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| Targets |
The primary target is BRD4, specifically the bromodomains (BD1 and BD2) of BRD4. BRD4 is an epigenetic reader that recognizes acetylated lysines on histones and transcription factors, regulating gene expression including oncogenes such as MYC and BCL2. By binding to BRD4, this moiety occupies the acetyl-lysine binding pocket, blocking BRD4's interaction with chromatin and transcriptional activation. In the context of PROTACs, this moiety serves as the warhead to recruit BRD4 to an E3 ligase for ubiquitination and degradation.
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| ln Vitro |
PROTAC BRD4 Degrader-2 is a potent PROTAC BRD4 degrader against BRD4 BD1, having an IC50 of 14.2 nM[1].
In vitro, PROTAC BRD4-binding moiety 1 (alone) inhibits BRD4 bromodomain binding with high affinity (typically IC₅0 in the low nanomolar range, e.g., 1-100 nM, depending on the specific scaffold). It may exhibit antiproliferative activity in cancer cell lines that depend on BRD4, such as acute myeloid leukemia (AML), multiple myeloma, and MYC-driven cancers. However, as a single agent, it is a BRD4 inhibitor, not a degrader. The moiety is used as a building block to synthesize PROTACs, which achieve more sustained target suppression via degradation. The solubility and permeability vary based on the exact structure; typically soluble in DMSO (>10 mM). |
| ln Vivo |
In vivo, the PROTAC BRD4-binding moiety 1 itself (as an inhibitor) may have activity in xenograft models, but it is more commonly used as part of a PROTAC. The PROTACs incorporating this moiety have demonstrated potent BRD4 degradation and tumor growth inhibition in mouse models of cancer, often at lower doses and with longer-lasting effects than the inhibitor alone. This moiety's specific PK properties depend on the full PROTAC structure. The isolated moiety is not typically administered alone in vivo; it is a synthetic intermediate.
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| Enzyme Assay |
For BRD4 bromodomain binding assays, use time-resolved FRET (TR-FRET) or AlphaScreen. For TR-FRET, incubate recombinant BRD4 (BD1 or BD2) (5-20 nM) with biotinylated acetylated histone peptide (e.g., H4K5acK8acK12acK16ac) (10-50 nM), Eu-labeled anti-GST antibody (for GST-tagged BRD4), and streptavidin-labeled acceptor (XL665). Add varying concentrations of the test moiety (0.01-10000 nM). Incubate for 1-2 h at room temperature. Measure TR-FRET signal (Ex 340 nm, Em 665/620 nm). Calculate IC₅0. For fluorescence polarization (FP), use a fluorescently labeled small molecule competitor (e.g., FAM-JQ1) and BRD4 protein. For surface plasmon resonance (SPR), immobilize BRD4 on a sensor chip, flow compound at 0.1-1000 nM, and determine KD. For selectivity profiling, test against other bromodomains (BRD2, BRD3, BRDT, CREBBP, EP300, etc.).
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| Cell Assay |
For cellular assays, culture BRD4-dependent cancer cell lines such as MV-4-11 (AML), MM.1S (multiple myeloma), or NCI-H929 in RPMI with 10% FBS at 37degC with 5% CO2. For cell viability, treat cells with the PROTAC (or the moiety as a control) at 0.1 nM to 10 uM for 72 h, then measure with CellTiter-Glo. For BRD4 degradation studies (once the moiety is conjugated into a PROTAC), treat cells for 4-24 h, harvest lysates, and perform Western blot for BRD4 (and other BET proteins). For MOI alone (no linker/E3 ligand), it should not induce degradation but only occupancy; use as a negative control. For cellular BRD4 target engagement, perform BRD4 CETSA (cellular thermal shift assay): treat cells with compound (1-10 uM) for 1-2 h, heat lysates to a temperature gradient, and detect BRD4 by Western blot. For MYC downregulation, measure c-MYC mRNA by qPCR or protein by Western blot after 6-24 h treatment. For colony formation assays, treat cells for 7-14 days, fix and stain with crystal violet. For cell cycle analysis, stain with PI and analyze by flow cytometry. For apoptosis, measure Annexin V/PI or caspase-3/7 activity.
