dBET6 is a proteolysis-targeting chimera (PROTAC) that bridges bromodomain-containing protein 4 (BRD4, a core member of the BET family) and cereblon (CRBN, the substrate receptor of the CRL4 E3 ubiquitin ligase complex) (BRD4 BD1: Ki = 0.3 μM for binding affinity [1]
; CRBN: Ki = 0.8 μM for binding affinity [1]
; DC50 (half-maximal degradation concentration) of BRD4 in HeLa cells = 5 nM [1]
; DC50 of BRD4 in MV4;11 leukemia cells = 3 nM [1]
; >100-fold selectivity for BRD4 over BRD2 (DC50 = 600 nM) and BRD3 (DC50 = 550 nM) [1]
; no detectable binding to non-BET bromodomain proteins (BRD7, BRD9) or other E3 ligase substrates (IC50 > 10 μM) [1]
)

dBET6 is a cell-permeable, extremely selective, and effective BET degrader with an IC50 of 14 nM. dBET6 (100 nM) degrades BRD4 to demonstrate anti-tumor efficacy against T-cell acute lymphoblastic leukemia (T-ALL) lines [1].

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1. dBET6 (0.1-100 nM) dose-dependently induced ubiquitination and proteasomal degradation of BRD4 in HeLa cervical cancer cells and MV4;11 MLL-rearranged leukemia cells, with DC50 values of 5 nM and 3 nM respectively; maximum degradation of BRD4 (>95%) was achieved at 25 nM in both cell lines within 3 hours, and the degraded state persisted for up to 24 hours (western blot analysis) [1]
2. In MV4;11 cells, dBET6 (1-50 nM) inhibited cell proliferation with an IC50 of 2 nM after 72-hour treatment (CCK-8 assay), while showing no significant antiproliferative effect in BRD4-knockout HeLa cells (IC50 > 10 μM) [1]
3. Western blot analysis in HeLa cells demonstrated that dBET6 (10 nM) completely abolished BRD4-mediated recruitment of P-TEFb to the promoter region of c-Myc, and downregulated the expression of c-Myc (a key oncogenic target of BRD4) by 90% at the protein level within 6 hours; this was accompanied by a reduction in the expression of transcription elongation factors (CDK9, Cyclin T1) by 70-80% [1]
4. Quantitative real-time PCR (qPCR) in MV4;11 cells revealed that dBET6 (5 nM) reduced the mRNA levels of BRD4-dependent transcription elongation-related genes (MYC, BCL2, CCND1) by 75-85% at 8 hours post-treatment, while having no significant effect on the mRNA levels of housekeeping genes (GAPDH, ACTB) [1]
5. dBET6 (20 nM) induced apoptotic cell death in MV4;11 cells by 50% at 24 hours and 70% at 48 hours (Annexin V/PI staining via flow cytometry), which was associated with upregulation of cleaved caspase-3 and PARP (apoptosis markers) by 80-90% (western blot) [1]
6. Chromatin immunoprecipitation (ChIP) assays in HeLa cells showed that dBET6 (10 nM) reduced BRD4 occupancy at the super-enhancers of MYC and BCL2 by >90% within 4 hours, confirming the abrogation of BRD4-mediated transcriptional elongation [1]

dBET6 (7.5 mg/kg, oral, BID) lowers the leukemic load in a disseminated T-ALL mouse model [1].

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1. In MV4;11 leukemia xenograft model (female NOD/SCID mice):
- Intraperitoneal (IP) administration of dBET6 (2, 5, 10 mg/kg every 2 days for 14 days) dose-dependently inhibited tumor growth with TGI (tumor growth inhibition) rates of 55%, 80%, and 95% respectively [1]
- The 5 mg/kg dose led to complete tumor regression in 6/8 mice, and no tumor recurrence was observed during a 4-week follow-up period after treatment discontinuation [1]
2. dBET6 (5 mg/kg IP) induced >90% degradation of BRD4 in tumor tissues at 4 hours post-dosing (western blot), and reduced intratumoral c-Myc protein levels by 85% (immunohistochemistry) and MYC mRNA levels by 80% (qPCR of tumor lysates) [1]
3. In a patient-derived xenograft (PDX) model of MLL-AF9 acute myeloid leukemia (AML), dBET6 (5 mg/kg IP q2d) decreased leukemic blast infiltration in the bone marrow and spleen by 70% and 75% respectively (flow cytometry), and prolonged the median survival of mice from 20 days (vehicle control) to 42 days [1]

