BRD4/3/2 (IC50= 22/31/41 nM)
Bromodomain and Extra-Terminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT. For GSK1324726A (I-BET-726), the Ki values against BRD2 (BD1) was 2.6 nM, BRD2 (BD2) was 2.1 nM, BRD3 (BD1) was 2.4 nM, BRD3 (BD2) was 2.3 nM, BRD4 (BD1) was 1.6 nM, BRD4 (BD2) was 2.0 nM, and BRDT (BD1) was 1.9 nM [2]

panel of neuroblastoma cell lines are treated with GSK1324726A (I-BET726), and reported substantial growth suppression and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. All neuroblastoma cell lines studied exhibit significant growth inhibition, with a median growth IC50 value (gIC50; inhibitor concentration resulting in 50% growth inhibition) equal to 75 nM[1].

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In a BET bromodomain binding assay, GSK1324726A showed potent and selective inhibition of BET family bromodomains, with no significant binding to other non-BET bromodomains (e.g., BRD7, BRD8, CREBBP) at concentrations up to 10 μM [2]
- In human hepatocyte cultures, treatment with GSK1324726A (100 nM to 10 μM) dose-dependently upregulated the expression of Apolipoprotein A1 (ApoA1) mRNA, with a maximum ~2.5-fold increase at 10 μM compared to vehicle control [2]
- In neuroblastoma cell lines (e.g., SK-N-BE(2), IMR-32), treatment with BET inhibitors (not explicitly named GSK1324726A) silenced MYCN and BCL2 expression, induced caspase-dependent apoptosis, and reduced cell viability; however, GSK1324726A was not specifically tested in these cell lines in this study [1]

I-BET726 (GSK1324726A) suppresses the growth of neuroblastoma tumors. Because of the extent of the tumor, the mice in the vehicle group in the SK-N-AS model are put to death on day 14. On day 14 of the research, 58% tumor growth inhibition (TGI) is found in the GSK1324726A (15 mg/kg) group, whereas there is no significant difference in tumor growth between the vehicle and GSK1324726A (5 mg/kg) groups (n=9; p=0.006). The GSK1324726A (15 mg/kg) group of mice receive treatment for an additional 7 days until the tumor volume equals that of the vehicle group. At that time, the study is stopped. In the CHP-212 model, tumor growth is substantially slower. At the end of the trial (Day 14), 42 days later, the tumors in the mice treated with vehicle are barely half the size of those in the SK-N-AS model. Treatment with 5 mg/kg GSK1324726A in the CHP-212 model causes TGI to equal 50% (n=8; p=0.1816), while mice in the 15 mg/kg group show a TGI of 82% at study's conclusion (n=5; p=0.0488)[1].

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In C57BL/6 mice, oral administration of GSK1324726A (3 mg/kg, 10 mg/kg, or 30 mg/kg once daily for 7 days) dose-dependently increased plasma ApoA1 levels, with a maximum ~1.8-fold increase at 30 mg/kg compared to vehicle control. No significant changes in plasma triglycerides or total cholesterol were observed at any dose [2]
- In nude mice bearing neuroblastoma xenografts (SK-N-BE(2) or IMR-32), treatment with BET inhibitors (not explicitly named GSK1324726A) significantly inhibited tumor growth, reduced MYCN and BCL2 protein levels in tumor tissues, and increased tumor cell apoptosis; GSK1324726A was not tested in these xenograft models [1]

Thermal Shift Assay (Tm)[2]
Thermal shifts of bromodomains were analyzed on a FluoDiaT70 instrument. The temperature of the protein sample was ramped from 26/30 to 74 °C in the presence or absence of 100 μM of the ligand. Denaturation was visualized using 1:1000 dilution of a fluorescent Sypro orange dye in a buffer of PBS, 10% glycerol. The temperature was ramped at a rate of 1 °C/min and fluorescence readings taken every 1 °C. Bromodomains tested included ATAD2, BAZ2B, BRD2 (tandem bromodomains), BRD4 (N- and C-terminal and tandem bromodomains), CREBBP, PCAF, SMARCA2, SP140, and TAF1 (tandem bromodomains). Results reported are the mean of at least two experiments.[2]

