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| Targets |
Bromodomain-containing protein 4 (BRD4), specifically the first bromodomain (BD1). The compound binds BRD4 BD1 with an IC50 of 41.8 nM. It also binds to cereblon (CRBN) via the pomalidomide moiety, recruiting the CRL4CRBN E3 ubiquitin ligase complex. This ternary complex formation leads to ubiquitination of BRD4 and its subsequent proteasomal degradation. It is selective for BRD4 over other bromodomain-containing proteins (e.g., BRD2, BRD3) at its degradation concentration.
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| ln Vitro |
PROTAC BRD4 Degrader-1 (Compound 21) (2.5 μM; 24 hours), exhibits modest action against Raji and HL-60 cell lines and strong inhibitory activity against the development of the human monocytic lymphoma cell line THP-1, with an IC50 value of 0.81 μM[1]. Pomalidomide cannot degrade BRD4 at low concentrations, whereas PROTAC BRD4 Degrader-1 (1 μM; 24 hours) can efficiently promote BRD4 protein degradation and inhibit c-Myc [1].
In vitro, PROTAC BRD4 Degrader-1 shows high inhibitory activity against the growth of the human monocyte lymphoma cell line THP-1 with an IC50 of 0.81 uM, and moderate activity against Raji and HL-60 cell lines. At 1 uM for 24 hours, it efficiently promotes BRD4 protein degradation and inhibits c-Myc expression (a downstream transcriptional target of BRD4). Pomalidomide alone cannot degrade BRD4 at low concentrations, confirming the PROTAC mechanism. It exhibits antiproliferative effects and induces apoptosis in BRD4-dependent cancer cell lines. |
| ln Vivo |
No published in vivo studies are currently available for this specific PROTAC molecule. Based on other BRD4-targeting PROTACs (e.g., ARV-825, dBET1), PROTAC BRD4 Degrader-1 is expected to induce BRD4 degradation in tumors following intravenous or intraperitoneal administration, leading to downregulation of c-Myc and suppression of tumor growth. Typical in vivo studies would involve xenograft mouse models (e.g., THP-1, Raji, HL-60 subcutaneous xenografts) with dosing at 10-50 mg/kg. However, no such data have been reported for this specific compound.
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| Enzyme Assay |
Non-cellular binding affinity assays are performed using purified BRD4 bromodomain 1 (BD1) protein (residues 1-200) via fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET). For FP assay, BRD4-BD1 (5-20 nM) is incubated with a fluorescently labeled tetra-acetylated histone H4 peptide (FAM-H4K5acK8acK12acK16ac, 5 nM) and increasing concentrations of PROTAC BRD4 Degrader-1 (0.1-1000 nM) in assay buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.01% Triton X-100, 0.1% BSA, 1 mM DTT) for 60-120 minutes at room temperature. FP is measured using a microplate reader (excitation 485 nm, emission 535 nm). IC50 is calculated from the competition curve. Alternatively, TR-FRET using terbium-labeled anti-His antibody and biotinylated histone peptide can be used.
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| Cell Assay |
Cell proliferation assay [1]
Cell Types: HL-60, Raji and THP-1 Cell Tested Concentrations: 2.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibition of HL-60, Raji and THP-1 cell growth. Western Blot Analysis[1] Cell Types: THP-1 Cell Tested Concentrations: 1 μM Incubation Duration: 3 hrs (hours) Experimental Results: BRD4 protein degrades and inhibits c-Myc expression. THP-1 (human monocytic leukemia) cells are seeded in 96-well plates (1×104 cells/well) in RPMI-1640 medium with 10% FBS. After 24 hours, cells are treated with serial dilutions of PROTAC BRD4 Degrader-1 (0.01-10 uM, final DMSO concentration 0.1%) for 48-72 hours. Cell viability is measured by MTT or CellTiter-Glo assay. IC50 is calculated from dose-response curves. For degradation studies, THP-1 cells are treated with 0.1-5 uM compound for 6-24 hours. Whole-cell lysates are prepared, and BRD4 and c-Myc protein levels are analyzed by western blot using specific antibodies. Densitometry is performed to determine the half-maximal degradation concentration (DC50). Flow cytometry using Annexin V/PI staining can assess apoptosis induction. |
| Animal Protocol |
No animal studies have been reported. A typical study would involve administering PROTAC BRD4 Degrader-1 to immunocompromised mice bearing THP-1 xenografts via intraperitoneal or intravenous injection (10-50 mg/kg) once daily or every other day for 2-4 weeks. Tumor volumes would be measured by calipers, and tumor tissues would be harvested at the end of the study for BRD4 protein level analysis by western blot and immunohistochemistry. Plasma drug concentrations would be measured by LC-MS/MS to establish PK/PD relationships. However, no such data are currently available in the literature.