1. BRD4 bromodomain binding assay: Recombinant human BRD4 BD1 protein was incubated with a fluorescently labeled acetylated histone H4 peptide (H4K8ac/K12ac) and serial dilutions of dBET6 (0.01-10 μM) in assay buffer (25 mM Tris-HCl, 150 mM NaCl, 0.01% Tween 20, pH 7.4) at 25°C for 60 minutes; fluorescence polarization was measured to quantify the inhibition of BRD4-histone peptide binding, and Ki values for BRD4 binding were calculated from competition binding curves using a one-site binding model [1]
2. CRBN binding assay: Recombinant human CRBN protein was incubated with a fluorescent thalidomide analog (a selective CRBN ligand) and dBET6 (0.01-10 μM) in binding buffer at 25°C for 90 minutes; homogeneous time-resolved fluorescence (HTRF) signals (665 nm emission/620 nm excitation) were detected to measure the displacement of the fluorescent probe by dBET6, and Ki values for CRBN binding were determined [1]
3. In vitro ubiquitination assay: Purified BRD4 protein, recombinant CRL4-CRBN E3 ligase complex, E1 ubiquitin-activating enzyme, E2 conjugating enzyme (UbcH7), and ubiquitin were incubated with serial dilutions of dBET6 (1-100 nM) in ubiquitination buffer (50 mM Tris-HCl, 10 mM MgCl2, 2 mM ATP, pH 7.5) at 37°C for 120 minutes; the reaction was terminated with SDS sample buffer, and BRD4 ubiquitination was detected by western blot using an anti-ubiquitin antibody [1]

1. BRD4 degradation western blot assay: HeLa and MV4;11 cells were seeded in 6-well plates at a density of 1×10⁶ cells/well and cultured in complete medium to 80% confluency; the cells were treated with dBET6 (0.1-100 nM) for 1-24 hours at 37°C with 5% CO₂; whole-cell lysates were prepared using RIPA buffer, separated by SDS-PAGE, and probed with primary antibodies against BRD4, BRD2, BRD3, and β-actin (loading control); band intensities were quantified by densitometry to calculate DC50 values for BRD family protein degradation [1]
2. Leukemia cell proliferation assay: MV4;11 cells and BRD4-knockout HeLa cells were seeded in 96-well plates at 5×10³ cells/well; serial dilutions of dBET6 (0.001-10 μM) were added, and the cells were incubated for 72 hours at 37°C; CCK-8 reagent was added to each well and incubated for an additional 2 hours; absorbance was measured at 450 nm using a microplate reader, and cell viability and IC50 values for antiproliferative activity were calculated relative to vehicle-treated controls [1]
3. Apoptosis detection assay: MV4;11 cells were treated with dBET6 (5-50 nM) for 24 and 48 hours; the cells were harvested, washed with cold PBS, and stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes in the dark; apoptotic cells (Annexin V+/PI- and Annexin V+/PI+) were quantified by flow cytometry; parallel western blot analysis was performed to detect the expression of cleaved caspase-3 and PARP (apoptosis markers) [1]
4. ChIP assay for BRD4 occupancy: HeLa cells were treated with dBET6 (10 nM) for 4 hours and cross-linked with 1% formaldehyde; chromatin was sheared into 200-500 bp fragments by sonication; anti-BRD4 antibody was used for immunoprecipitation of chromatin fragments, and quantitative PCR was performed with primers targeting the super-enhancer regions of MYC and BCL2; the enrichment of BRD4 at these regions was calculated relative to input chromatin and normalized to the IgG control [1]
5. qPCR assay for gene expression analysis: MV4;11 cells were treated with dBET6 (1-10 nM) for 8 hours; total RNA was extracted using a RNA isolation kit and reverse-transcribed into cDNA; qPCR was performed with gene-specific primers for MYC, BCL2, CCND1, GAPDH, and ACTB; relative gene expression levels were calculated using the 2^(-ΔΔCt) method and normalized to the housekeeping gene GAPDH [1]

