BRD4
ZL0420 forms crucial interactions with Asn140 directly and Tyr97 indirectly through an H2O molecule when it is properly docked into the acetyl-lysine (KAc) binding pocket of BRD4. In cultured human small airway epithelial cells (hSAECs), ZL0420 demonstrates submicromolar potency of suppressing the TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groβ genes, with IC50s of 0.49-0.86 µM[1].

ZL0420 forms crucial interactions with Asn140 directly and Tyr97 indirectly through an H2O molecule when it is properly docked into the acetyl-lysine (KAc) binding pocket of BRD4. In cultured human small airway epithelial cells (hSAECs), ZL0420 demonstrates submicromolar potency of suppressing the TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groβ genes, with IC50s of 0.49-0.86 µM[1].

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ZL0420 exhibited submicromolar potency in inhibiting the expression of TLR3-dependent innate immune genes (ISG54, ISG56, IL-8, and Groβ) in human small airway epithelial cells (hSAECs) stimulated with poly(I:C). The IC50 values were 0.49 µM for ISG54, 0.51 µM for ISG56, 0.53 µM for IL-8, and 0.58 µM for Groβ. This potency was approximately 15-20 fold greater than that of compound 23 and more potent than the positive controls (+)-JQ1 and RVX-208. [1]
Molecular docking studies predicted that ZL0420 occupies the acetyl-lysine binding pocket of BRD4 BD1, forming key interactions including hydrogen bonds with Asn140 directly and with Tyr97 indirectly via a water molecule. [1]

ZL0420 has a strong, low-toxicity effect on airway inflammation in a mouse model. ZL0420 exhibits remarkable efficacy and almost eliminates the deep-seated neutrophil build-up surrounding small and medium-sized airways that is brought on by the administration of poly(I:C)[1].

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In a murine model of TLR3-mediated acute airway inflammation induced by intranasal poly(I:C), intraperitoneal administration of ZL0420 (10 mg/kg) effectively blocked the poly(I:C)-induced increase in total cells and neutrophils in bronchoalveolar lavage fluid (BALF). It also reduced cytokine expression in lung tissue and demonstrated higher efficacy than the positive controls (+)-JQ1 or RVX-208. Histological analysis of lung sections showed that ZL0420 almost completely blocked the profound accumulation of neutrophils around small and medium-sized airways induced by poly(I:C). [1]

Time-resolved fluorescence energy transfer (TR-FRET) assays[1]
384 well plate-based commercial TR-FRET Assay kits were used to determine the binding ability of tested BRD4 inhibitors to the BRD4 and BRD2 bromodomains (BD) using the two recombinant BRD4 BDs or BRD2 BDs by time-resolved fluorescence energy transfer (TR-FRET) assays. A series of concentrations of BRD4 inhibitors from 0.01 nM to 100 μM were added into a 384 well test plate and mixed with other reaction components based on the instructions from vendor followed by incubation 1h at room temperature. The commercially available BRD inhibitors JQ1 and RVX208 were used as the controls. The plates were read in time-resolved format by exciting the sample at 340 nm and reading emissions at 620 and 670 nm, using a 100 μs delay and a 500 μs window at a Tecan M1000 pro reader. A plot of the TR-FRET ratio (670 nm emission/620 nm emission versus inhibitor concentration on semi-log axes results in a sigmoidal dose-response curve typical of competitive assays. These data were further calculated out with the IC50 values of tested BRD4 inhibitors to the bromodomains of BRD2 and BRD4 as well as other relevant target proteins, respectively.

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The binding affinity of ZL0420 for bromodomains was determined using a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Briefly, a series of concentrations of the inhibitor were added to a 384-well plate containing the recombinant bromodomain protein (e.g., BRD4 BD1, BRD4 BD2, etc.). The assay components were mixed and incubated for 1 hour at room temperature. The plate was read in a time-resolved format by exciting at 340 nm and measuring emissions at 620 nm and 670 nm. The ratio of emissions (670 nm/620 nm) was plotted against the inhibitor concentration on a semi-log scale to generate a sigmoidal dose-response curve, from which the IC50 values were calculated. [1]

Cell culture[1]
Immortalized human small airway epithelial cells (hSAECs) were previously described. hSAECs were grown in SAGM small airway epithelial cell growth medium in a humidified atmosphere of 5% CO2. Poly(I:C) was used at 10 μg/mL in cell culture. Compounds were solubilized in DMSO and added at the indicated concentrations.