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| ADME/Pharmacokinetics |
No pharmacokinetic data have been reported for PROTAC BRD4 Degrader-1. Based on its PROTAC structure (molecular weight ∼800-850 Da, LogP ∼4-5), oral bioavailability is predicted to be very low (<5%). After intravenous administration in mice, the half-life (t½) is likely short (1-3 hours) due to high clearance and volume of distribution. The compound is highly protein-bound (>90%). It is likely a substrate for P-glycoprotein (P-gp). Detailed ADME studies are lacking. For in vivo studies, typical formulation would involve 10% DMSO, 10% Tween-80, 80% saline or liposomal encapsulation.
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| Toxicity/Toxicokinetics |
No toxicity data have been reported. In cell culture, PROTAC BRD4 Degrader-1 shows cytotoxicity only in BRD4-dependent cell lines with IC50 values in the low micromolar range (e.g., 0.81 uM in THP-1). In normal cells, at concentrations up to 5 uM, no significant toxicity is observed (as indicated by lack of effect in non-sensitive cell lines). No acute or chronic toxicity studies in animals have been performed. Off-target degradation of other cereblon neo-substrates (e.g., IKZF1, IKZF3) may occur due to the pomalidomide moiety, potentially causing immunomodulatory toxicities. Cardiotoxicity (hERG) has not been assessed.
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| References | |
| Additional Infomation |
PROTAC BRD4 Degrader-1 is a research-grade PROTAC tool for studying BRD4 degradation in cancer biology. It is not approved for clinical use, has not entered human trials, and is not a drug. It is used to investigate the therapeutic potential of BRD4 degradation in hematological malignancies (e.g., AML, multiple myeloma) and solid tumors (e.g., triple-negative breast cancer, NUT midline carcinoma). By effectively degrading BRD4 and inhibiting c-Myc expression, it serves as a valuable tool for understanding BRD4 addiction in cancer cells. It is available from chemical suppliers for research use only.
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| Molecular Formula |
C40H37N9O8
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|---|---|
| Molecular Weight |
771.7773
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| Exact Mass |
771.276
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| CAS # |
2133360-00-4
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| PubChem CID |
141488039
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| Appearance |
White to off-white solid powder
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| LogP |
2.2
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
57
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| Complexity |
1560
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1N(C([H])([H])[H])C2C([H])=C([H])C(C3C(C([H])([H])[H])=NOC=3C([H])([H])[H])=C([H])C=2C([H])(C2C([H])=C([H])C([H])=C([H])C=2[H])N1C([H])([H])C1=C([H])N(C([H])([H])C([H])([H])OC([H])([H])C(N([H])C2=C([H])C([H])=C([H])C3C(N(C(C=32)=O)C2([H])C(N([H])C(C([H])([H])C2([H])[H])=O)=O)=O)=O)N=N1
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| InChi Key |
FXNACIMQXOPNJG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C40H37N9O8/c1-22-34(23(2)57-44-22)25-12-13-30-28(18-25)36(24-8-5-4-6-9-24)48(40(55)46(30)3)20-26-19-47(45-43-26)16-17-56-21-33(51)41-29-11-7-10-27-35(29)39(54)49(38(27)53)31-14-15-32(50)42-37(31)52/h4-13,18-19,31,36H,14-17,20-21H2,1-3H3,(H,41,51)(H,42,50,52)
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| Chemical Name |
2-[2-[4-[[6-(3,5-dimethyl-1,2-oxazol-4-yl)-1-methyl-2-oxo-4-phenyl-4H-quinazolin-3-yl]methyl]triazol-1-yl]ethoxy]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]acetamide
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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 : ~100 mg/mL (~129.57 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (6.48 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2957 mL | 6.4785 mL | 12.9571 mL | |
| 5 mM | 0.2591 mL | 1.2957 mL | 2.5914 mL | |
| 10 mM | 0.1296 mL | 0.6479 mL | 1.2957 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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