IC50: 27 nM (BRD4 BD1), 32 nM (BRD4 BD2)[1]
ZL0420 specifically targets BRD4 (Bromodomain-containing protein 4), a key epigenetic reader that recognizes acetylated lysine residues on histones. It inhibits both the first (BD1) and second (BD2) bromodomains of BRD4, with reported IC50 values of 27 nM for BD1 and 32 nM for BD2. By binding to the acetyl-lysine binding pocket, it disrupts BRD4-mediated transcriptional activation of inflammatory genes.

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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In vitro, ZL0420 potently suppresses the TLR3-dependent innate immune gene program. In cultured human small airway epithelial cells (hSAECs), it inhibits the expression of interferon-stimulated genes such as ISG54, ISG56, IL-8, and Grobeta, with IC50 values ranging from 0.49 to 0.86 uM. Docking studies confirm that ZL0420 fits well into the KAc binding pocket of BRD4, forming critical interactions, including direct hydrogen bonds with Asn140.

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 vivo, ZL0420 exhibits strong, low-toxicity efficacy against airway inflammation in a mouse model. It almost completely blocks the profound accumulation of neutrophils around the small and medium-sized airways induced by poly(I:C) administration, demonstrating remarkable efficacy. This suggests its potential for treating acute inflammatory conditions of the lung.

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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A cell-free time-resolved fluorescence energy transfer (TR-FRET) assay is used to determine the binding affinity of ZL0420 to BRD4 bromodomains. Purified recombinant BRD4 BD1 or BD2 proteins are incubated with a biotinylated acetylated histone H4 peptide and a Europium-labeled anti-Histone antibody. The compound is added to the reaction, and the TR-FRET signal is measured to calculate the IC50 value.

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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In cellular assays, hSAECs are seeded in a 96-well plate and allowed to adhere overnight. They are pre-treated with serial dilutions of ZL0420 (0.01 nM to 100 uM) for 24 hours. Poly(I:C) at 10 ug/mL is then added to the culture medium to stimulate the TLR3 pathway, and the cells are incubated for an additional 4 hours. After harvesting, total RNA is extracted, and the expression levels of innate immune genes (e.g., ISG54, IL-8) are measured by RT-qPCR.

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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C57BL/6 mice are pre-treated with a single intraperitoneal (IP) dose of ZL0420 (10 mg/kg) or vehicle control one day prior to poly(I:C) stimulation. The next day, the mice receive another dose of the compound, followed immediately by intranasal (i.n.) administration of poly(I:C) (300 ug) or PBS. One day later, the animals are euthanized. Bronchoalveolar lavage fluid (BALF) is collected to measure neutrophil infiltration, and lung tissues are harvested for histopathological analysis.

Specific ADME data for ZL0420 is not detailed in the provided literature. However, its in vivo efficacy via IP injection (10 mg/kg) demonstrates that it is well-absorbed and reaches sufficient concentrations in the lungs to exert its pharmacological effect. The compound has a molecular weight of 296.32 and is soluble in DMSO (30 mg/mL), facilitating its use in both in vitro and in vivo applications.

The provided literature describes ZL0420 as having a "low-toxicity" effect in the mouse model. At the efficacious dose of 10 mg/kg, the compound was well-tolerated, enabling the complete blockage of neutrophil accumulation without apparent adverse effects. However, no detailed systemic toxicological profile is provided. As a BRD4 inhibitor, long-term effects would need to be evaluated for therapeutic development.

[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 that BRD4 inhibitors were significantly effective in reducing airway inflammation in mouse models with low toxicity, suggesting that BRD4 inhibitors may have therapeutic potential in blocking virus-induced airway inflammation. [1]

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ZL0420 is a chemical probe that highlights the role of BRD4 in inflammatory signaling, specifically the TLR3-dependent pathway. Its high selectivity for BRD4 over other BET family members and its potent in vivo efficacy make it a valuable tool for studying acute airway inflammation. The compound is in the preclinical stage and is not approved for human use. It is a lead candidate for potential treatment of conditions like steroid-resistant asthma.

C16H16N4O2

296.32

296.127

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

2230496-80-5

(E/Z)-ZL0420;2229039-45-4

137285011

Typically exists as Yellow to orange solids at room temperature

2.2

3

5

2

22

442

0

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)

6-[(2-amino-4-hydroxy-5-methylphenyl)diazenyl]-3,4-dihydro-1H-quinolin-2-one

6-[(2-amino-4-hydroxy-5-methylphenyl)diazenyl]-3,4-dihydro-1H-quinolin-2-one; 2230496-80-5; CHEMBL4176038; (E/Z)-ZL0420; 6-((2-Amino-4-hydroxy-5-methylphenyl)diazenyl)-3,4-dihydroquinolin-2(1H)-one; 6-[(2-Amino-4-hydroxy-5-methylphenyl)diazenyl]-3,4-dihydroquinolin-2(1H)-one;

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: 125 mg/mL (421.84 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.02 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 20.8 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: ≥ 2.08 mg/mL (7.02 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)