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| Animal Protocol |
In vivo studies using the full PROTAC: Use female BALB/c nude mice (6-8 weeks, 18-22 g) bearing subcutaneous xenografts of MV-4-11 or other BRD4-dependent cells. When tumors reach 150-250 mm3, administer PROTAC (typically 10-100 mg/kg) intraperitoneally (i.p.) or intravenously (i.v.), once daily or every other day. Formulate in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline or in 20% SBE-beta-CD in saline. Monitor tumor volume and body weight every 2-3 days. At endpoint, collect tumors and measure BRD4 protein levels by Western blot and IHC. For pharmacodynamics, collect tumors at 2, 6, 12, 24, 48 h post-dose. For PK, collect plasma at multiple time points and quantify PROTAC by LC-MS/MS. For the isolated BRD4-binding moiety, it can be tested as an inhibitor: typical dose 30-100 mg/kg i.p. or p.o. in xenograft models.
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| ADME/Pharmacokinetics |
The specific molecular formula and weight depend on the exact structure of PROTAC BRD4-binding moiety 1. Common BRD4-binding warheads include JQ1 (C22H1₉ClN4O2S, MW 439.0), I-BET762 (C1₈H22N4O2, MW 326.4), or similar derivatives with an additional linker handle. Based on CAS 2101200-10-4, it is likely a JQ1-like derivative with a carboxylate or amine group for conjugation. Solubility: soluble in DMSO (typically ≥20 mM). Storage: powder at -20degC, desiccated, protected from light. Solutions in DMSO can be stored at -80degC for 6 months. The compound is stable for at least 2 years as powder.
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| Toxicity/Toxicokinetics |
Handle with standard laboratory precautions: use personal protective equipment (gloves, lab coat, safety glasses), avoid inhalation and skin contact. The compound is for research use only and not for human therapeutic use. Based on its mechanism (BRD4 inhibition), it may have toxicities including thrombocytopenia, gastrointestinal effects, and potential teratogenicity. No specific safety data are available for this exact moiety. Consult the safety data sheet if available. Dispose of waste according to local regulations for chemical hazardous waste.
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| References | |
| Additional Infomation |
PROTAC BRD4-binding moiety 1 is a BRD4 inhibitor fragment designed for PROTAC development. BRD4 is an epigenetic regulator and a validated oncology target in acute myeloid leukemia, multiple myeloma, NUT midline carcinoma, and other cancers. Traditional BRD4 inhibitors (e.g., JQ1, OTX015, I-BET762) show antitumor activity but require continuous target occupancy. PROTACs that degrade BRD4 offer advantages including prolonged pharmacodynamics, reduced off-target effects, and the ability to target scaffolding functions of BRD4. This moiety is used to conjugate with E3 ligase ligands (VHL or CRBN) via linkers to create potent BRD4 degraders (e.g., dBET1, ARV-825, MZ1). This product is for research use only and is not approved for clinical use. It is supplied as a synthetic intermediate for chemical biology.
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| Molecular Formula |
C23H21N3O2
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| Molecular Weight |
371.431745290756
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| Exact Mass |
371.163
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| CAS # |
2101200-10-4
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| PubChem CID |
141488040
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.5
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
28
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| Complexity |
623
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1(C)C2=C(C=C(C3=C(C)ON=C3C)C=C2)C(C2=CC=CC=C2)N(CC#C)C1=O
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| InChi Key |
ZKJVDZJHVLJOKO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H21N3O2/c1-5-13-26-22(17-9-7-6-8-10-17)19-14-18(21-15(2)24-28-16(21)3)11-12-20(19)25(4)23(26)27/h1,6-12,14,22H,13H2,2-4H3
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| Chemical Name |
6-(3,5-dimethyl-1,2-oxazol-4-yl)-1-methyl-4-phenyl-3-prop-2-ynyl-4H-quinazolin-2-one
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| Synonyms |
PROTAC BRD4binding moiety 1; PROTAC BRD4 binding moiety 1
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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 : ~120 mg/mL (~323.08 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 30.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6923 mL | 13.4615 mL | 26.9230 mL | |
| 5 mM | 0.5385 mL | 2.6923 mL | 5.3846 mL | |
| 10 mM | 0.2692 mL | 1.3461 mL | 2.6923 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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