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| Targets |
P300/CBP-IN-3 targets p300 histone acetyltransferase (HAT) (IC50 = 0.18 μM) [1]
P300/CBP-IN-3 targets CBP histone acetyltransferase (HAT) (IC50 = 0.25 μM) [1] P300/CBP-IN-3 targets c-MYC (inhibits c-MYC transcriptional activity with EC50 = 0.8 μM in c-MYC reporter assay) [1] |
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| ln Vitro |
- p300/CBP HAT inhibitory activity: P300/CBP-IN-3 potently inhibited the acetyltransferase activity of recombinant human p300 and CBP HAT domains in a dose-dependent manner, with IC50 values of 0.18 μM and 0.25 μM respectively. It showed no significant inhibition against other histone acetyltransferases (GCN5, PCAF) at 10 μM [1]
- Anti-proliferative activity: The compound inhibited the proliferation of various cancer cell lines dependent on p300/CBP or c-MYC. IC50 values were 1.2 μM (A549, lung cancer), 0.9 μM (MCF-7, breast cancer), 1.5 μM (HCT116, colon cancer), and 0.7 μM (NCI-H929, multiple myeloma). It had minimal cytotoxicity to normal human bronchial epithelial cells (BEAS-2B, IC50 > 20 μM) [1] - Inhibition of c-MYC transcriptional activity: In c-MYC luciferase reporter assay, P300/CBP-IN-3 (0.1-5 μM) dose-dependently suppressed c-MYC-mediated transcription, with EC50 = 0.8 μM. RT-PCR confirmed that it reduced mRNA levels of c-MYC target genes (Cyclin D1, c-Myc, Survivin) by 65%, 72%, and 58% respectively at 2 μM in A549 cells [1] - Reduction of histone acetylation: Western blot analysis showed that P300/CBP-IN-3 (0.5-2 μM) decreased acetylation levels of histone H3 (H3Ac) and histone H4 (H4Ac) in MCF-7 cells, with maximum inhibition of 70% (H3Ac) and 65% (H4Ac) at 2 μM. It did not affect total H3 or H4 protein levels [1] - Inhibition of clonogenicity: P300/CBP-IN-3 (0.3-1 μM) suppressed colony formation of A549 and NCI-H929 cells. At 1 μM, colony formation rates were reduced by 78% (A549) and 82% (NCI-H929) compared to control [1] - Induction of apoptosis: Flow cytometry analysis showed that P300/CBP-IN-3 (2 μM) induced apoptosis in NCI-H929 cells, with apoptotic rate increased from 5% (control) to 38%. Western blot detected increased cleavage of caspase-3 and PARP [1] |
| ln Vivo |
- Antitumor efficacy in xenograft models: In A549 lung cancer xenograft-bearing nude mice, oral administration of P300/CBP-IN-3 (15 mg/kg, 30 mg/kg, once daily for 21 days) resulted in tumor growth inhibition rates of 56% and 73% respectively. In NCI-H929 multiple myeloma xenografts, intraperitoneal administration of 20 mg/kg (once daily for 14 days) achieved a tumor growth inhibition rate of 68% [1]
- Mechanism in vivo: Tumor tissues from treated mice (30 mg/kg, oral) showed reduced H3Ac and H4Ac levels (by 62% and 58% respectively) and downregulated expression of Cyclin D1 and Survivin (by 65% and 59% respectively) compared to vehicle control [1] - Tolerability: No significant body weight loss (< 8%) or obvious toxic signs (lethargy, diarrhea) were observed in treated mice at effective doses [1] |
| Enzyme Assay |
- p300/CBP HAT activity assay: Recombinant human p300 or CBP HAT domain was mixed with histone H3 substrate, acetyl-CoA, and P300/CBP-IN-3 at gradient concentrations (0.01-5 μM) in reaction buffer (pH 7.4). The mixture was incubated at 37°C for 1 hour, and acetylated histone H3 was detected by a colorimetric assay based on antibody recognition. IC50 values were calculated by plotting inhibition rate against drug concentration [1]
- Histone acetyltransferase selectivity assay: Recombinant GCN5 and PCAF enzymes were separately mixed with their corresponding substrates, acetyl-CoA, and P300/CBP-IN-3 (10 μM) in reaction buffer. After 37°C incubation for 1 hour, enzyme activity was detected by colorimetric assay to evaluate selectivity [1] - c-MYC reporter assay: HEK293 cells stably transfected with c-MYC luciferase reporter plasmid were seeded into 96-well plates, treated with P300/CBP-IN-3 (0.1-5 μM) for 24 hours. Luciferase activity was measured and normalized to protein concentration to calculate EC50 for inhibiting c-MYC transcriptional activity [1] |
| Cell Assay |
- Cell viability assay: Cancer cells (A549, MCF-7, HCT116, NCI-H929) and BEAS-2B cells were seeded into 96-well plates at 4×10³ cells/well, treated with P300/CBP-IN-3 (0.05-20 μM) for 72 hours. Cell viability was measured by tetrazolium salt-based assay, and IC50 values were calculated [1]
- Western blot assay: MCF-7 cells were treated with P300/CBP-IN-3 (0.5-2 μM) for 24 hours, lysed, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against H3Ac, H4Ac, total H3, total H4, cleaved caspase-3, cleaved PARP, and GAPDH. Band intensities were quantified by densitometry [1] - RT-PCR assay: A549 cells were treated with P300/CBP-IN-3 (0.5-2 μM) for 16 hours, total RNA was extracted and reverse-transcribed to cDNA. Quantitative PCR was performed to detect mRNA levels of Cyclin D1, c-Myc, and Survivin, with GAPDH as internal control [1] - Clonogenic assay: A549 and NCI-H929 cells were treated with P300/CBP-IN-3 (0.3-1 μM) for 24 hours, then seeded into 6-well plates at 400 cells/well and incubated for 14 days. Colonies were stained with crystal violet, counted, and inhibition rate was calculated relative to control [1] - Apoptosis assay: NCI-H929 cells were treated with P300/CBP-IN-3 (2 μM) for 48 hours, stained with Annexin V-FITC and propidium iodide (PI), and apoptotic cells were analyzed by flow cytometry [1] |
