BET/bromodomain and extra terminal domain
Bromodomain and Extra-Terminal (BET) Family Proteins (BRD4 Bromodomain 1: Ki=2.3 nM for (+)-JQ1 carboxylic acid binding; BRD4 Bromodomain 2: Ki=3.1 nM) [1]

On the surface of B16F10 cells, JQ-1 carboxylic acid reduces the expression of PD-L1 [1].

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(+)-JQ1 carboxylic acid is a BET bromodomain-targeting ligand, serving as the warhead in trivalent PROTACs for specific BET protein degradation [1]
- BET protein binding selectivity: Binds to BRD4 Bromodomain 1 (BD1) and Bromodomain 2 (BD2) with Ki values of 2.3 nM and 3.1 nM, respectively; shows >100-fold selectivity over non-BET bromodomains (e.g., BRD7, BRD9) with Ki>500 nM [1]
- Supports PROTAC-mediated BET degradation: When conjugated to trivalent PROTACs via its carboxylic acid functional group, (+)-JQ1 carboxylic acid maintains BET-binding affinity, enabling the PROTAC to induce dose-dependent degradation of BRD4 in MV4;11 leukemia cells (DC₅₀=120 nM for PROTAC, western blot) [1]

(+)-JQ1 (50 mg/kg) inhibits tumors growth in mice with NMC 797 xenografts. (+)-JQ1 (50 mg/kg) results in effacement of NUT nuclear speckles in mice with NMC 797 xenografts, consistent with competitive binding to nuclear chromatin. (+)-JQ1 (50 mg/kg) induces strong (grade 31) keratin expression in NMC 797 xenografts. (+)-JQ1 (50 mg/kg) promotes differentiation, tumor regression and prolonged survival in mice models of NMC xenografts. (+)-JQ1 (50 mg/kg) results in a significant prolongation in overall survival of SCID-beige mice orthotopically xenografted after intravenous injection with MM.1S-luc+ cells compared to vehicle-treated animals. (+)-JQ1 (50 mg/kg i.p.) leads to a highly significant increase in survival of mice bearing Raji xenografts.

BET bromodomain binding assay (AlphaScreen): Recombinant human BRD4 BD1/BD2 domains are diluted in assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 mM DTT). Serial 3-fold dilutions of (+)-JQ1 carboxylic acid (0.1 nM–1 μM) are mixed with BRD4 domains and biotinylated acetylated histone H4 peptide (substrate) in 384-well plates. Streptavidin-conjugated donor beads and anti-GST acceptor beads (for GST-tagged BRD4) are added, and the mixture is incubated at room temperature for 1 hour. AlphaScreen signal is measured, and Ki values are calculated using nonlinear regression analysis [1]

PROTAC-mediated BRD4 degradation assay: MV4;11 leukemia cells are seeded in 6-well plates (2×10⁶ cells/well) and treated with trivalent PROTACs containing (+)-JQ1 carboxylic acid (0.01–1 μM) for 24 hours. Cells are lysed in RIPA buffer, and proteins are separated by SDS-PAGE. Membranes are probed with primary antibodies against BRD4 and GAPDH (loading control), followed by HRP-conjugated secondary antibodies. Band intensities are quantified by densitometry to determine BRD4 degradation efficiency [1]
- Cell proliferation assay (MTT): MV4;11 cells are seeded in 96-well plates (5×10³ cells/well) and treated with trivalent PROTACs containing (+)-JQ1 carboxylic acid (0.01–5 μM) for 72 hours. MTT reagent is added, incubated at 37°C for 4 hours, and absorbance at 570 nm is measured. IC₅₀ values are calculated for the PROTACs (no standalone data for (+)-JQ1 carboxylic acid) [1]

In vivo formulations used (reported):
1. Dissolved in 5% dextrose; 50 mg/kg; i.p. injection; Nature. 2010 Dec 23;468(7327):1067-73
2. Dissolved in 10% DMSO and 90% of a 10% 2-hydroxypropyl-β-cyclodextrin solution; Leukemia. 2017 Oct;31(10):2037-2047
3. Dissolved in 1% DMSO+5% Glucose+ddH2O; Cell. 2018 Sep 20;175(1):186-199.e19
4. Dissolved in 20% hydroxypropyl-β-cyclodextrin, 5% DMSO, 0.2% Tween-80 in saline; Mol Cancer Ther. 2016 Jun;15(6):1217-26
5. Dissolved in 1:1 propylene glycol:water; J Biol Chem. 2016 Nov 4;291(45):23756-23768
6. Dissolved in 5% DMSO in 10% 2-hydroxypropyl-β-cyclodextrin solution; Cancer Lett. 2017 Aug 28;402:100-109

[1]. Design, Synthesis, and Evaluation of Trivalent PROTACs Having a Functionalization Site with Controlled Orientation. Bioconjug Chem. 2022 Jan 19;33(1):142-151.

[2]. Dual drugs decorated bacteria irradiate deep hypoxic tumor and arouse strong immune responses. Biomaterials. 2022 Jul;286:121582.

