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Purity: ≥98%
JQ-EZ-05 (also abbreviated as JQEZ5) is a novel, potent, specific and reversible inhibitor of EZH2 (overexpressed in tumor cells such as lung cancer). Epigenetic targets are exciting new avenues for the discovery of targeted anticancer drugs. Zhang etc. have designed the open-source EZH2 inhibitor JQEZ5 which showed potent antitumor efficacy in vitro and in vivo in preclinical studies in murine and human lung adenocarcinoma models expressing high levels of EZH2. EZH2 is a lysine methyltransferase and acts as master regulator of chromatin function, it orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive.
| Targets |
JQEZ5 targets Enhancer of Zeste Homolog 2 (EZH2) (SAM competitive binding, KD = 87 nM for PRC2 five-component complex) [1]
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| ln Vitro |
JQEZ5 inhibits PRC2's enzymatic functioning with a biochemical IC50 of 80nM. H661 cells treated with increasing dosages of JQEZ5 show markedly lower levels of H3K27me3 without changing H3K27 mono- or di-methylation. After 4 days of treatment, JQEZ5 inhibits the proliferation of EZH2-overexpressing H661 and H522 cells while having no effect on the proliferation of cell lines that were previously thought to be resistant to EZH2 knockdown [1].
1. In a five-component PRC2 complex radiometric Scintillation Proximity Assay (SPA) using radiolabeled S-adenosyl methionine (SAM), JQEZ5 exhibited inhibitory activity against EZH2; the IC50 was determined under increasing SAM concentrations to confirm its SAM competitive binding activity [1] 2. Treatment of H661 and H292 human lung cancer cells with increasing concentrations of JQEZ5 led to changes in protein expression (detected by Western blotting), including alterations in methylation levels (H3K27me3) in H661 cells after 48 h or 72 h of treatment; JQEZ23 (negative control) had no such effect on methylation levels in H661 cells after 72 h of treatment [1] 3. MTS assay showed that JQEZ5 inhibited the relative cell growth of H661 and H292 human lung cancer cells in a concentration-dependent manner; H661 cells infected with shEZH2 also showed altered sensitivity to JQEZ5 in the MTS assay (error bars represent SD, n=3) [1] |
| ln Vivo |
During the course of the three-week treatment, there is a noticeable and swift reduction in tumor size with JQEZ5 (75 mg/kg; intraperitoneal injection; daily). Additionally, the treatment significantly lowers H3K27me3, supporting JQEZ5's on-target effect in mice[1].
1. Actin-Cre;LSL-EZH2 mice bearing lung tumors were treated with JQEZ5 at 75 mg/kg daily for 1–3 weeks; MRI scans showed regression of lung tumors (relative tumor volume quantified by 3D Slicer, mean ± SEM, n=2), and immunostaining of lung sections revealed reduced H3K27me3 levels in treated mice [1] 2. Nude mice subcutaneously injected with 2×10⁶ H661 human NSCLC cells (tumors ~200 mm³) were randomized and treated with vehicle or JQEZ5 (75 mg/kg/d, i.p.) for 18 days; Western blot analysis of tumors and lung tissues from treated mice showed changes in protein expression/methylation levels, and caliper measurements demonstrated that JQEZ5 inhibited tumor growth (mean ± SEM, n=3/vehicle, n=6/JQEZ5) [1] 3. Nude mice were subcutaneously injected with H661 and H292 human NSCLC cells infected with control (NT) or shEZH2 lentivirus; when the largest tumor reached ~150 mm³, mice were euthanized, and tumor size was quantified relative to shNT tumors (mean ± SEM, n=3/treatment) [1] |
| Enzyme Assay |
1. A radiometric Scintillation Proximity Assay (SPA) was conducted to measure the inhibitory activity of JQEZ5, JQEZ23, GSK-126 and UNC1999 on the five-component PRC2 complex. The assay utilized radiolabeled S-adenosyl methionine (SAM) as a substrate to evaluate the inhibitory effect of the compounds on EZH2 activity within the PRC2 complex [1]
2. Single-cycle kinetics runs with four concentrations of the PRC2 five-component complex were performed using Biacore SPR. A biotinylated derivative of JQEZ5 (JQEZ6) was immobilized on a streptavidin SPR chip, and the affinity (KD) of PRC2 for JQEZ6 was determined to be 87 nM [1] 3. The IC50 of JQEZ5 was measured with increasing concentrations of SAM to verify its SAM competitive binding mode to EZH2 [1] |
| Cell Assay |
1. Human NSCLC cell lines H661 and H292 were transfected with non-targeting control shRNA (NT) or two different shRNAs targeting EZH2 (shEZH2-A and shEZH2-B); EZH2 expression levels were analyzed by Western blotting, and the expression levels between H661 and H292 were compared via Western blotting [1]
2. MTS assay was used to assess the relative cell growth of H661, H522 and H292 cells expressing NT or shEZH2 (shEZH2-A and shEZH2-B), with error bars representing S.E.M. (n=3, p < 0.001) [1] 3. H661 and H292 cells were incubated with increasing concentrations of JQEZ5; cell lysates were prepared and subjected to SDS-PAGE, followed by Western blotting with specific antibodies to detect protein expression and methylation levels (H3 was used as a loading control for methylation analysis in H661 cells treated for 48 h/72 h or with JQEZ23 for 72 h) [1] 4. MTS assay was performed to evaluate the relative cell growth of H661 cells (infected with NT or shEZH2 lentivirus) treated with increasing concentrations of JQEZ5, with error bars representing SD (n=3) [1] |
| Animal Protocol |
JQEZ5 was dissolved in DMSO and then diluted 1:10 in 10% (2-Hydroxypropyl)-β-cyclodextrin; 75 mg/kg/d, i.p.
