BRD4 (1/2) (IC50= 77/33 nM)

(+)-JQ-1 represents a strong, highly selective and Kac-competitive inhibitor of the bromodomain BET family. (+)-JQ-1 (100 nM, 48 h) increases squamous development, evidenced by cell spindle formation, flattening, and enhanced keratin expression. (+)-JQ-1 (250 nM) stimulates fast expression of keratin in treated NMC 797 cells compared to (-)-JQ1 (250 nM) and vehicle control, as evaluated by quantitative immunohistochemistry of. (+)-JQ-1 (relative to (-)-JQ1 (250 nM)) causes time-dependent strong (3+) keratin staining in treated NMC 797 cells [1]. Derepression of autophagy genes was seen almost immediately upon addition of (+)-JQ-1 [2]. (+)-JQ-1 is a strong thiophenediazepine inhibitor (Kd=90 nM) of the BET family coactivator protein BRD4, which participates in the development of cancer through the transcriptional regulation of the MYC oncogene. Dose-ranging experiments of (+)-JQ-1 indicated efficient suppression of H4Kac4 binding, with IC50 values of 10 nM for mouse BRDT (1) and 11 nM for human BRDT (1) [3].

Matching mouse cohorts with tumors that had already developed were randomized to receive intraperitoneal injections of either vehicle or (+)-JQ1 (50 mg/kg) every day. FDG-PET imaging was used to assess the mice both four days post-treatment and before to randomization. FDG uptake was shown to be significantly reduced when (+)-JQ1 was administered. Tumor growth was inhibited by JQ1 treatment, as demonstrated by assessments of tumor volume. CD1 mice were used for pharmacokinetic studies of (+)-JQ1 following oral and intravenous dosing. Time profile of the mean plasma concentration of (+)-JQ1 following intravenous injection (5 mg/kg). The half-life (T1/2) of intravenous (+)-JQ1 was approximately one hour, and its pharmacokinetic characteristics demonstrated good drug exposure (AUC=2090 hr*ng/mL). After oral dosage (10 mg/kg), a mean plasma concentration-time profile of (+)-JQ1 was created. Oral (+)-JQ1 pharmacokinetic parameters showed good drug exposure (AUC=2090 hr*ng) /mL), peak plasma concentration (Cmax=1180 ng/mL), and oral bioavailability (F=49%)[1].

Acetyl-Histone Binding Assay. [1]
Assays were performed as described previously51 with minor modifications from the manufacturer’s protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150 – 0 μM and 4 μl transferred to low-volume 384-well plates, followed by 4 μl of His-tagged protein (BRD4(1), 250 nM, BRD4(2) and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, HSGRGK( Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH; peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 μg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader using an AlphaScreen 680 excitation/570 emission filter set. IC50 values were calculated in Prism 5 (GraphPad Software, USA) after normalization against corresponding DMSO controls and are given as the final concentration of compound in the 20 μl reaction volume.

Cell Proliferation Assay. [1]
Cells were seeded into white, 384-well microtiter plates (Nunc) at 500 cells per well in a total volume of 50 μl media. The 797, TT and TE10 cells were grown in DMEM containing 1 % penicillin/streptomycin and 10 % FBS. The Per403 cells were grown in DMEM containing 1 % penicillin/streptomycin and 20 % FBS. Patient-derived NMC 11060 cells were grown in RPMI with 10 % FBS and 1% penicillin/streptomycin. Compounds were delivered to microtiter assay plates by robotic pin transfer (PerkinElmer JANUS equipped with a V&P Scientific 100 nl pin tool). Following a 48 h incubation at 37 ºC, cells were lysed and wells were assessed for total ATP content using a commercial proliferation assay. Replicate measurements were analyzed with respect to dose and estimates of IC50 were calculated by logistic regression. [1]

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Cell Growth Assay.[1]
Cells were seeded in 6-well tissue culture dishes at a concentration of 1.5 x 104 cells per well. Cells were grown in 2 ml of either DMEM (797) or RPMI (11060) containing 10 % fetal bovine serum, 1 % penicillin/streptomycin and either 250 nM (+)-JQ1 or the equivalent volume of DMSO (0.025 %). One half of the media in each well was replaced daily. On days 0, 1, 3, 7 and 10, dishes of cells assigned to each time point were trypsinized, mixed in a 1:1 ratio with 0.4 % trypan blue and counted using a Countess automated cell counter.

