BRD4 (DC50 = 0.15 nM); bivalent glue

- IBG1 resulted in potent BRD4 degradation with a half-maximal degradation concentration (DC50) of 0.15 nM and pronounced growth inhibition in various cancer cell lines. [1]
- In HEK293 cells, treatment with IBG1 for 6 hours led to potent and specific degradation of BRD2 and BRD4 compared to their paralogue BRD3, as quantified by immunoblot. [1]
- Whole-proteome changes in KBM7 cells after 6 hours of treatment with 1 nM IBG1 showed specific degradation of BRD2 and BRD4. [1]
- The proteasome inhibitor MG132 and the neddylation inhibitor MLN4924 blocked IBG1-induced BET protein degradation and/or ubiquitination, indicating that the compound functions via CRL-mediated ubiquitination and proteasomal degradation. [1]
- IBG1 functioned independently of DCAF15. Instead, members of the CRL4-DCAF16 complex (CUL4A, RBX1, DDB1, and DCAF16) were identified as required for IBG1 function. Invalidation of CRL4-DCAF16 complex subunits prevented degradation of BRD4-BFP as well as endogenous BRD2 and BRD4. [1]
- Knockout of DCAF16 prevented the induction of apoptosis by IBG1 and led to enhanced tolerance of KBM7 cells. [1]
- IBG1 fragments containing truncations of the sulfonamide moiety (compounds 1e-g) did not promote BRD4 degradation despite efficient binding to BRD4, suggesting that the E7820 moiety is required for IBG1 activity. [1]
- In cellular assays using BRD4-BFP reporter KBM7 cell lines, isolated BRD4 bromodomains (BD1 and BD2) were not degraded by IBG1, whereas a construct containing both bromodomains (BRD4Tandem) was sufficient for degradation. [1]
- Disruption of the JQ1 binding sites in either bromodomain via single asparagine to phenylalanine changes (N140F or N433F) was sufficient to prevent degradation by IBG1. [1]
- IBG1 selectively degraded BRD2 and BRD4 but not BRD3 in the BRD4-BFP reporter assay. [1]

- Isothermal Titration Calorimetry (ITC): The formation of a ternary complex between IBG1, DCAF16, and BRD4Tandem was observed by ITC, yielding a dissociation constant (Kd) of 567 nM. ITC titrations of DCAF16 into unbound versus IBG1-bound BRD4Tandem revealed that IBG1 strengthens (Kd of 0.6 µM vs. 4 µM) and thermodynamically alters the BRD4-DCAF16 interaction, changing the binding from exothermic to endothermic (ΔH of -8 kJ mol⁻¹ vs. 38 kJ mol⁻¹). The entropic term (TΔS) becomes much more favourable in the presence of IBG1 (TΔS of 22.5 kJ mol⁻¹ vs. 73.9 kJ mol⁻¹), leading to a greater binding energy (ΔG of -35.7 vs. -30.6 kJ mol⁻¹) and a favourable binding energy change (ΔΔG) of -5.1 kJ mol⁻¹. [1]
- Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET): A ternary complex formation assay showed that a complex formed between DCAF16 and BRD4Tandem in a dose-dependent manner upon IBG1 titration, with a half-maximal effective concentration (EC50) of 44 nM. A complementary TR-FRET-based complex-stabilization assay confirmed an interaction upon titrating DCAF16 into BRD4Tandem in the presence of IBG1 (Kd = 712 nM). An intrinsic affinity of DCAF16 to BRD4Tandem was also observed in the absence of IBG1 (Kd = 1 µM by TR-FRET and Kd = 4 µM by ITC). [1]
- Size-Exclusion Chromatography (SEC): SEC experiments showed that DCAF16 and BRD4Tandem co-eluted in the absence of compound, and this interaction was stabilized by IBG1. No interaction was observed with isolated BD1 and BD2. Increased retention of IBG1-bound BRD4Tandem was observed compared with unbound or JQ1-bound BRD4Tandem, indicating a decreased hydrodynamic radius consistent with compaction through intramolecular dimerization. [1]
- AlphaLISA Displacement Assay: In alphaLISA displacement assays, the affinity of IBG1 to BRD4Tandem was significantly enhanced in the presence of DCAF16 (IC50 = 12.8 nM) compared with IBG1 and BRD4Tandem alone (IC50 = 462 nM), with a calculated cooperativity (α) of 36. DCAF16 did not induce the binding of IBG1 to isolated BRD4-BD1. [1]
- Fluorescence Polarization Assay: A dose-dependent binding of a FITC-labelled E7820 probe to recombinant DCAF15 was observed, but it showed no affinity for DCAF16.
[1]