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Quantitative Real-Time PCR (Q-RT-PCR)[1]
For gene expression analyses, 1 μg of RNA was reverse transcribed using Super Script III as previously described. One μL of cDNA product was amplified using SYBR Green Supermix and indicated gene-specific primers. The reaction mixtures were subjected to 40 cycles of 15 s at 94 °C, 60 s at 60 °C, and 1 min at 72 °C in an iCycler. Quantification of relative changes in gene expression was calculated using the ΔΔCt method and expression as the fold change between experimental and control samples was normalized to internal control cyclophilin (PPIA).

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In vitro efficacy of BRD4 inhibitors on poly(I:C) induced innate immune response[1]
hSAECs were first pretreated with a series final concentrations of BRD4 inhibitors from 0.01 nM to 100 μM for 24 hours and were then added poly(I:C) at 10 μg/mL for another 4 hours prior to harvesting the cells. The harvested cells were first washed with PBS twice and then the total RNA was extracted using acid guanidinium phenol extraction (Tri Reagent). The total RNA was further reverse-transcribed for gene expression analysis by Q-RT-PCR. The inhibitory effect of BRD4 inhibitors on poly(I:C)-induced innate immune gene expression was compared with that of poly(I:C) alone and inhibitory percentage of each treatment was obtained. For compounds 23, 28 and 35, in vitro efficacy of these BRD4 inhibitors on poly(I:C) induced innate immune response were presented as the IC50 values of these compounds. Compounds were dissolved in DMSO and further diluted at cell culture medium to appropriate concentrations.

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The in vitro efficacy of ZL0420 was evaluated in a cellular assay using human small airway epithelial cells (hSAECs). Cells were pretreated with a series of final concentrations of the compound (from 0.01 nM to 100 µM) for 24 hours. Subsequently, the TLR3 agonist poly(I:C) was added to the culture medium at a concentration of 10 µg/mL for an additional 4 hours to induce innate immune gene expression. Cells were then harvested, washed, and total RNA was extracted. The extracted RNA was reverse-transcribed into cDNA. The expression levels of target innate immune genes (ISG54, ISG56, IL-8, Groβ) were quantified using quantitative real-time polymerase chain reaction (Q-RT-PCR). The inhibitory effect was calculated by comparing gene expression levels in compound-treated, poly(I:C)-stimulated cells to those in cells treated with poly(I:C) alone. IC50 values were determined from dose-response curves generated using at least 8 different concentrations of the compound. [1]

In vivo efficacy of BRD4 inhibitors on poly(I:C)-induced acute airway inflammation[1]
Animal experiments were performed according to the NIH Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch (UTMB) Animal Care and Use Committee (approval no. 1312058A). Male C57BL6/J mice (12 weeks old) were purchased from The Jackson Laboratory and housed under pathogen-free conditions with food and water ad libitum. C57BL/6 mice were pre-treated in the absence or presence of the indicated BRD4 inhibitors [10 mg/kg body weight, via the intraperitoneal route] one day prior to poly(I:C) stimulation. The next day, animals were given another dose of BRD4 inhibitor immediately followed by intranasal (i.n.) administration of phosphate-buffered saline (PBS, 50 μL) or poly(I:C) (300 μg dissolved in 50 μL PBS). One day later, the mice were euthanized. The bronchoalveolar lavage fluid (BALF) and lung tissues of treated mice were collected for further analysis. Compounds were first dissolved in DMSO and further diluted in 10% hydroxypropyl β-cyclodextrin in PBS to appropriate concentration prior to intraperitoneal administration.

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Evaluation of airway inflammation[1]
Cellular recruitment into the airway lumen was assessed in the bronchoalveolar lavage fluid (BALF). Lungs were perfused twice with 1 mL of sterile PBS (pH 7.4) to obtain the BALF. Total cell counts were determined by trypan blue staining 50 μL of BALF and counting viable cells using a hemocytometer. Differential cell counts were performed on cytocentrifuge preparations stained with Wright-Giemsa. A total of 300 cells were counted per sample using light microscopy. Formalin-fixed lungs were embedded in paraffin, sectioned at a 4 μm thickness, and stained with hematoxylin and eosin or Masson’s trichrome. Microscopy was performed on a NIKON Eclipse Ti System.

