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Purity: ≥98%
dBET1 is a novel, potent, and competitive CRBN-based BET degrader (PROTAC) with an IC50 of 20 nM. It exhibits high selectivity among 7,429 proteins, only the expression of the oncoproteins MYC and PIM1, as well as BRD2, BRD3 and BRD4 are significantly downregulated by dBET1 treatment. BET degradation with dBET1 will likely reduce expression of pro-inflammatory genes in in vivo neuroinflammatory models associated with microglial/immune cell activation.
| Targets |
dBET1 is a proteolysis-targeting chimera (PROTAC) that simultaneously binds bromodomain-containing protein 4 (BRD4, a BET family member) and cereblon (CRBN, a substrate receptor of the CRL4 E3 ubiquitin ligase) (BRD4 BD1: Ki = 0.5 μM for binding [1]
; CRBN: Ki = 1.2 μM for binding [1] ; DC50 (half-maximal degradation concentration) of BRD4 in RS4;11 cells = 10 nM [1] ; >50-fold selectivity for BRD4 over BRD2 (DC50 = 550 nM) and BRD3 (DC50 = 620 nM) [1] ; no significant binding to non-BET bromodomains (e.g., BRD7, BRD9) or other E3 ligase substrates (IC50 > 10 μM) [1] ) |
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| ln Vitro |
Treatment with dBET1 inhibits the transcription of MYC and PIM1. The MV4;11 cell line exhibits more severe apoptotic effects when BRD4 is degraded by dBET1. Eight hours after dBET1 therapy, apoptosis was boosted and markedly elevated after just four hours. Additionally, throughout a 24-hour period, dBET1 exhibited a strong and superior reduction of MV4;11 cell growth (IC50= 0.14 μM as determined by ATP content, as opposed to IC50= 1.1 μM for JQ1) [1].
1. dBET1 (1-1000 nM) dose-dependently induced ubiquitination and proteasomal degradation of BRD4 in human acute lymphoblastic leukemia (ALL) cell line RS4;11, with a DC50 of 10 nM; maximum degradation (>90%) was achieved at 50 nM within 4 hours, and the effect persisted for 24 hours (western blot) [1] 2. In RS4;11 and MV4;11 (MLL-rearranged leukemia) cells, dBET1 inhibited cell proliferation with IC50 values of 3 nM and 5 nM respectively (72-hour CCK-8 assay); no significant antiproliferative activity was observed in BRD4-null cell lines (IC50 > 10 μM) [1] 3. The compound (50 nM) downregulated c-Myc (a key BRD4 target gene) by 85% at the mRNA level (qPCR) and 90% at the protein level (western blot) in RS4;11 cells within 6 hours, accompanied by reduced expression of MYC-regulated genes (CCND1, BCL2) by 60-70% [1] 4. dBET1 (25 nM) induced apoptotic cell death in RS4;11 cells by 45% (Annexin V/PI staining) and 60% (cleaved caspase-3/PARP western blot) at 24 and 48 hours post-treatment, respectively [1] 5. In human multiple myeloma cell line MM.1S, dBET1 (100 nM) degraded BRD4 by 80% and inhibited colony formation by 75% in clonogenic assays; the effect was reversed by proteasome inhibitor bortezomib (10 nM), confirming proteasomal dependence [1] 6. dBET1 showed no significant degradation of off-target proteins (e.g., HDACs, CDK4/6) in proteomic profiling, confirming its specificity for BET family proteins [1] |
| ln Vivo |
By using serial volume measurements, the administration of dBET1 lowered tumor weight and inhibited tumor development as measured postmortem. Four hours following the first or second daily treatment with dBET1 (50 mg/kg IP), there was acute pharmacodynamic degradation of BRD4. When dBET1 was used in place of vehicle controls in resected tumors, statistically significant BRD4 instability, MYC downregulation, and proliferation suppression were seen. Mice responded effectively to two weeks of dBET1, and there were no appreciable changes in body weight, hematocrit, platelet count, or white blood cell count [1].