Xenograft Efficacy Studies.[1]
NMC 797 xenografts were established by injecting NMC 797 cells (107) in 30 % Matrigel (BD Biosciences) into the flank of 6 week-old female NCr nude mice. Twelve days after injection, mice with measureable tumors were divided into cohorts to be treated with JQ1 at 50 mg kg-1 IP or vehicle (5 % DMSO in 5 % dextrose). For FDG-PET studies, mice with established tumors measuring approximately 1 cm in the largest linear dimension underwent baseline CT/PET imaging 1 h after injection of 250 μCi of FDG (Pre-treatment). Mice were then treated with four daily doses of 50 mg kg-1 of racemic JQ1 by intraperitoneal injection. Two hours after the fourth dose of JQ1 or vehicle, mice underwent repeat FDG-PET imaging (Post-treatment). The integrated signal encompassed within the entire tumor volume is expressed as the percent of injected dose per gram (% ID/gm). Tumors were fixed in 10 % buffered formalin for histopathological analysis. For tumor caliper studies, the average size of tumors in the JQ1 treatment group (n = 8) and vehicle group (n = 7) were similar (63.8 ± 17.1 and 73.6 ± 14.4 mm3 respectively) at the start of treatment. Animals were followed daily for clinical symptoms. Tumor measurements were assessed by caliper measurements, and volume calculated using the formula Vol = 0.5 x L x W2. After 2 weeks of treatment, all mice were humanely euthanized, and tumors were fixed in 10 % formalin for histopathological examination. Statistical significance of tumor volumes was calculated by two-sided Students t-test.[1]

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Primary NMC Xenograft Studies.[1]
A primary xenograft model of NMC was established by injecting NCr nude mice with primary cells (107 cells in 100 μl of 30 % Matrigel in 70 % PBS) collected from malignant pleural fluid obtained with IRB approval and informed consent from a patient at the Dana-Farber Cancer Institute and Brigham & Women’s Hospital. As above, four mice with established tumors measuring approximately 1 cm in the largest linear dimension underwent baseline CT/PET imaging 1 h after injection of 250 μCi of FDG (Pre-treatment). Mice were then treated with four daily doses of 50 mg kg-1 of (+)-JQ1 by intraperitoneal injection. Animals were followed daily for clinical symptoms. Two hours after the fourth dose of (+)-JQ1 or vehicle, mice underwent repeat FDG-PET imaging (Post-treatment). The integrated signal encompassed within the entire tumor volume is expressed as the percent of injected dose per gram (%ID/gm). Tumors were fixed in 10 % buffered formalin for histopathological analysis. At the conclusion of the study, all mice were humanely euthanized, and tumors were fixed in 10 % formalin for histopathological examination. Survival (30 days) studies performed with NMC Per403 and 11060 xenografts were initiated as above. For these studies, (+)-JQ1 was administered at a dose of 50 mg kg-1 by daily intraperitoneal injection. The average size of tumors in the (+)-JQ1 treatment group (n = 10) and vehicle group (n = 10) were similar at the start of treatment. Animals were followed daily for clinical symptoms. Tumor measurements were assessed by caliper measurements, and volume calculated using the formula Vol = 0.5xLxW2. Statistical significance of tumor volumes was calculated by two-sided Students t-test. Comparative survival analysis was performed using the Log-rank (Mantel-Cox) Test, and data were presented as a Kaplain-Meier plot annotated with a measure of statistical significance (pvalue). All animal studies were approved by the IACUC of the DFCI.