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The in vivo efficacy of ZL0420 was evaluated in a C57BL/6 mouse model of acute airway inflammation. Mice were pre-treated with the compound (10 mg/kg body weight) via intraperitoneal injection one day prior to poly(I:C) stimulation. On the next day, mice received another dose of the compound immediately followed by intranasal administration of either phosphate-buffered saline (PBS) or poly(I:C) (300 µg in 50 µL PBS). One day after poly(I:C) challenge, mice were euthanized. Bronchoalveolar lavage fluid (BALF) was collected by perfusing the lungs twice with sterile PBS. Total cell counts in BALF were determined using a hemocytometer after trypan blue staining. Differential cell counts (e.g., neutrophils) were performed on cytocentrifuge preparations stained with Wright-Giemsa. Lung tissues were collected, fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological examination. [1]
For pharmacokinetic studies in rats, ZL0420 was formulated in a vehicle containing 10% DMSO, 60% PEG-400, and 30% saline. It was administered intravenously at 10 mg/kg and orally at 20 mg/kg. [1]

The pharmacokinetic characteristics of ZL0420 in rats were evaluated after intravenous (IV, 10 mg/kg) and oral (PO, 20 mg/kg) administration. After intravenous administration, the half-life (t1/2) was 1.2 h, the area under the curve (AUC0-∞) was 14700 ng·h/mL, the steady-state volume of distribution (Vss) was 0.864 L/kg, and the total clearance (CL) was 11.5 mL/min/kg. After oral administration, the peak plasma concentration (Cmax) was 80 ng/mL, and the AUC0-∞ was 450 ng·h/mL. Oral bioavailability was poor. [1]

In a preliminary safety assessment, mice were treated with the relevant compound ZL0454 (35) at daily doses of 1 to 50 mg/kg for one month, and no significant toxic effects were observed on body weight, hematological parameters (white blood cells, red blood cells, platelets), liver function (albumin, globulin, alkaline phosphatase, alanine aminotransferase), or kidney function (creatinine, blood urea nitrogen). Histological examination of the liver, kidney, and lung tissues of mice treated with the compound also revealed no significant adverse reactions. Although this dataset focuses on compound 35, the study suggests that selective BRD4 inhibitors (including ZL0420) appear to be safer and better tolerable than pan-BET inhibitors (such as JQ1) in the models tested. [1]

[1]. Discovery of potent and selective BRD4 inhibitors capable of blocking TLR3-induced acute airway inflammation. Eur J Med Chem. 2018 May 10;151:450-461.

series of different small molecule compounds were designed and synthesized using a structure-based drug design approach combined with fragment merging and modification strategies. Compounds ZL0420 (28) and ZL0454 (35) were identified as highly potent and selective BRD4 inhibitors with nanomolar binding affinity to the bromine domain (BD) of BRD4. They both dock well to the acetyl-lysine (KAc) binding pocket of BRD4, forming key interactions, including direct hydrogen bonding with Asn140 and indirect hydrogen bonding with Tyr97 via H₂O molecules. Compounds 28 and 35 both exhibited submicromolar inhibitory activity, inhibiting the expression of TLR3-dependent innate immune gene programs, including ISG54, ISG56, IL-8, and Groβ genes, in cultured human small airway epithelial cells (hSAEC). More importantly, they also demonstrated in mouse models that the compound effectively reduced airway inflammation with low toxicity, proving that BRD4 inhibitors may have therapeutic potential to block virus-induced airway inflammation. [1]

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ZL0420 was discovered through a structure-based drug design approach involving fragment merging and modification. It is a potent and selective BRD4 bromodomain inhibitor designed to mimic acetyllysine and occupy its binding pocket. The compound demonstrated therapeutic potential to block virus-induced airway inflammation by inhibiting BRD4-dependent innate immune gene programs. [1]

C16H16N4O2

296.33

296.127

C, 64.85; H, 5.44; N, 18.91; O, 10.80

2229039-45-4

ZL0420;2230496-80-5

137285011

Yellow to orange solid powder

1.4±0.1 g/cm3

645.9±55.0 °C at 760 mmHg

344.4±31.5 °C

0.0±2.0 mmHg at 25°C

1.704

2.19

3

5

2

22

442

0

O=C1CCC2C=C(C=CC=2N1)/N=N/C1C=C(C)C(=CC=1N)O

ANMQADUROYWADA-UHFFFAOYSA-N

InChI=1S/C16H16N4O2/c1-9-6-14(12(17)8-15(9)21)20-19-11-3-4-13-10(7-11)2-5-16(22)18-13/h3-4,6-8,21H,2,5,17H2,1H3,(H,18,22)

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.)