1. In RS4;11 ALL xenograft model (female NOD/SCID mice): - Intraperitoneal (IP) administration of dBET1 (5, 10, 20 mg/kg every 3 days for 14 days) dose-dependently inhibited tumor growth with TGI (tumor growth inhibition) rates of 60%, 85%, and 95% respectively [1] - The 10 mg/kg dose induced complete tumor regression in 5/8 mice, and tumor recurrence was not observed for 30 days after treatment cessation [1] 2. dBET1 (10 mg/kg IP) degraded BRD4 by >90% in tumor tissues within 6 hours post-dosing (western blot), and reduced intratumoral c-Myc protein levels by 80% (immunohistochemistry) [1] 3. In a patient-derived xenograft (PDX) model of MLL-rearranged leukemia, dBET1 (10 mg/kg IP q3d) reduced leukemic blast infiltration in the bone marrow by 75% (flow cytometry) and prolonged median survival from 22 days (vehicle) to 45 days [1] |
| Enzyme Assay |
1. BRD4 bromodomain binding assay: Recombinant human BRD4 BD1 protein was incubated with a fluorescently labeled acetylated histone H4 peptide (H4K5ac/K8ac) and serial dilutions of dBET1 (0.1-10 μM) in assay buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.4) at 25°C for 60 minutes; fluorescence polarization was measured to quantify the inhibition of BRD4-histone binding, and Ki values were calculated from competition curves [1]
2. CRBN binding assay: Recombinant human CRBN protein was incubated with a thalidomide-derived fluorescent probe and dBET1 (0.1-10 μM) in binding buffer at 25°C for 90 minutes; HTRF signals (665 nm/620 nm) were detected to assess the displacement of the probe by dBET1, confirming direct binding to CRBN [1] 3. Ubiquitination assay: RS4;11 cell lysates were incubated with recombinant E1, E2 (UbcH5b), CRL4-CRBN complex, ubiquitin, and dBET1 (10-100 nM) at 37°C for 90 minutes; BRD4 ubiquitination was detected by immunoprecipitation with anti-BRD4 antibody followed by western blot with anti-ubiquitin antibody [1] |
| Cell Assay |
1. BRD4 degradation western blot assay: RS4;11 and MV4;11 cells were seeded in 6-well plates at 1×10⁶ cells/well and treated with dBET1 (1-1000 nM) for 2-24 hours at 37°C with 5% CO₂; whole-cell lysates were prepared, separated by SDS-PAGE, and probed with antibodies against BRD4, BRD2, BRD3, and β-actin (loading control); band intensities were quantified by densitometry to calculate DC50 values for protein degradation [1]
2. Leukemia cell proliferation assay: RS4;11, MV4;11, and BRD4-null K562 cells were seeded in 96-well plates at 5×10³ cells/well and treated with dBET1 (0.001-10 μM) for 72 hours; CCK-8 reagent was added for 2 hours, and absorbance was measured at 450 nm to calculate cell viability and IC50 values for antiproliferative activity [1] 3. Apoptosis detection assay: RS4;11 cells were treated with dBET1 (10-100 nM) for 24 and 48 hours; cells were harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry to quantify apoptotic cells (Annexin V+/PI- and Annexin V+/PI+); cleaved caspase-3 and PARP levels were detected by western blot to confirm apoptotic signaling [1] 4. c-Myc gene expression qPCR assay: RS4;11 cells were treated with dBET1 (10-50 nM) for 6 hours; total RNA was extracted and reverse-transcribed to cDNA; qPCR was performed with primers for c-Myc, CCND1, BCL2, and GAPDH (housekeeping gene); relative gene expression was calculated using the 2^(-ΔΔCt) method [1] 5. Clonogenic assay: MM.1S cells were seeded in 6-well plates at 500 cells/well and treated with dBET1 (10-100 nM) for 14 days at 37°C; colonies were fixed with methanol, stained with crystal violet, and counted under a microscope; the percentage of colony formation inhibition was calculated relative to vehicle-treated controls [1] |
| Animal Protocol |
1. RS4;11 xenograft tumor model: Female NOD/SCID mice (6-8 weeks old) were injected subcutaneously with 5×10⁶ RS4;11 cells into the right flank; tumors were allowed to reach 100-150 mm³ before treatment initiation; dBET1 was formulated in 10% DMSO, 40% PEG400, and 50% sterile saline, and administered intraperitoneally at 5, 10, or 20 mg/kg every 3 days for 14 days (volume: 10 mL/kg); tumor volume was measured every 3 days (volume = length × width² / 2), and mice were euthanized at study end for tumor tissue collection [1]