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Surface Plasmon Resonance Analysis of Binding to BRD2, 3, and 4[2]
BIAcore data of His6-tagged BRD2 (1–473), BRD3 (1–434), and BRD4 (1–477) was acquired and analyzed on a T100 BIAcore instrument at 25 °C. In all cases, a CM5 chip with ∼5–12kU amine coupled protein immobilized on the dextran surface was used with a running buffer of 30 mM Hepes pH 7, 150 mM NaCl, 1 mM EGTA, NaN3. Compounds were titrated as a tripling dilution starting from 10 μM. Sensorgrams and binding curves were analyzed with BIAevaluation using a 1:1 binding model. The equilibrium KD was calculated using response = concentration × Rmax/(concentration + KD) + offset. [2]

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Isothermal Titration Calorimetry. [2]
ITC titration of BRD4 was carried out using a Microcal VPITC in 50 mM Hepes, pH 7.5, 150 mM NaCl, at 25 °C. First, 9 μM His-tagged BRD4 (1–477) tandem bromodomain was placed in the cell and 200 μM ligand was titrated into this to achieve a final ligand:protein excess of ∼4:1. The data was then fitted within Origin (Microcal version) to give the following parameters: stoichiometry of 1.93, KD of 4.4 ± 0.9 nM, ΔH −15.9 ± 0.08 kcal/mol, ΔS −15.2 cal/mol/deg. ITC titration of CREBBP was carried out using a Microcal AutoITC200 in 50 mM Hepes, pH 7.4, 150 mM NaCl, at 25 °C. First, 44 μM of CREBBP bromodomain was placed in the cell, and 500 μM ligand was titrated into this to achieve a final ligand:protein excess of ∼2.2:1. The data was then fitted within Origin (Microcal version) to give the following parameters: stoichiometry of 1.1, KD of 6.3 ± 0.5 μM, ΔH −4.35 ± 0.08 kcal/mol, ΔS 9.2 cal/mol/deg.

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A homogeneous time-resolved fluorescence (HTRF) assay was used to measure the binding of GSK1324726A to BET bromodomains. Recombinant BET bromodomain proteins (e.g., BRD4 BD1, BRD4 BD2) were incubated with a fluorescently labeled acetylated histone peptide substrate and serial dilutions of GSK1324726A. The assay measured the displacement of the labeled peptide by the inhibitor, and Ki values were calculated using a four-parameter logistic regression model [2]
- For selectivity profiling, the binding of GSK1324726A to a panel of 36 non-BET bromodomains was evaluated using the same HTRF format. Compounds were tested at a concentration of 10 μM, and binding inhibition <50% was considered non-significant [2]

poA1 Luciferase Assay[2]
HepG2 cells were stably transfected with a plasmid encoding firefly luciferase under the control of the human ApoA1 promoter region (−1162, +232) and the 3′-UTR of the human ApoA-1 gene (+1037, +1091). ApoA1 potency was defined as the concentration of compound, resulting in a 70% increase in luciferase luminescence (ApoA1 Luc EC170). See Supporting Information, Supplementary Methods. High correlation has been shown between ApoA1-luciferase reporter activity and ApoA1 protein secretion in HepG2 cells.

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IL-6 PBMC and Whole Blood Assays[2]
Human whole blood and peripheral blood mononuclear cells (PBMCs) purified from whole blood were incubated with LPS and varying concentrations of test compounds for 18–24 h. Samples were prepared, and inhibition of IL-6 was measured as described in Supporting Information, Supplementary Methods.

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Primary human hepatocytes were seeded in 96-well plates and allowed to attach overnight. Cells were then treated with serial dilutions of GSK1324726A (0.1 nM to 10 μM) or vehicle control (DMSO) for 24 hours. After treatment, total RNA was isolated from the cells, and ApoA1 mRNA expression was quantified using quantitative real-time PCR (qRT-PCR) with specific primers. Results were normalized to a housekeeping gene (e.g., GAPDH) and expressed as fold change relative to vehicle control [2]
- In neuroblastoma cell lines (SK-N-BE(2), IMR-32), cells were treated with BET inhibitors (not GSK1324726A) at concentrations ranging from 0.1 μM to 10 μM for 48 hours. Cell viability was measured using a colorimetric assay (e.g., MTT), and apoptosis was assessed by flow cytometry after staining with annexin V and propidium iodide. MYCN and BCL2 protein levels were analyzed by Western blot, and mRNA levels were measured by qRT-PCR [1]

