dBRD9 targets BRD9 (bromodomain-containing protein 9, a subunit of the human BAF/SWI/SNF nucleosome remodeling complex) and cereblon (CRBN, a component of the E3 ubiquitin ligase complex). . [1]

Actin, BRD4, and BRD7 expression are not significantly affected by dBRD9 (0.5-5000 nM; 4 h), although it does concentrate the reduction of BRD9 expression in MOLM-13 cells [1]. In MOLM-13 cells, dBRD9 (100 nM; 2 h) is selective for BRD9 [1]. EOL-1 and MOML-13 cell lines exhibit anti-proliferative effects from dBRD9 (7 d) [1].

---

1. dBRD9 is a heterobifunctional ligand that mediates the degradation of BRD9 through bridging BRD9 and the CRBN-DDB1 E3 ubiquitin ligase complex. It exhibits markedly enhanced potency (10- to 100-fold) compared to parental BRD9-targeting ligands. [1]

2. In MOLM-13 acute myeloid leukemia (AML) cells, dBRD9 induces dose-dependent degradation of BRD9 after 4 hours of treatment, as detected by immunoblotting (actin as a loading control). [1]

3. dBRD9 shows high biochemical and cellular selectivity: it does not significantly affect the expression of BRD7 or BRD4 (other bromodomain-containing proteins) in MOLM-13 cells, as confirmed by immunoblotting. Bromoscan analysis (phage-displayed bromodomain displacement assay) demonstrates its selectivity for BRD9 among bromodomains. [1]

4. Mechanistic studies in MM.1S cells show that dBRD9-induced BRD9 degradation is dependent on the ubiquitin-proteasome pathway: pre-treatment with the proteasome inhibitor carfilzomib, the NEDD8-activating enzyme inhibitor MLN-4924, or the CRBN ligand lenalidomide abrogates BRD9 degradation; pre-treatment with the BRD9 bromodomain inhibitor I-BRD9 also blocks this effect. Additionally, dBRD9 fails to degrade BRD9 in CRBN-knockout (MM.1S^CRBN−/−) cells, confirming CRBN dependence. [1]

5. dBRD9 induces time-dependent BRD9 degradation in MOLM-13 cells: significant BRD9 reduction is observed after treatment with 100 nM dBRD9 for indicated time points (detected by immunoblotting). [1]

6. Whole-cell lysate proteomics analysis (quintuplicate replicates) of MOLM-13 cells treated with 100 nM dBRD9 for 2 hours (versus DMSO vehicle) confirms its selectivity: only BRD9 shows a significant fold change in relative abundance, with no major alterations in 7325 other quantified proteins. [1]

7. dBRD9 affects the viability of cultured human leukemia cell lines: treatment of EOL-1 and MOLM-13 cells with dBRD9 for 7 days reduces cell viability, as measured by ATP-Lite assay (quadruplicate means ± SEM). This effect is rescued by transduction of recombinant BRD9 alleles in MOLM-13 AML cells (vector control as reference). [1]

8. dBRD9 promotes the formation of a ternary complex between recombinant BRD9 bromodomain (BRD9(bd)) and CRBN-DDB1, as detected by AlphaScreen assay (quadruplicate means ± SEM). [1]

1. BRD9 bromodomain displacement assay (AlphaScreen): Recombinant BRD9 bromodomain (BRD9(bd)) is used to evaluate the displacement activity of dBRD9. The assay is performed in quadruplicate, and results are presented as vehicle-normalized displacement values with ± SEM. This assay measures the binding of dBRD9 to BRD9(bd). [1]

2. CRBN-DDB1 ternary complex formation assay (AlphaScreen): Recombinant BRD9(bd) and CRBN-DDB1 proteins are incubated with dBRD9, and the formation of the ternary complex is detected by AlphaScreen. The assay is conducted in quadruplicate, with results expressed as vehicle-normalized values ± SEM. A parallel assay with recombinant BRD4(1) bromodomain is used to confirm selectivity (no significant ternary complex formation with BRD4(1)). [1]

3. Bromodomain selectivity assay (Bromoscan): Phage-displayed bromodomains are used to assess the selectivity of dBRD9. The assay measures the displacement of dBRD9 from various bromodomains to confirm its preferential binding to BRD9. [1]

Western Blot Analysis[1]
Cell Types: MOLM-13 cells.
Tested Concentrations: 0.5, 5, 50, 500 and 5000 nM.
Incubation Duration: 4 hrs (hours).
Experimental Results: The expression of BRD9 was diminished.

