blot confirmed dose-dependent increase of H3K18 acetylation in in vitro reaction mixtures with p300/CBP and nucleosomes [1]
5. I-CBP112 enhanced H3K18 acetylation in LNCaP (prostate cancer) and KG1a (acute leukemia) cells in a concentration range matching its antiproliferative effects; IC50 for LNCaP cell proliferation ([³H]thymidine incorporation, 72 h) was 5.5 ± 1.1 μM (Hill slope = 1.3 ± 0.1, n=3), and 9.1 ± 1.2 μM for KG1a cells (Hill slope = 0.82 ± 0.09, n=4) [1]
6. I-CBP112 impaired colony formation of human/mouse leukemic cell lines (MLL-CBP immortalized murine bone marrow cells, KASUMI-1, SEM, MOLM13 human AML cell lines, primary AML patient blast cells) in a dose-dependent manner, induced cellular differentiation, and had no significant cytotoxicity (WST1 assay) [2]
7. I-CBP112 reduced leukemia-initiating potential of MLL-AF9⁺ acute myeloid leukemia (AML) cells in vitro (extreme limiting dilution analysis, ELDA: stem cell frequency decreased from 1/2.3 to 1/13, p<0.00001); it also altered cell cycle phase distribution of KASUMI-1 cells (G1 phase changes, two-way ANOVA, p<0.01, p<0.001, p<0.0001) [2]
8. I-CBP112 showed synergistic cytotoxic effects with BET inhibitor JQ1 and doxorubicin in KASUMI-1, SEM, MOLM13 cells (combination index CI<1); it had minimal effects on clonogenic growth of CD34⁺ cells from healthy donors [2]
9. I-CBP112 selectively modulated transcription of genes in leukemic cells, with changes in gene expression confirmed by q-RTPCR (time-dependent regulation of selected genes, three biological replicates) [2]

In both vitro and vivo settings, I-CBP112 dramatically decreased the mLL-AF9+ AML cells' capacity to initiate leukemia in a dose-dependent manner. I-CBP112's synergy with both established standard therapies (doxorubicin) and newer approaches to management (BET inhibition) opens up new avenues for the combined treatment of leukemia and possibly other cancers [2].

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1. I-CBP112 (5µM ex vivo treatment for 3 days) significantly reduced the leukemia-initiating potential of MLL-AF9⁺ AML cells in syngeneic secondary mouse recipients; Kaplan-Meier analysis showed significantly extended survival of mice injected with I-CBP112-treated leukemic cells (50,000/10,000/5,000/500 cells per mouse, log-rank test, n=4/5 per group) [2]

1. ITC (Isothermal Titration Calorimetry) assay: Binding affinities of I-CBP112 to CBP bromodomain, p300 bromodomain, BRD4(1) and BRD4(2) bromodomains were measured; raw titration heats and normalized binding heats were recorded, non-linear least squares fits were applied to generate binding isotherms, and fitted parameters were calculated (no cross-reactivity with BRD4 bromodomains) [2]
2. AlphaScreen assay: The in vitro IC50 value for I-CBP112 inhibiting CBP bromodomain-H3K56ac peptide interaction was determined by measuring the competitive binding of I-CBP112 to CBP bromodomain against acetylated histone peptide [2]
3. Acetyltransferase activity assay (p300/CBP): Reaction mixtures contained p300/CBP enzyme, nucleosome/histone H3 substrate, [¹⁴C]acetyl-CoA, and varying concentrations of I-CBP112; autoradiograph images of acetylation products were obtained, and acetylation levels were quantified (average ± span/SEM); time course and concentration-dependent acetylation were analyzed, with curves fitted by standard binding isotherm/Boltzmann equation [1]
4. SPOT assay: Binding of CBP bromodomain to a library of acetylated histone peptides (including mono/poly-acetylated peptides) was assessed to identify peptide substrates for subsequent ITC experiments; non-acetylated peptides were used as control [2]