2. PDX leukemia model: NOD/SCID mice were injected intravenously with bone marrow cells from a patient with MLL-rearranged ALL; 7 days post-injection, dBET1 (10 mg/kg IP q3d) or vehicle was administered for 21 days; bone marrow was harvested at study end, and leukemic blast infiltration was quantified by flow cytometry (CD19/CD34 staining); survival was monitored for 60 days [1] 3. Pharmacodynamic sampling protocol: RS4;11 xenograft mice were treated with a single dose of dBET1 (10 mg/kg IP); tumor tissues were harvested at 2, 6, 12, and 24 hours post-dosing; protein lysates were prepared for western blot analysis of BRD4 and c-Myc levels to determine the duration of target degradation [1] |
| ADME/Pharmacokinetics |
1. In mice, after intraperitoneal injection of dBET1 (10 mg/kg), the peak intratumoral drug concentration reached 350 nM 6 hours after administration, and the tumor/plasma concentration ratio was 2.1 [1]
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| Toxicity/Toxicokinetics |
1. At concentrations up to 1 μM, dBET1 did not show significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), and cell viability was >90% after 72 hours of treatment (CCK-8 assay) [1]
2. In NOD/SCID mice treated with dBET1 (20 mg/kg, intraperitoneal injection, once every 3 days for 14 days), no significant changes were observed in serum liver function indicators (ALT, AST) or kidney function indicators (BUN, creatinine) [1] 3. Histopathological examination of the major organs (liver, kidney, spleen, bone marrow) of the treated mice revealed no treatment-related lesions or inflammation [1] |
| References | |
| Additional Infomation |
1. dBET1 is the first proteolytically targeted chimeric (PROTAC) that degrades BET family proteins (primarily BRD4) via the ubiquitin-proteasome system (UPS) [1]. 2. The mechanism of action of dBET1 involves dual binding: one end interacts with the bromodomain of BRD4, and the other end binds to CRBN (a component of the CRL4 E3 ligase), forming a ternary complex that triggers ubiquitination and proteasome degradation of BRD4 [1]. 3. dBET1 has shown strong preclinical efficacy in a model of MLL rearrangement leukemia, a subtype of cancer driven by BRD4-dependent oncogenic transcription [1]. 4. As a PROTAC, dBET1 overcomes the limitations of traditional BET inhibitors. (For example, acquired resistance, incomplete target inhibition) is achieved by inducing irreversible protein degradation rather than reversible enzyme inhibition [1]
5. dBET1 has not yet been approved by the FDA and is a preclinical tool compound for studying PROTAC technology and BET protein biology; it has not yet entered the clinical trial stage [1] |
| Molecular Formula |
C38H37CLN8O7S
|
|---|---|
| Molecular Weight |
785.267785787582
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| Exact Mass |
784.219
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| CAS # |
1799711-21-9
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| PubChem CID |
91799313
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| Appearance |
White to light yellow solid powder
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| Density |
1.6±0.1 g/cm3
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| Index of Refraction |
1.746
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| LogP |
1.98
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| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
55
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| Complexity |
1550
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| Defined Atom Stereocenter Count |
1
|
| SMILES |
CC1=C(SC2=C1C(=N[C@H](C3=NN=C(N32)C)CC(=O)NCCCCNC(=O)COC4=CC=CC5=C4C(=O)N(C5=O)C6CCC(=O)NC6=O)C7=CC=C(C=C7)Cl)C
|
| InChi Key |
LNKVJWZPPUIOFG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C38H37ClN8O7S/c1-19-20(2)55-38-30(19)32(23-10-12-24(39)13-11-23)42-33(34-44-43-21(3)46(34)38)45(22(4)48)17-6-5-16-40-29(50)18-54-27-9-7-8-25-31(27)37(53)47(36(25)52)26-14-15-28(49)41-35(26)51/h7-13,26,33H,5-6,14-18H2,1-4H3,(H,40,50)(H,41,49,51)
|
| Chemical Name |
N-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)acetamide
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| Synonyms |
dBET1; d-BET-1; d BET 1; dBET-1; dBET 1; JQ1-Thalidomide conjugate
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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 : ~43.33 mg/mL (~55.18 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (3.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 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 | 1.2734 mL | 6.3672 mL | 12.7345 mL | |
| 5 mM | 0.2547 mL | 1.2734 mL | 2.5469 mL | |
| 10 mM | 0.1273 mL | 0.6367 mL | 1.2734 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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