- Cell Viability Assay: MV4;11, HCT-116, or KBM7 cells were plated in 96-well plates at densities of 0.5 x 10⁶ (MV4;11 and HCT-116) or 0.1 x 10⁶ (KBM7) cells per ml in 50 µl cell suspension per well. The following day, 2x stocks of compounds were added for a final volume of 100 µl. Cells were treated for 24 h (MV4;11), 72 h (KBM7), or 96 h (HCT-116). CellTiterGlo reagent was added, and luminescence was measured. Results were normalized to DMSO controls. [1]
- Degradation Assays and Western Blotting: HEK293 and HCT-116 cells were plated in 6-well plates at varying densities (0.2 to 0.6 x 10⁶ cells per ml). Media was changed prior to compound treatment. For collection, cells were washed with cold PBS and lysed in RIPA buffer. Protein concentration was determined, and 20-30 µg of lysate was run on NuPAGE 4-12% bis-tris gels. Proteins were transferred to nitrocellulose membranes, blocked, and incubated with primary antibodies (BRD2, BRD3, BRD4, etc.) overnight at 4°C. Membranes were washed and incubated with fluorescent or HRP-conjugated secondary antibodies before imaging. [1]
- HiBiT Degradation Assays: Endogenously tagged HiBiT cells were plated in 96-well plates at 0.5 x 10⁶ cells per ml (50 µl/well). The next day, 2x compound stocks were added to a final volume of 100 µl. Cells were treated for 5, 6, or 24 h before lysis using HiBiT lytic assay buffer. Luminescence was measured on a plate reader. Treated wells were normalized to a DMSO-only control to derive DC50 and maximal degradation (Dmax) values. [1]
- Kinetic Ubiquitination and Degradation Assays: For ubiquitination, HiBiT-tagged HEK293 cells were transfected with LgBiT and Halo-Ub cDNA. The next day, cells were seeded in 96-well plates with or without HaloTag NanoBRET ligand. After overnight incubation, media was replaced with OptiMEM containing Vivazine substrate. 10x compound stocks were added, and NanoBRET signals were measured kinetically for 6 h. For degradation, HiBiT-tagged cells with LgBiT were incubated in Endurazine substrate for 2.5 h before 10x compound addition, with luminescence measured every 15 min for 24 h. [1]
- NanoBRET Bromodomain Conformational Sensor Assay: HEK293 cells were transfected with a dual NanoLuc and Halo-Tagged BRD4Tandem plasmid. The next day, cells were seeded in 96-well plates with or without HaloTag NanoBRET ligand. The following morning, media was replaced with media containing MG132 (10 µM final) for 1 h, then cells were incubated with test compounds for 3 h. NanoBRET substrate solution was added, and the plate was read on a plate reader with a NanoBRET filter. [1]
- Flow Cytometric BRD4 Reporter Assay: KBM7 iCas9 cells stably expressing SFFV-BRD4(S)-mTagBFP-P2A-mCherry reporter were treated with DMSO or IBG1 (1 nM) for 6 h. For genetic perturbations, cells were transduced with lentiviral sgRNA and/or transgene expression vectors. Cas9 expression was induced with doxycycline (0.4 µg ml⁻¹ for 3 days), followed by 6 h of degrader treatment. Cells were stained for sgRNA expression with an APC-conjugated anti-mouse Thy1.1 antibody. Cells were washed and analyzed on an LSR Fortessa. BRD4 abundance was calculated as the ratio of background-subtracted BFP to mCherry mean fluorescence intensity. [1]
- Quantitative Proteomics: KBM7 iCas9 cells (50 x 10⁶ per condition) were treated with DMSO or IBG1 (1 nM) for 6 h in biological triplicates. Cells were lysed, and proteins were processed using filter-aided sample preparation. Peptides were labelled with TMTpro 16plex reagent. Labelled peptides were fractionated by high-pH reverse-phase HPLC and analyzed by LC-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Data were processed using Proteome Discoverer. [1]