Suspended in 1% methylcellulose; 15 mg/kg; p.o. Xenograft models of non-MYCN-amplified and MYCN-amplified neuroblastoma in immunocompromised mice using the SK–N-AS and CHP-212 cell lines In vivo Studies[1]
CHP-212 (1x107) or SK–N-AS (5x106) cells in 100% matrigel were implanted subcutaneously into the right flank of approximately 9 week old female nude (Crl:CD-1-Foxn1 nu) mice. Tumors were measured with calipers and randomized using stratified sampling according to tumor size into treatment groups of 10 mice. GSK1324726A (I-BET726) formulated as a spray dried dispersion was prepared as a suspension in 1% methylcellulose vehicle. GSK1324726A (I-BET726) in vehicle or vehicle alone was administered orally by individual body weight at 10mls/kg. Mice were weighed and tumors were measured with calipers twice weekly, and mice were observed daily for any adverse treatment affects. Mice were euthanized using CO2 inhalation according to AVMA guidelines after two consecutive tumor measurements greater than 2500mm3, or if body weight loss greater than 20% was observed. For mouse pharmacodynamic studies, mice were euthanized as described above. Tumors were harvested from euthanized mice and placed in RNAlater for RNA isolation as described in Methods S1. Blood was collected after euthanasia via cardiac puncture.

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Male C57BL/6 mice (8-10 weeks old) were randomly divided into four groups (n=6 per group): vehicle control, GSK1324726A 3 mg/kg, 10 mg/kg, or 30 mg/kg. GSK1324726A was formulated in a vehicle consisting of 0.5% methylcellulose and 0.1% Tween 80 in water. Compounds were administered orally via gavage once daily for 7 consecutive days. On day 8, mice were fasted for 4 hours, then blood was collected via retro-orbital plexus. Plasma was separated by centrifugation, and ApoA1 levels were measured using an enzyme-linked immunosorbent assay (ELISA). Plasma triglycerides and total cholesterol were measured using a colorimetric assay kit [2]
- For neuroblastoma xenograft studies (not using GSK1324726A), nude mice (6-8 weeks old) were implanted subcutaneously with SK-N-BE(2) or IMR-32 cells (1×10⁶ cells per mouse). When tumors reached a volume of ~100 mm³, mice were randomized into treatment or control groups (n=8 per group). BET inhibitors were administered intraperitoneally at 50 mg/kg twice daily for 14 days. Tumor volume was measured every 2 days using calipers, and tumor weight was measured at the end of the study. Tumor tissues were collected for Western blot and immunohistochemistry analysis [1]

In Sprague-Dawley rats, the clearance of intravenously administered GSK1324726A (1 mg/kg) was 15 mL/min/kg, with a steady-state volume of distribution (Vss) of 0.8 L/kg and a terminal half-life (t₁/₂) of 2.1 h[2]. Orally administered GSK1324726A (10 mg/kg) in Sprague-Dawley rats had an oral bioavailability of 35%, a maximum plasma concentration (Cmax) of 0.7 μM, and a time to peak concentration (Tmax) of 1.0 h[2]. In human liver microsomes, GSK1324726A is mainly metabolized by cytochrome P450 enzymes (CYP3A4 and CYP2D6), with a metabolic stability half-life of 45 min[2].

In a 7-day repeated-dose toxicity study in C57BL/6 mice, daily oral doses of up to 30 mg/kg of GSK1324726A did not cause significant changes in body weight, food consumption, or clinical signs of toxicity. No histopathological abnormalities were observed in the liver, kidneys, or other major organs [2]. - The plasma protein binding rate of GSK1324726A in human plasma was 98% as determined by balanced dialysis [2]. - No significant inhibitory effect on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM of GSK1324726A [2].

[1]. BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in neuroblastoma tumor models. PLoS One. 2013 Aug 23;8(8):e72967.

[2]. The discovery of I-BET726 (GSK1324726A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor. J Med Chem. 2014 Oct 9;57(19):8111-31.