We designed, synthesized, and evaluated trivalent PROTACs with controllable orientation functionalization sites. Based on the X-ray crystal structure of the BRD protein degrader MZ1 (1) complex with human VHL and BRD4BD2, we anticipated that the 1,2-disubstituted ethyl group near the JQ-1 moiety of MZ1 (1) could be substituted with a planar benzene ring, serving as a platform for further functionalization. To verify this hypothesis, we first designed six divalent MZ1 derivatives, 2a-c and 3a-c, obtained by combining three substitution modes (1,2-, 1,3-, and 1,4-substitution) on the benzene ring with two combinations of ethylene glycol unit numbers (2 or 1). We then tested the degradation activity of each synthesized compound against BRD4. As expected, we found that the activity of the MZ1 derivative 1,2D-EG2-MZ1 (2a), with two ethylene glycol units on the 1,2-disubstituted benzene ring, was similar to that of MZ1 (1). Based on the structure of 2a, we synthesized and evaluated four isomeric trivalent MZ1 derivatives 15a-15d with tert-butyl ester units on the benzene ring, which can serve as linking groups for further functionalization. Among these four isomers, 1,2,5T-EG2-MZ1 (15c) retained similar BRD4 depletion activity to 2a and no obvious hook effect was observed, and its BRD4 depletion kinetics were the same as those of MZ1 (1). The other isomers also showed BRD4 depletion activity. Therefore, the trivalent PROTACs we synthesized can serve as a high-efficiency platform for further applications. [1]

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The tumor intratumoral environment is a hypoxic, non-inflammatory "cold" state, where many drugs are difficult to accumulate and activate the immune system. Facultative anaerobic Salmonella VNP20009 can itself target hypoxic areas of tumors, invade tumor cells and exert immune effects. We constructed a bio-hybrid NVJ by modifying the bacterial surface with a newly synthesized heptamethrin dye NHS-N782 and a JQ-1 derivative, thereby enabling deep tumor-targeted photothermal therapy and enhanced immunotherapy. Due to the mitochondrial targeting capability of NHS-N782, NVJ is highly sensitive to temperature increases after reaching the tumor. This synergistic strategy promotes systemic immunity by constructing an inflammatory “hot” tumor state through three different dimensions, including the inherent immunogenicity of the bacteria, near-infrared laser-triggered tumor antigens, and downregulation of PD-L1 expression. All of these methods can generate effective and durable T-cell immune responses, thereby suppressing the progression of local and distant tumors in the long term. Using attenuated bacteria to deliver dual drugs to tumor tissue to construct a self-synthesized vaccine provides a new paradigm for enhancing bacterial-mediated cancer immunotherapy. [2]

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(+)-JQ1 carboxylic acid is a carboxyl-functionalized derivative of the potent BET bromine domain inhibitor (+)-JQ1, designed as a targeted warhead for PROTAC synthesis. [1]
- Structural features: The carboxylic acid group (-COOH) serves as a coupling site, which can be linked to PROTAC linkers and effector molecules to form trivalent PROTACs with controllable orientation. [1]
- Mechanism of action (as a PROTAC warhead): It binds to the acetyllysine binding pocket of the BET bromine domain (mainly BRD4), mediating PROTAC-induced ubiquitination and degradation of BET proteins. [1]
- Applications: Specifically designed as an intermediate/functional component in the development of trivalent PROTACs for potential anticancer therapies. [1]

C19H17CLN4O2S

400.88

400.076

C, 56.93; H, 4.27; Cl, 8.84; N, 13.98; O, 7.98; S, 8.00

202592-23-2

(+)-JQ-1;1268524-70-4;(R)-(-)-JQ1 Enantiomer; 1268524-71-5; 202592-23-2 (free); 1426257-60-4 (HCl); 2069219-37-8 (TFA); 2230314-61-9 (xTFA);

66828107

Typically exists as Off-white to yellow solids

1.5±0.1 g/cm3

661.6±65.0 °C at 760 mmHg

353.9±34.3 °C

0.0±2.1 mmHg at 25°C

1.737

2.79

1

6

3

27

613

1

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2C(C([H])([H])[H])=C(C([H])([H])[H])SC=2N2C(C([H])([H])[H])=NN=C2C([H])(C([H])([H])C(=O)O[H])N=1

LJOSBOOJFIRCSO-AWEZNQCLSA-N

InChI=1S/C19H17ClN4O2S/c1-9-10(2)27-19-16(9)17(12-4-6-13(20)7-5-12)21-14(8-15(25)26)18-23-22-11(3)24(18)19/h4-7,14H,8H2,1-3H3,(H,25,26)/t14-/m0/s1

2-[(9S)-7-(4-chlorophenyl)-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]acetic acid

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: >120 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.24 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (6.24 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 3: ≥ 1.39 mg/mL (3.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 13.9 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 4: ≥ 1.39 mg/mL (3.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 13.9 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 5: ≥ 1.39 mg/mL (3.47 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 13.9 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.)