[1]. Medicine containing BRD4 protein degradation inducer. JP2023024381.

[2]. Targeted protein degradation via intramolecular bivalent glues. Nature. 2024 Mar;627(8002):204-211.

- Mechanism of Action: IBG1 functions as an intramolecular bivalent glue. Instead of connecting target and ligase in trans like PROTACs, it simultaneously engages and connects two adjacent bromodomains (BD1 and BD2) of the BRD4 target protein in cis. This conformational change 'glues' BRD4 to the E3 ligase DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of the compound. [1]
- Cryo-EM Structure: The structure of the ternary complex formed between BRD4Tandem, IBG1, and DCAF16-DDB1(ΔBPB)-DDA1 was solved by cryo-electron microscopy at a resolution of approximately 3.77 Å. The structure shows that the JQ1 moiety of IBG1 binds canonically to the acetyllysine pocket of BD2, while the E7820 moiety unexpectedly binds to the equivalent pocket of BD1. DCAF16 encloses the hydrophobic dimethylthiophene and phenyl groups of the JQ1 moiety and the linker phenyl, shielding them from solvent. [1]
- Rational Design of Improved Degrader: Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders, such as IBG3, which contains two JQ1 moieties and shows degradation in a low picomolar range (DC50 = 6.7 pM for BRD4). [1]

C44H38N8O5S2

822.953126430511

822.24

C, 64.22; H, 4.65; N, 13.62; O, 9.72; S, 7.79

2684292-71-3

156822078

White to yellow solid powder

6.4

3

11

11

59

1690

1

CC1=C2C(=CNC2=C(C=C1)NS(=O)(=O)C3=CC=C(C=C3)CNC(=O)C4=CC=C(C=C4)C5=CC=C(C=C5)C6=N[C@H](C7=NN=C(N7C8=C6C(=C(S8)C)C)C)CC(=O)OC)C#N

HHSZFSOLNUHGMP-BHVANESWSA-N

InChI=1S/C44H38N8O5S2/c1-24-6-19-35(41-38(24)33(21-45)23-46-41)51-59(55,56)34-17-7-28(8-18-34)22-47-43(54)32-15-11-30(12-16-32)29-9-13-31(14-10-29)40-39-25(2)26(3)58-44(39)52-27(4)49-50-42(52)36(48-40)20-37(53)57-5/h6-19,23,36,46,51H,20,22H2,1-5H3,(H,47,54)/t36-/m0/s1

methyl 2-[(9S)-7-[4-[4-[[4-[(3-cyano-4-methyl-1H-indol-7-yl)sulfamoyl]phenyl]methylcarbamoyl]phenyl]phenyl]-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetate

IBG1; PROTAC BRD4 Degrader-19; IBG 1; 2684292-71-3; IBG-1; methyl 2-[(9~{S})-7-[4-[4-[[4-[(3-cyano-4-methyl-1~{H}-indol-7-yl)sulfamoyl]phenyl]methylcarbamoyl]phenyl]phenyl]-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^{2,6}]trideca-2(6),4,7,10,12-pentaen-9-yl]ethanoate; orb1982275;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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