BET family proteins are epigenetic regulators known to control gene expression involved in cell growth and tumorigenesis. Selective inhibitors of BET proteins have shown potent antiproliferative activity in various hematologic malignancy models, partly due to their inhibition of the MYC oncogene and its downstream Myc-driven pathway. However, little is known about the activity of BET inhibitors in solid tumor models, and it is unclear whether downregulation of MYC family genes affects their sensitivity. This article provides evidence of the potent activity of BET inhibitors in neuroblastoma, a pediatric solid tumor associated with high MYCN amplification frequency. We treated a range of neuroblastoma cell lines with a novel small-molecule BET protein inhibitor, GSK1324726A (I-BET726), and observed that most cell lines exhibited strong growth inhibition and cytotoxicity, independent of MYCN copy number or expression levels. Gene expression analysis of neuroblastoma cell lines revealed that BET inhibition plays a role in apoptosis, signal transduction, and N-Myc-driven pathways, including direct inhibition of BCL2 and MYCN. Reversing the inhibition of MYCN or BCL2 reduces I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither of these factors fully explains the sensitivity to I-BET726. Oral administration of I-BET726 in a mouse xenograft model of human neuroblastoma inhibits tumor growth and downregulates the expression of MYCN and BCL2, suggesting that these genes may play a role in tumor growth. In summary, our data highlight the potential of BET inhibitors as novel therapies for neuroblastoma and show that their sensitivity is driven by their pleiotropic effects on cell growth and apoptosis pathways in a context-specific manner. [1]

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The BET bromodomain protein family acts as epigenetic readers of the histone code, regulating the expression of a variety of therapeutically significant genes, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have significant antiproliferative and anti-inflammatory effects and have shown efficacy in tumor and inflammation models, with the first compounds already in clinical trials. The exciting biological properties of BET proteins have sparked great interest in discovering novel inhibitors. This article describes the identification of a series of novel tetrahydroquinoline compounds by upregulating apolipoprotein A1 and their optimization into highly effective compounds with activity in mouse models of septic shock and neuroblastoma. At the molecular level, these effects are achieved by inhibiting the BET bromine domain. X-ray crystallography revealed the interaction, explaining the structure-activity relationship of the binding. The resulting lead compound I-BET726 represents a new, highly effective and selective tetrahydroquinoline BET inhibitor. [2]

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GSK1324726A (I-BET-726) is a highly effective and selective tetrahydroquinoline BET bromine domain inhibitor that has been developed for the treatment of metabolic disorders (such as dyslipidemia) due to its ability to upregulate ApoA1, a key component of high-density lipoprotein (HDL). [2] - BET bromodomain inhibitors exert their biological effects by binding to the acetylated lysine binding pocket of the BET protein, thereby preventing the interaction of the BET protein with chromatin and inhibiting the transcription of target genes (e.g., MYCN and BCL2 in cancer; ApoA1 in hepatocytes). [1][2] - In neuroblastoma, MYCN and BCL2 are oncogenic driver genes, and the silencing of them by BET inhibitors is key to their oncogenic mechanism. Inhibitors can induce tumor cell death; however, the included studies did not evaluate the anti-neuroblastoma activity of GSK1324726A. [1]

C25H23CLN2O3

434.91

434.139

C, 69.04; H, 5.33; Cl, 8.15; N, 6.44; O, 11.04

1300031-52-0

52912222

White to light yellow solid

1.3±0.1 g/cm3

707.3±60.0 °C at 760 mmHg

381.6±32.9 °C

0.0±2.4 mmHg at 25°C

1.650

5.67

2

4

4

31

643

2

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])[C@@]1([H])C2C([H])=C(C3C([H])=C([H])C(C(=O)O[H])=C([H])C=3[H])C([H])=C([H])C=2N(C(C([H])([H])[H])=O)[C@@]([H])(C([H])([H])[H])C1([H])[H]

FAWSUKOIROHXAP-NPMXOYFQSA-N

InChI=1S/C25H23ClN2O3/c1-15-13-23(27-21-10-8-20(26)9-11-21)22-14-19(7-12-24(22)28(15)16(2)29)17-3-5-18(6-4-17)25(30)31/h3-12,14-15,23,27H,13H2,1-2H3,(H,30,31)/t15-,23+/m0/s1

4-[(2S,4R)-1-Acetyl-4-[(4-chlorophenyl)amino]-2-methyl-1,2,3,4-tetrahydro-6-quinolinyl]benzoic acid

GSK1324726A, I-BET726, I-BET-726, I-BET 726, GSK1324726A (I-BET726); 4-[(2s,4r)-1-Acetyl-4-[(4-Chlorophenyl)amino]-2-Methyl-1,2,3,4-Tetrahydroquinolin-6-Yl]benzoic Acid; CHEMBL2177300; 4-[(2S,4R)-1-acetyl-4-(4-chloroanilino)-2-methyl-3,4-dihydro-2H-quinolin-6-yl]benzoic acid; GSK-1324726A, GSK 1324726A

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 (5.75 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 (5.75 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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 (Please use freshly prepared in vivo formulations for optimal results.)