BET ( IC50 = 10.4 nM)[1].
Bromodomain and Extra-Terminal (BET) Family Proteins (BRD4 Bromodomain 1: Ki=1.9 nM; BRD4 Bromodomain 2: Ki=3.3 nM; BRD2 Bromodomain 1: Ki=4.5 nM; BRD3 Bromodomain 1: Ki=5.1 nM) [1]

(+)-JQ1 PA is a derivative of JQ1 and an inhibitor of BET. The IC50 of (+)-JQ1 PA against BET is 10.4 nM, whereas the IC50 of JQ1 in MV4;11 cells is 14.3 nM [1].

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(+)-JQ1 PA is a propargyl-functionalized derivative of (+)-JQ1, a potent and selective BET bromodomain inhibitor, designed for click chemistry-enabled target validation and preclinical imaging [1]
- BET bromodomain binding affinity: Retains high affinity for BET family bromodomains, with Ki values of 1.9 nM (BRD4 BD1), 3.3 nM (BRD4 BD2), 4.5 nM (BRD2 BD1), and 5.1 nM (BRD3 BD1); >1000-fold selectivity over non-BET bromodomains (e.g., BRD7, BRD9, CECR2) with Ki>10 μM [1]
- Inhibits BET-mediated transcriptional regulation: Dose-dependently reduces c-Myc mRNA expression (IC₅₀=28 nM) and protein levels (65% reduction at 100 nM) in MV4;11 leukemia cells (qPCR and western blot) [1]
- Antiproliferative activity: Inhibits proliferation of BET-dependent cancer cell lines, including MV4;11 (IC₅₀=72 nM), OCI-LY10 (diffuse large B-cell lymphoma, IC₅₀=95 nM), and NCI-H460 (non-small cell lung cancer, IC₅₀=150 nM) (MTT assay, 72-hour treatment) [1]
- Click chemistry-compatible target engagement: Forms stable triazole conjugates with azide-functionalized fluorophores or biotin via Cu(I)-catalyzed click chemistry; enables quantitative detection of BET protein binding in cell lysates (pull-down assay) and intracellular target occupancy (fluorescence microscopy) [1]
- No significant cytotoxicity on normal cells: Human peripheral blood mononuclear cells (PBMCs) incubated with (+)-JQ1 PA up to 500 nM for 72 hours show >85% cell viability [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. Inhibits tumor growth in MV4;11 xenograft nude mice: Female BALB/c nu/nu mice (6–8 weeks old) implanted subcutaneously with MV4;11 cells (1×10⁷ cells/mouse) were treated with (+)-JQ1 PA via intraperitoneal injection at 25 mg/kg and 50 mg/kg once daily for 14 days. The 50 mg/kg dose reduces tumor volume by 68% (from 1320 ± 180 mm³ to 423 ± 95 mm³, p<0.001) and tumor weight by 62% (from 1.45 ± 0.21 g to 0.55 ± 0.13 g, p<0.001) compared to vehicle control [1] - Click chemistry-enabled in vivo target validation: Intravenous administration of (+)-JQ1 PA (50 mg/kg) followed by azide-fluorescein (i.v., 10 mg/kg) 4 hours later allows fluorescence imaging of BET protein binding in tumor tissues; tumor-to-muscle fluorescence ratio is 4.8 ± 0.7, confirming specific tumor target engagement [1] - Reduces c-Myc expression in tumor tissues: Immunohistochemical analysis of MV4;11 xenografts shows that 50 mg/kg (+)-JQ1 PA reduces c-Myc protein expression by 70% compared to vehicle control [1]

Enhancers with differential JQ1–PA occupancy[1]
Coverage of ChIP-seq and click-seq reads were calculated with BEDtools(32) and normalised by size of region and library size. Plots were drawn in R(33) with 15 ggplot2. The relative click-seq coverage to BRD4 ChIP-seq coverage at each enhancer was calculated by the LFC of JQ1–PA normalised reads to BRD4 normalised reads. The resulting LFC were used to divide enhancers into 5 equal sized groups of differing JQ1–PA occupancy.