| Animal Protocol |
- A549 lung cancer xenograft model: Female nude mice (6-7 weeks old) were subcutaneously injected with A549 cells (4×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were randomly divided into vehicle control, 15 mg/kg, and 30 mg/kg P300/CBP-IN-3 groups (n=7 per group). The compound was dissolved in a mixture of DMSO, PEG400, and sterile water (volume ratio 1:2:7) to prepare oral suspension, administered once daily for 21 days. Tumor volume was measured every 3 days, and body weight was recorded weekly [1]
- NCI-H929 multiple myeloma xenograft model: Female nude mice were subcutaneously injected with NCI-H929 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were divided into vehicle control and 20 mg/kg P300/CBP-IN-3 groups (n=6 per group). The compound was dissolved in DMSO + PEG400 + sterile water (1:2:7) and administered intraperitoneally once daily for 14 days. Tumor volume and body weight were monitored as above [1] - Tumor tissue analysis: At the end of treatment, mice were sacrificed, tumors were excised, weighed, and stored at -80°C. Tumor lysates were used for Western blot analysis of H3Ac, H4Ac, Cyclin D1, and Survivin [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: The plasma protein binding rate of P300/CBP-IN-3 in human plasma was 90.2 ± 2.1% as determined by equilibrium dialysis [1]
- In vitro metabolic stability: The compound showed good metabolic stability in human liver microsomes with a half-life (t1/2) of 6.4 hours and a metabolic clearance rate of 0.28 mL/min/mg protein [1] - Pharmacokinetics in mice: After a single oral administration of 30 mg/kg, the Cmax was 8.9 μM, the AUC₀₋₂₄h was 56.3 μM·h, the elimination half-life (t1/2) was 5.7 hours, and the oral bioavailability (F) was 46.8%. Following a single intraperitoneal injection of 20 mg/kg, the Cmax was 11.2 μM, the AUC₀₋₂₄h was 62.5 μM·h, and the t1/2 was 6.1 hours [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or obvious toxic symptoms were observed in mice after a single oral dose of up to 300 mg/kg of P300/CBP-IN-3, and the maximum tolerated dose (MTD) was > 300 mg/kg [1]. Subacute toxicity: No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, blood urea nitrogen) after mice were treated with P300/CBP-IN-3 (30 mg/kg, orally, once daily for 28 days). No abnormal lesions were found in the histopathological examination of the major organs (heart, liver, spleen, lungs, kidneys) [1].
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| References | |
| Additional Infomation |
Chemical Classification: P300/CBP-IN-3 is a small molecule inhibitor belonging to the class of 5-(1H-benzo[d]imidazol-2-yl)pyridine-2-amine or 5-(3H-imidazol[4,5-b]pyridine-6-yl)pyridine-2-amine derivatives [1] Mechanism of Action: This compound binds to the acetyl-CoA binding pocket of the p300/CBP histone acetyltransferase (HAT) domain, competitively inhibiting its acetyltransferase activity. This reduces histone acetylation levels, inhibits the transcriptional activity of c-MYC and the expression of its target genes (cyclin D1, Survivin), thereby inhibiting tumor cell proliferation and inducing apoptosis [1] Target Background: p300 and CBP are homologous histone acetyltransferases that regulate gene transcription by acetylation of histones and transcription factors (such as c-MYC). Abnormal activation of p300/CBP and overexpression of c-MYC are associated with the development and progression of various cancers [1] - Therapeutic potential: P300/CBP-IN-3 is a potent, selective and orally bioavailable p300/CBP and c-MYC inhibitor that has shown therapeutic potential for treating various cancers such as lung cancer, breast cancer, colon cancer and multiple myeloma [1].
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| Molecular Formula |
C24H29N7O
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|---|---|
| Molecular Weight |
431.533364057541
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| Exact Mass |
431.243
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| CAS # |
2299226-01-8
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| PubChem CID |
138466007
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.23±0.1 g/cm3(Predicted)
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
32
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| Complexity |
615
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
LYVJDLHFTGYNAV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H29N7O/c1-5-29(6-2)22-11-9-18(15-26-22)23-27-20-14-17(24(32)25-4)8-10-21(20)31(23)16-19-12-13-30(7-3)28-19/h8-15H,5-7,16H2,1-4H3,(H,25,32)
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| Chemical Name |
2-[6-(diethylamino)pyridin-3-yl]-1-[(1-ethylpyrazol-3-yl)methyl]-N-methylbenzimidazole-5-carboxamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~579.33 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.82 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.82 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3173 mL | 11.5867 mL | 23.1734 mL | |
| 5 mM | 0.4635 mL | 2.3173 mL | 4.6347 mL | |
| 10 mM | 0.2317 mL | 1.1587 mL | 2.3173 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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