1. Western blot assay (in vitro and cell-based): For in vitro experiments, reaction mixtures with p300/CBP, nucleosomes, acetyl-CoA, and varying I-CBP112 concentrations were prepared, and H3K18 acetylation levels were detected by Western blot (quantified as average ± standard error of the fit, n=3); for cell-based experiments, LNCaP/KG1a cells were treated with I-CBP112 (4 h/6 h), total H3 and H3K18ac levels were detected, and fold change relative to no ligand was calculated (average ± span, n=2) [1]
2. Cell proliferation assay ([³H]thymidine incorporation): LNCaP/KG1a cells were treated with I-CBP112 for 72 h, [³H]thymidine incorporation was measured to assess cell proliferation; IC50 and Hill slope were calculated (n=3 for LNCaP, n=4 for KG1a) [1]
3. Cytotoxicity assay (WST1): Murine MLL-CBP immortalized bone marrow cells and MLL-AF9⁺ leukemic blasts were treated with I-CBP112 at different concentrations, WST1 reagent was used to detect cell viability, data were normalized to vehicle-treated controls (mean ± SD), and statistical analysis was performed by ANOVA with Dunnett multiple comparison (p<0.01, p<0.0001, n=4) [2]
4. Clonogenic growth/replating assay: Murine MLL-CBP immortalized bone marrow progenitors, MLL-AF9⁺ leukemic blasts, human leukemic cell lines (KASUMI-1, SEM, MOLM13), primary AML patient blast cells, and healthy donor CD34⁺ cells were cultured in methylcellulose with varying I-CBP112 concentrations; colony formation was scored, data were normalized to vehicle-treated controls (mean ± SD), and statistical analysis was performed by ANOVA with Dunnett multiple comparison (p<0.01, p<0.001, p<0.0001, n=2-5) [2]
5. Flow cytometry (cell cycle analysis): KASUMI-1 cells were treated with increasing doses of I-CBP112 (medium/compound renewed after 3 days), cell cycle phase distribution was analyzed by flow cytometry at different time points; data were normalized to vehicle-treated controls (mean ± SD), and statistical analysis was performed by two-way ANOVA with Turkey multiple comparison (p<0.01, p<0.001, p<0.0001, n=2-4) [2]
6. FRAP (Fluorescence Recovery After Photobleaching) assay: Full-length CBP/GFP, CBP bromodomain mutant (N1168F)/GFP, and 3xCBP_{BRD}/GFP constructs were transfected into cells; half-recovery times of fluorescent signals were measured to assess CBP bromodomain-chromatin interaction, with SAHA (2.5 µM) treated cells as control [2]
7. NanoBRET assay: NanoLuc Luciferase fusion full-length CBP, isolated CBP bromodomain, and full-length GFP-BRD4 were expressed in cells; dose-dependent displacement of bromodomains from chromatin by I-CBP112 was measured, and cellular IC50 for CBP bromodomain displacement was calculated (600 ± 50 nM) [2]
8. q-RTPCR assay: Leukemic cells treated with I-CBP112 were collected at different time points, RNA was extracted, and expression of selected genes was quantified by q-RTPCR; average values of three biological replicates were reported [2]

5 μM of I-CBP112
Mice with Leukemic blasts expressing MLL-AF9

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1. Murine AML model: Bone marrow cells retrovirally expressing MLL-AF9 fusion oncogene were transplanted into syngeneic mice to induce AML; leukemic blasts from diseased mice were ex vivo treated with 5 µM I-CBP112 or DMSO vehicle for 3 days, then transplanted into secondary syngeneic recipients (50,000/10,000/5,000/500 cells per mouse); survival of recipient mice was monitored by Kaplan-Meier analysis (n=4/5 per group) [2]

1. At concentrations that inhibit colony formation and induce differentiation, I-CBP112 did not show significant cytotoxicity to human/mouse leukemia cell lines (WST1 assay) [2]. 2. I-CBP112 had minimal effect on the colony formation and growth of CD34⁺ cells derived from healthy donors, indicating low toxicity to normal hematopoietic cells [2].

[1]. Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain LigandI-CBP112. Biochemistry. 2016 Jul 12;55(27):3727-34.

[2]. Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy. Cancer Res. 2015 Dec 1;75(23):5106-19.

1. I-CBP112 is a specific and potent competitive protein-protein interaction inhibitor of acetyllysine that targets the CBP/p300 bromodomain; it activates p300/CBP-mediated nucleosome acetylation (different from CBP30) through bromodomain interaction allosteric activation [1]
2. I-CBP112 is a selective small molecule inhibitor that inhibits the CBP/p300 bromodomain and does not significantly bind to the BRD4 bromodomain (BET family); it displaces the CBP bromodomain from chromatin in a dose-dependent manner [2]
3. I-CBP112 inhibits abnormal self-renewal and induces differentiation of leukemia cells, and has a synergistic effect with JQ1 (BET inhibitor) and doxorubicin (chemotherapeutic drug), providing an opportunity for combination therapy of leukemia [2]
4. p300/CBP is a histone acetyltransferase (HAT) and transcriptional coactivator that is dysregulated in cancer (e.g., leukemia-associated chromosomal translocations); I-CBP112 regulates p300/CBP-mediated histone acetylation (particularly H3K18), a key epigenetic regulatory mechanism [1,2].

C27H36N2O5

468.59

468.262

1640282-31-0

90488984

White to off-white solid powder

1.1±0.1 g/cm3

623.5±55.0 °C at 760 mmHg

330.9±31.5 °C

0.0±1.8 mmHg at 25°C

1.547

3.29

0

6

7

34

649

1

CCC(=O)N1CCOC2=C(C1)C=C(C=C2OC[C@H]3CCCN(C3)C)C4=CC(=C(C=C4)OC)OC

YKNAKDFZAWQEEO-IBGZPJMESA-N

InChI=1S/C27H36N2O5/c1-5-26(30)29-11-12-33-27-22(17-29)13-21(20-8-9-23(31-3)24(14-20)32-4)15-25(27)34-18-19-7-6-10-28(2)16-19/h8-9,13-15,19H,5-7,10-12,16-18H2,1-4H3/t19-/m0/s1

1-[7-(3,4-dimethoxyphenyl)-9-[[(3S)-1-methylpiperidin-3-yl]methoxy]-3,5-dihydro-2H-1,4-benzoxazepin-4-yl]propan-1-one

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 (5.34 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 (5.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (5.34 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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 (Please use freshly prepared in vivo formulations for optimal results.)