H661 Tumor-bearing GEMMs mice 1. For Actin-Cre;LSL-EZH2 mice with lung tumors: JQEZ5 was administered at a dose of 75 mg/kg daily for 1–3 weeks; MRI scans were performed at baseline (t=0) and after treatment to monitor tumor volume, and lung sections were prepared for immunostaining of H3K27me3 post-treatment [1] 2. For nude mice bearing H661 xenografts: 2×10⁶ H661 human NSCLC cells were subcutaneously injected into the flank of nude mice; when tumors reached approximately 200 mm³, mice were randomized into vehicle and JQEZ5 treatment groups, with JQEZ5 administered intraperitoneally (i.p.) at 75 mg/kg per day for 18 days; tumor volume was measured by caliper, and tumors/lung tissues were collected for Western blot analysis [1] 3. For nude mice injected with shRNA-modified NSCLC cells: H661 and H292 cells infected with NT or shEZH2 lentivirus were subcutaneously injected into nude mice; when the largest tumor reached ~150 mm³, mice were euthanized, and tumor size was quantified relative to the NT group (n=3 per treatment group) [1] |
| References | |
| Additional Infomation |
1. JQEZ5 is a potent open-source EZH2 inhibitor developed specifically for the treatment of EZH2-dependent lung cancer; 1. A negative control compound (JQEZ23) with different chemical structures was used in parallel experiments [1]
2. Based on the reported model [1], a computational docking model was constructed to predict the binding mode of JQEZ5 to EZH2. 3. EZH2-driven mouse lung cancer (induced by EZH2 overexpression) is similar to human non-small cell lung cancer (NSCLC) with high EZH2 expression and low levels of phosphorylated AKT and ERK, which can serve as biomarkers for JQEZ5 sensitivity [1] 4. JQEZ5 promotes EZH2-driven tumor regression in vivo, confirming the oncogenic dependence of EZH2 in established tumors and providing a theoretical basis for epigenetic therapy of some lung cancers [1] |
| Molecular Formula |
C30H38N8O2
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| Molecular Weight |
542.675125598907
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| Exact Mass |
542.311
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| CAS # |
1913252-04-6
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| Related CAS # |
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| PubChem CID |
121322599
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
40
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| Complexity |
988
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C1C=C(C2=CN=C(C=C2)N2CCN(C)CC2)N=C2C=1C=NN2C(C)C)NCC1C(NC(C)=CC=1CCC)=O
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| InChi Key |
LQTWDAYNGMMHLV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H38N8O2/c1-6-7-21-14-20(4)34-30(40)24(21)17-32-29(39)23-15-26(35-28-25(23)18-33-38(28)19(2)3)22-8-9-27(31-16-22)37-12-10-36(5)11-13-37/h8-9,14-16,18-19H,6-7,10-13,17H2,1-5H3,(H,32,39)(H,34,40)
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| Chemical Name |
N-[(6-methyl-2-oxo-4-propyl-1H-pyridin-3-yl)methyl]-6-[6-(4-methylpiperazin-1-yl)pyridin-3-yl]-1-propan-2-ylpyrazolo[3,4-b]pyridine-4-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8427 mL | 9.2135 mL | 18.4271 mL | |
| 5 mM | 0.3685 mL | 1.8427 mL | 3.6854 mL | |
| 10 mM | 0.1843 mL | 0.9214 mL | 1.8427 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
JQEZ5 Inhibits Lung Cancer GrowthIn Vivo.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
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JQEZ5 Inhibits Lung Cancer Growth of EZH2 Overexpressed Human Lung Cancer Cell.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
Small Molecule EZH2 Inhibitor Development.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
 A Subset of Human NSCLC Cells are Dependent on EZH2 Overexpression.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
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EZH2 Overexpression Establishes a Unique and Conserved Super Enhancer-Associated Transcriptional Landscape.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
 EZH2-Driven Lung Cancer as a Molecularly Distinct Entity.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
 ZH2 Overexpression Induces Murine Lung Cancer.Cancer Discov. 2016 Sep; 6(9): 1006–1021. |
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 Structure of PRC2 bound to an inhibitor (Inhibitor 1).Cancer Discov. 2016 Sep; 6(9): 949–952. |
 EZH2 inhibitors. Each of the preclinical and clinical molecules shown has a pyridine amide core except CPI-1205. The compounds in clinical studies are tazemetostat, GSK281626, and CPI-1205.Cancer Discov. 2016 Sep; 6(9): 949–952. |
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