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Pharmacokinetic Studies in Mice.[1]
Male CD1 mice (24 – 29 gm) were treated with a single dose of (+)-JQ1 at 5 mg kg-1 for intravenous tail vein injection studies and 10 mg kg-1 for oral gavage studies. Approximately 150 μl of blood were taken from animals by retro-orbital puncture under anesthesia with Isoflurane into EDTA tubes at pre-specified time intervals: 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 5, 8 and 24 hours. Three animals were analyzed per time point. Blood samples were put on ice and centrifuged to obtain plasma samples (2000 x.g, 5 min under 4 °C) within 15 minutes post-sampling. Plasma samples were stored at approximately -70 °C until analysis was performed. Mice were provided free access to food and water throughout the study. Compound was formulated for intravenous injection in 10 % DMSO and 10 % HP-β-CD. Pharmcokinetic studies and pharmacologic assay development was performed at ChemPartner (Shanghai, CHINA). Data were analyzed by J.E.B. using Microsoft Excel and GraphPad Prism 5.02.

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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.

The bromodomain inhibitor (+)-JQ1 is a highly validated chemical probe; however, it exhibits poor in vivo pharmacokinetics.
The large differences between individual mice in this small study may lead to questions about the significance of the modest average differences obtained from this pharmacokinetic analysis. Given that our main goal was to assess the relative effects of deuteration on clearance of (+)-JQ1 rather than determining the absolute pharmacokinetic parameters of (+)-JQ1 and (+)-JQ1-D, we examined the isotopomeric ratios for (+)-JQ1 and for its major metabolite M19 from the pharmacokinetic time course for evidence of the effects of deuteration. In both male and female mice, the (+)-JQ1/(+)-JQ1-D ratio drops steadily over the examined time course (Figure 7), indicating that (+)-JQ1 is cleared more rapidly than (+)-JQ1-D.[https://pmc.ncbi.nlm.nih.gov/articles/PMC10788937/]

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A plasma pharmacokinetic study of JQ1 was performed in non-tumor bearing mice dosed with 30 mg/kg JQ1 intraperitoneally. All JQ1 concentrations collected after 6 h post-dose fell under the LOQ. JQ1 total plasma concentration-time data were well captured with a linear one-compartment model, in which the intraperitoneal absorption was assumed immediate and total (Figure 6). The model parameter estimates are reported in Table 7. The limited sampling model determined the following informative time-points for plasma sampling during the cerebral microdialysis study: 15 min, 1 h, and 6 h post-dose. [https://pmc.ncbi.nlm.nih.gov/articles/PMC8384680/]

[1]. Selective inhibition of BET bromodomains. Nature. 2010 Dec 23;468(7327):1067-73.

[2]. Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and LysosomalFunction. Mol Cell. 2017 May 18;66(4):517-532.e9.

[3]. Small-molecule inhibition of BRDT for male contraception. Cell. 2012 Aug 17;150(4):673-84.

JQ1 is a member of the class of thienotriazolodiazepines that is the tert-butyl ester of [(6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetic acid. An inhibitor of bromodomain-containing protein 4 that exhibits anti-cancer and cardioprotective properties. It has a role as a bromodomain-containing protein 4 inhibitor, a cardioprotective agent, an antineoplastic agent, an anti-inflammatory agent, an angiogenesis inhibitor, an apoptosis inducer and a ferroptosis inducer. It is a thienotriazolodiazepine, an organochlorine compound, a carboxylic ester and a tert-butyl ester.

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Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.[1]

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Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.[2]

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A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.[3]

C23H25CLN4O2S

456.99

456.138

C, 60.45; H, 5.51; Cl, 7.76; N, 12.26; O, 7.00; S, 7.02

1268524-70-4

(R)-(-)-JQ1 Enantiomer;1268524-71-5;JQ-1 (carboxylic acid);202592-23-2

46907787

White to yellow solid

1.3±0.1 g/cm3

610.4±65.0 °C at 760 mmHg

322.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.657

4.49

0

6

5

31

706

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=C2[C@]([H])(C([H])([H])C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N=1

DNVXATUJJDPFDM-KRWDZBQOSA-N

InChI=1S/C23H25ClN4O2S/c1-12-13(2)31-22-19(12)20(15-7-9-16(24)10-8-15)25-17(11-18(29)30-23(4,5)6)21-27-26-14(3)28(21)22/h7-10,17H,11H2,1-6H3/t17-/m0/s1

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.47 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 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 (5.47 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 25.0 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 3: ≥ 2.5 mg/mL (5.47 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5mg/mL

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