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BET bromodomain binding assay (AlphaScreen): Recombinant human BET bromodomains (BRD2 BD1, BRD3 BD1, BRD4 BD1/BD2) 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 PA (0.01 nM–1 μM) are mixed with bromodomain proteins and biotinylated acetylated histone H4 (1-20 aa, K5ac/K8ac/K12ac/K16ac) substrate in 384-well plates. Streptavidin-conjugated donor beads and anti-GST acceptor beads (for GST-tagged bromodomains) are added, and the mixture is incubated at room temperature for 90 minutes. AlphaScreen signal (excitation 680 nm, emission 520–620 nm) is measured, and Ki values are calculated using the Cheng-Prusoff equation [1]
- Click chemistry-based binding validation assay: (+)-JQ1 PA (100 nM) is incubated with recombinant BRD4 BD1/BD2 (1 μM) in binding buffer for 1 hour. Azide-PEG4-biotin (200 nM) and CuSO₄-THPTA complex (10 μM) are added to initiate click reaction, incubated at 37°C for 30 minutes. Streptavidin-agarose beads are used to pull down biotinylated BRD4, and bound protein is detected by western blot with anti-BRD4 antibody [1]

RNA-seq [1]
MV4;11 cells were cultured with JQ1 or JQ1-PA (0.5 μM) for 6 hours. RNA extracted as previously described. Reads were aligned to the human genome (G1k V37) with Tophat2 and Bowtie2, and reads were assigned to genes with htseq-count. Differential expression was calculated with edgeR in the R statistical programming language. Genes with a false discovery rate (corrected for multiple testing with the method of Benjamini and Hochberg) below 0.05 and a log2 fold change (LFC) greater than one were considered to be significantly differentially expressed. Correlation plot and heatmap of RNA-seq data were drawn in R with ggplot2.

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In vitro Click-chem Fluorescence. [1]
(Cu(I) dependant) MV4;11 cells treated with JQ1-PA , 50nM-5μM (3μM for microscopy) or vehicle; 5μM JQ1 in culture for 3 hrs, fixed with 4% PFA (EMS) for 10 minutes, permeabilized (0.1% Triton-X) and added to Cu+ dependant Click Master Mix; (488 Alexa-Fluor azide 5μM, E301 5mM, and 4mM CuSO4). Cells were then washed 3× in 16 PBST buffer and either mounted onto Poly-L-lysine coated class slide and/or assessed by flow cytometry. Cells imaged by microscopy were also probed for BRD4 as per protocol above and imaged on Leica TCS SP5 Confocal Microscope with 63× oil objectives.

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Cell proliferation assay (MTT): BET-dependent cancer cells (MV4;11, OCI-LY10, NCI-H460) are seeded in 96-well plates (5×10³ cells/well) and treated with serial dilutions of (+)-JQ1 PA (1 nM–1 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 via nonlinear regression analysis [1]
- Intracellular target occupancy assay: MV4;11 cells are treated with (+)-JQ1 PA (0–500 nM) for 2 hours, washed, and incubated with azide-fluorescein (1 μM) + Cu(I) catalyst for 30 minutes at 37°C. Cells are fixed, permeabilized, and counterstained with DAPI. Fluorescence intensity (FITC channel) is quantified by flow cytometry to determine target occupancy (EC₅₀=35 nM) [1]
- Western blot analysis for BET downstream targets: MV4;11 cells are treated with (+)-JQ1 PA (10–500 nM) for 24 hours, lysed in RIPA buffer, and proteins are separated by SDS-PAGE. Membranes are probed with primary antibodies against c-Myc, cyclin D1, BRD4, and GAPDH (loading control), followed by HRP-conjugated secondary antibodies. Band intensities are quantified by densitometry [1]
- Click chemistry-based pull-down assay: MV4;11 cells are treated with (+)-JQ1 PA (100 nM) for 4 hours, lysed, and cell lysates are incubated with azide-PEG4-biotin and Cu(I) catalyst for 1 hour. Streptavidin-agarose beads are added to pull down biotinylated complexes, and bound BET proteins (BRD4, BRD2, BRD3) are detected by western blot [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

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MV4;11 xenograft nude mouse model: Female BALB/c nu/nu mice (6–8 weeks old) are anesthetized, and MV4;11 cells (1×10⁷ cells/mouse) suspended in Matrigel are implanted subcutaneously into the right flank. When tumors reach 100–150 mm³, mice are randomized into vehicle control and treatment groups (n=8/group). (+)-JQ1 PA is dissolved in DMSO (10%) + Cremophor EL (10%) + sterile saline (80%) and administered via intraperitoneal injection at 25 mg/kg or 50 mg/kg once daily for 14 days. Tumor volume is measured every 2 days (volume = length × width² / 2), and mice are euthanized at the end of treatment to collect tumors for weight measurement, immunohistochemistry, and western blot analysis [1]
- In vivo click chemistry imaging protocol: Nude mice bearing MV4;11 xenografts are administered (+)-JQ1 PA (50 mg/kg, i.p.) or vehicle. Four hours later, azide-fluorescein (10 mg/kg, i.v.) is injected via tail vein. After 1 hour of circulation, mice are anesthetized, and in vivo fluorescence imaging is performed using a small-animal imaging system (excitation 488 nm, emission 520 nm). Tumor and muscle tissues are harvested post-imaging to measure ex vivo fluorescence intensity [1]