---

1. BRD9 degradation detection by immunoblotting: MOLM-13 or MM.1S cells are treated with dBRD9 at indicated concentrations or time points (100 nM for time-course experiments). Cell lysates are prepared, and BRD9 protein levels are analyzed by immunoblotting, with actin as a loading control. For selectivity testing, BRD7 and BRD4 protein levels are also detected by immunoblotting in MOLM-13 cells. [1]

2. Mechanistic validation assay: MM.1S cells are pre-treated with vehicle, I-BRD9 (BRD9 inhibitor), lenalidomide (CRBN ligand), carfilzomib (proteasome inhibitor, 30-minute pre-treatment), or MLN-4924 (NEDD8-activating enzyme inhibitor) for 4 hours, followed by 2 hours of treatment with 100 nM dBRD9. BRD9 degradation is evaluated by immunoblotting (actin as control). Additionally, MM.1S^wt and MM.1S^CRBN−/− cells are treated with dBRD9 at indicated doses for 4 hours, and BRD9 levels are detected by immunoblotting. [1]

3. Cell viability assay: EOL-1 and MOLM-13 leukemia cells are treated with dBRD9 at indicated concentrations for 7 days. Cell viability is measured using the ATP-Lite assay, with results presented as quadruplicate means ± SEM. For rescue experiments, MOLM-13 cells are transduced with recombinant BRD9 alleles or vector control prior to dBRD9 treatment, and viability is assessed similarly. [1]

4. Whole-cell proteomics analysis: MOLM-13 cells are treated with 100 nM dBRD9 or DMSO vehicle for 2 hours (quintuplicate replicates). Whole-cell lysates are prepared, and 7326 proteins are quantified by proteomics. The fold change in relative abundance of each protein and corresponding q-values are calculated to assess the selectivity of dBRD9. [1]

[1]. Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands. Angew Chem Int Ed Engl. 2017 May 15;56(21):5738-5743.

1. dBRD9 is the first chemical degrader targeting BRD9, designed through iterative optimization of a heterobifunctional ligand. [1] 2. The mechanism of action of dBRD9 is to recruit CRBN E3 ubiquitin ligase to BRD9, leading to BRD9 ubiquitination, followed by degradation via the proteasome pathway. [1] 3. dBRD9 solves the problem of multiple pharmacological aspects of the bromine domain in BRD9-targeting drugs and can serve as a powerful tool for studying other functions of BRD9 besides acetyllysine recognition. [1] 4. BRD9 has become a highly attractive target in cancer treatment, especially in acute myeloid leukemia. [1] 5. dBRD9 belongs to the naphthidinone class of BRD9 degraders and has higher biochemical and cellular selectivity compared to earlier thienopyridinone degraders. [1]

C40H45N7O10

783.826209783554

783.322

2170679-45-3

dBRD9 dihydrochloride;2341840-98-8

135397681

Light yellow to yellow solid powder

0.9

3

13

18

57

1500

0

O=C1C(CCC(N1)=O)N1C(C2C=CC=C(C=2C1=O)NCCOCCOCCNC(CN(C)CC1C(=CC(=CC=1OC)C1=CN(C)C(C2C=NC=CC1=2)=O)OC)=O)=O

AIOCFZJGGGEWDK-UHFFFAOYSA-N

InChI=1S/C40H45N7O10/c1-45(21-29-32(54-3)18-24(19-33(29)55-4)28-22-46(2)38(51)27-20-41-11-10-25(27)28)23-35(49)43-13-15-57-17-16-56-14-12-42-30-7-5-6-26-36(30)40(53)47(39(26)52)31-8-9-34(48)44-37(31)50/h5-7,10-11,18-20,22,31,42H,8-9,12-17,21,23H2,1-4H3,(H,43,49)(H,44,48,50)

2-((2,6-Dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)(methyl)amino)-N-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)acetamide

dBRD-9; dBRD 9; dBRD9

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 : ~100 mg/mL (~127.58 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)