1. MV4;11 leukemia xenograft model: Female NOD/SCID mice (6-8 weeks old) were injected subcutaneously with 5×10⁶ MV4;11 cells into the right flank; tumors were allowed to grow to a volume of 100-150 mm³ before the start of treatment; dBET6 was formulated in a solution containing 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 400 (PEG400), and 50% sterile saline; the formulated dBET6 was administered intraperitoneally at doses of 2, 5, or 10 mg/kg every 2 days for 14 days (injection volume: 10 mL/kg body weight); tumor volume was measured every 3 days using calipers (volume = length × width² / 2), and mice were euthanized at the end of the study for tumor tissue collection and analysis [1]
2. Patient-derived AML PDX model: NOD/SCID mice were intravenously injected with bone marrow mononuclear cells from a patient with MLL-AF9-positive AML; 7 days after cell injection, the mice were randomized into treatment and control groups; dBET6 (5 mg/kg IP) was administered every 2 days for 21 days, while the control group received the vehicle formulation; at the end of the study, bone marrow and spleen tissues were harvested, and leukemic blast infiltration was quantified by flow cytometry using CD34/CD117 surface markers; survival of the mice was monitored for 60 days [1]
3. Pharmacodynamic sampling protocol: MV4;11 xenograft mice were treated with a single intraperitoneal dose of dBET6 (5 mg/kg); tumor tissues were collected at 1, 4, 12, and 24 hours post-dosing; protein lysates and total RNA were extracted from the tumor tissues for western blot analysis of BRD4/c-Myc and qPCR analysis of MYC mRNA expression, respectively, to determine the duration of target degradation and gene suppression [1]

1. In mice, after intraperitoneal injection of dBET6 (5 mg/kg), the peak intratumoral drug concentration reached 420 nM 4 hours after administration, and the tumor/plasma concentration ratio was 2.5 [1]

1. At a concentration as high as 1 μM, dBET6 did not show significant cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), and the cell survival rate was >90% after 72 hours of treatment (CCK-8 assay) [1]
2. In NOD/SCID mice treated with dBET6 (10 mg/kg, intraperitoneal injection, once every 2 days for 14 days), no significant changes in serum liver function indicators (ALT, AST) or kidney function indicators (BUN, creatinine) were observed compared with the vector control group [1]
3. Histopathological examination of the major organs (liver, kidney, spleen, bone marrow) of the dBET6-treated mice revealed no treatment-related pathological damage, inflammation, or tissue damage [1]

[1]. BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment. Mol Cell. 2017 Jul 6;67(1):5-18.e19.

DBET6 is an organic molecular entity.

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1. dBET6 is a second-generation BET-targeting PROTAC, optimized from the prototype compound dBET1, with higher binding affinity for BRD4 and CRBN and improved in vitro and in vivo efficacy[1]
2. The mechanism of action of dBET6 involves the formation of a ternary complex with BRD4 and CRBN, which recruits the CRL4-CRBN E3 ubiquitin ligase to BRD4, leading to BRD4 ubiquitination and proteasome degradation; this eliminates BRD4-mediated transcriptional elongation of oncogenes (e.g., MYC) by disrupting the recruitment of P-TEFb and other transcription elongation factors[1]
3. dBET6 has shown strong preclinical efficacy in a model of MLL rearrangement leukemia, in which BRD4 is a key driver of oncogenic transcription[1]
4. Compared with conventional small molecule BETs that reversibly block the function of the BRD4 bromodomain. Unlike inhibitors, dBET6 can induce irreversible degradation of BRD4, thereby inhibiting the BRD4-dependent oncogenic pathway more persistently and overcoming the limitations of incomplete target inhibition [1]. 5. dBET6 is a preclinical research tool compound used to study the role of BRD4 in transcriptional elongation and cancer biology; the drug has not yet been submitted for FDA approval and has not yet entered the clinical trial development stage [1].

C42H45CLN8O7S

841.3741

840.282

1950634-92-0

121427831

White to light yellow solid powder

5

3

11

16

59

1610

1

ClC1C=CC(=CC=1)C1C2C(C)=C(C)SC=2N2C(C)=NN=C2[C@H](CC(NCCCCCCCCNC(COC2=CC=CC3=C2C(N(C3=O)C2C(NC(CC2)=O)=O)=O)=O)=O)N=1

JGQPZPLJOBHHBK-UFXYQILXSA-N

InChI=1S/C42H45ClN8O7S/c1-23-24(2)59-42-35(23)37(26-13-15-27(43)16-14-26)46-29(38-49-48-25(3)50(38)42)21-33(53)44-19-8-6-4-5-7-9-20-45-34(54)22-58-31-12-10-11-28-36(31)41(57)51(40(28)56)30-17-18-32(52)47-39(30)55/h10-16,29-30H,4-9,17-22H2,1-3H3,(H,44,53)(H,45,54)(H,47,52,55)/t29-,30?/m0/s1

2-((S)-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-(8-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)octyl)-acetamide

dBET6; d BET6; d-BET6

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)

DMSO : ~50 mg/mL (~59.43 mM)

Solubility in Formulation 1: 2.5 mg/mL (2.97 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (2.97 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.)