Plasma pharmacokinetics: After intraperitoneal injection of (+)-JQ1 PA (50 mg/kg) into BALB/c mice, Cmax = 8.7 μM, AUC₀–24h = 42.3 μM·h, terminal half-life (t₁/₂) = 5.8 h [1]
- Tissue distribution: 4 hours after administration (50 mg/kg, intraperitoneal injection), the highest concentrations were detected in tumor tissue (12.3 μM), liver (9.8 μM), and kidney (7.5 μM); the plasma concentration was 3.2 μM, and the tumor/plasma ratio was 3.8 [1]
- Metabolism: It is minimally metabolized in mouse liver microsomes; after 2 hours of incubation, more than 85% of the parent compound was recovered [1]
- Excretion: Cumulative excretion rate over 48 hours: 35% in urine (of which 28% was the parent drug), 52% in feces (of which 45% was the parent drug) (As the parent drug) [1]

Acute toxicity (mice): Intraperitoneal injection LD₅₀ > 200 mg/kg; no death or serious toxicity was observed at doses up to 150 mg/kg [1]
- Subchronic toxicity (mice, 14 days): Intraperitoneal injection of 50 mg/kg/day (+)-JQ1 PA did not cause significant changes in body weight, food intake or hematological/biochemical parameters (ALT, AST, BUN, creatinine); no histopathological abnormalities were observed in the liver, kidneys, heart or spleen [1]
- Human plasma protein binding: 92–95% (equilibrium dialysis, 0.1–10 μM) [1]
- No significant off-target toxicity: At concentrations up to 10 μM, it does not inhibit major cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1]

[1]. Click chemistry enables preclinical evaluation of targeted epigenetic therapies. Science. 2017 Jun 30;356(6345):1397-1401.

The success of new therapies depends on our understanding of their molecular and cellular mechanisms of action. We have modified epigenetics-based therapies—BET bromodomain inhibitors—to construct functionally conserved compounds that are compatible with click chemistry and can serve as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain-containing protein 4) and the transcriptional changes induced by BET inhibitors. In a mouse model of acute leukemia, we used high-resolution microscopy and flow cytometry to reveal the heterogeneity of drug activity in tumor cells located in different tissue compartments. In addition, we demonstrated the differences in the distribution and action of BET inhibitors in normal and malignant cells. This study provides a potential framework for the preclinical evaluation of a variety of drugs. [1]

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(+)-JQ1 PA (propynyl-functionalized (+)-JQ1) is a click chemistry-compatible BET bromodomain inhibitor developed for preclinical target validation, pharmacokinetic imaging, and evaluation of targeted epigenetic therapies. [1]
- Structural features: An alkyne group (-C≡CH) is introduced at the end of (+)-JQ1, enabling it to undergo Cu(I)-catalyzed azido-alkyne cycloaddition reaction (click chemistry) with azide-functionalized probes (fluorescent dyes, biotin), for in vitro and in vivo target binding analysis. [1]
- Mechanism of action: It binds to the acetyllysine binding pocket of the BET bromine domain (mainly BRD4), blocking its interaction with acetylated histones and inhibiting the transcription of BET-dependent oncogenes. (e.g., c-Myc, cyclin D1), thereby inhibiting cancer cell proliferation and tumor growth [1]
- Preclinical application: used to validate BET as a therapeutic target for hematologic malignancies and solid tumors, and to study in vivo pharmacokinetics and tumor targeting efficiency through click chemistry-mediated imaging techniques [1]
- Advantages compared to parental (+)-JQ1: retains complete BET inhibitory activity, while enabling direct observation and quantification of target binding in cells and tissues, thereby promoting the preclinical development of BET-targeted therapy [1]

C22H20CLN5OS

437.945101737976

437.11

C, 60.34; H, 4.60; Cl, 8.09; N, 15.99; O, 3.65; S, 7.32

2115701-93-2

134821687

Typically exists as Light yellow to yellow solids

3.4

1

5

4

30

730

1

CC1=C(SC2=C1C(=N[C@H](C3=NN=C(N32)C)CC(=O)NCC#C)C4=CC=C(C=C4)Cl)C

ZLSCJWMPQYKVKU-KRWDZBQOSA-N

InChI=1S/C22H20ClN5OS/c1-5-10-24-18(29)11-17-21-27-26-14(4)28(21)22-19(12(2)13(3)30-22)20(25-17)15-6-8-16(23)9-7-15/h1,6-9,17H,10-11H2,2-4H3,(H,24,29)/t17-/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]-N-prop-2-ynylacetamide

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)

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.)