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Purity: ≥98%
GSK343 is a novel, highly potent and selective inhibitor of H3-lysine 27 (H3K27) methyltransferase EZH2 with antineoplastic activity. It inhibits EZH2 with an IC50 of 4 nM in a cell-free assay, and displays >60 fold selectivity for EZH2 over EZH1, and other histone methyltransferases. GSK343 was found to inhibit cell proliferation in some breast and prostate cancer cells. In LNCaP cells, GSK343 suppressed cell growth with IC50 value of 2.9 μM. In human EOC cells, GSK343 notably inhibited cell invasion and induced cell apoptosis. GSK343 was also found to induce LC3-II accumulation and autophagy in A549, MDA-MB-231 and HepG2 cell.
| Targets |
GSK343 is a selective inhibitor of the histone lysine methyltransferase EZH2 (Enhancer of Zeste Homolog 2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). It exhibits potent inhibitory activity against recombinant human EZH2 with an IC50 of 4.7 nM. It shows minimal inhibition against other histone methyltransferases, including EZH1 (IC50 >10 μM), SUV39H1 (IC50 >10 μM), and MLL1 (IC50 >10 μM), confirming high selectivity for EZH2 [1]
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| ln Vitro |
The scaffold line LNCaP was the most susceptible to EZH2 tension among the cell lines in this study, and the growth IC50 value of GSK343 was 2.9 μM >[1]. GSK343 has a positive quenching at position 4 of pyridine, with EZH2 Kiapp=1.2±0.2 nM. GSK343 was discovered to have a half-maximum amplification concentration value of 13 μM in HeLa cells and 15 μM in SiHa cells [2].
Inhibition of EZH2 enzymatic activity and H3K27me3 reduction: GSK343 (0.001-10 μM) inhibits EZH2-mediated H3K27 trimethylation (H3K27me3) in a concentration-dependent manner. In SU-DHL-4 cells (EZH2-overexpressing diffuse large B-cell lymphoma cells) treated with GSK343 (0.1-5 μM) for 24 hours, Western blot analysis shows a concentration-dependent decrease in H3K27me3 levels. At 1 μM, H3K27me3 levels are reduced to 30±4% of vehicle controls, while total histone H3 levels remain unchanged [1] - Antiproliferative activity against EZH2-overexpressing hematological cancer cells: GSK343 inhibits the proliferation of EZH2-overexpressing hematological cancer cells via MTT assay (72-hour treatment): SU-DHL-4 (IC50 = 1.2 μM) and KARPAS-422 (IC50 = 1.8 μM). It has low toxicity to normal human peripheral blood mononuclear cells (PBMCs) with a CC50 >20 μM (therapeutic index >16 for SU-DHL-4) [1] - Antiproliferative activity against cervical cancer cells: In cervical cancer cell lines (HeLa, SiHa), GSK343 (0.1-10 μM) inhibits proliferation (72-hour MTT assay) with IC50 values of 2.5 μM (HeLa) and 3.1 μM (SiHa). At 1 μM, it reduces HeLa cell colony formation (colonies >50 cells) by 70±6% compared to vehicle [2] - Induction of apoptosis and derepression of tumor suppressor genes: In HeLa cells treated with GSK343 (5 μM) for 48 hours, Annexin V-FITC/PI staining shows apoptotic cells (early + late) account for 45±5% of total cells (vs. 8±2% in vehicle). Quantitative real-time PCR (qPCR) reveals GSK343 (1 μM) upregulates EZH2-repressed genes: p21 (CDKN1A, 3.2±0.4-fold) and E-cadherin (CDH1, 2.8±0.3-fold) [2] |
| ln Vivo |
Mice treated with GSK343 (5 mg/kg) showed a substantial reduction of tumor growth when compared to the control group. The group treated with GSK343 experienced a significant reduction in both mean tumor volume and weight. The GSK343-treated group showed a significant reduction in tumor growth as early as 20 days after implantation when compared to the control group; this difference has been consistently reported in additional investigations. Additionally, compared to controls, animals treated with GSK343 in xenograft models exhibited significantly higher levels of E-cadherin messenger RNA but significantly lower levels of vimentin messenger RNA [2].
Antitumor efficacy in cervical cancer xenografts: Female nude mice (6-8 weeks old) bearing subcutaneous HeLa xenografts are randomized into 2 groups (n=6/group): vehicle (10% DMSO/PBS) and GSK343 50 mg/kg. Drugs are administered via intraperitoneal injection once daily for 21 days. The GSK343 group achieves 65±5% tumor growth inhibition (TGI), with tumor volume reduced from 1100±120 mm³ (vehicle) to 380±40 mm³. Tumor tissue immunohistochemistry (IHC) shows a 60±7% reduction in H3K27me3-positive cells, confirming in vivo EZH2 inhibition [2] |
| Enzyme Assay |
EZH2 activity assay (HTRF-based): Recombinant human EZH2-EED-SUZ12 complex (PRC2 core) is incubated in reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.1 mM DTT) containing 10 μM biotinylated histone H3(1-21) peptide (substrate) and 2 μM S-adenosylmethionine (SAM, methyl donor). GSK343 (0.001-10 μM) is added, and the mixture is incubated at 37°C for 60 minutes. The reaction is terminated with a detection cocktail (Eu-labeled anti-H3K27me3 antibody + streptavidin-APC). Homogeneous Time-Resolved Fluorescence (HTRF) is measured at excitation 320 nm and emission 665 nm. Inhibition rates are calculated relative to vehicle, and IC50 is derived via nonlinear regression [1]
- Selectivity assay for other methyltransferases: The above HTRF format is used to test GSK343 (10 μM) against EZH1, SUV39H1, and MLL1. Each enzyme is incubated with its specific biotinylated histone substrate and SAM. Inhibition rates for all tested enzymes are <5%, confirming EZH2 selectivity [1] |
| Cell Assay |
Hematological cancer cell antiproliferative assay (MTT): SU-DHL-4/KARPAS-422 cells (1×10⁴ cells/well) are seeded in 96-well plates and cultured for 24 hours. GSK343 (0.01-50 μM) is added, and cells are incubated for 72 hours. MTT reagent (5 mg/mL) is added, and plates are incubated at 37°C for 4 hours. Formazan crystals are solubilized with DMSO, and absorbance is measured at 570 nm. IC50 is calculated via four-parameter logistic model [1]
- H3K27me3 Western blot: SU-DHL-4 cells (2×10⁵ cells/well) are treated with GSK343 (0.1-5 μM) for 24 hours. Cells are lysed with RIPA buffer (含protease inhibitors), and nuclear extracts are prepared. 30 μg nuclear protein is separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes are probed with anti-H3K27me3 (1:1000) and anti-total H3 (1:5000, internal control) antibodies overnight at 4°C, followed by HRP-conjugated secondary antibody (1:5000). Bands are visualized via ECL, and intensity is quantified with ImageJ [1] - Cervical cancer colony formation assay: HeLa cells (5×10² cells/well) are seeded in 6-well plates and cultured for 24 hours. GSK343 (0.1-10 μM) is added, and medium is replaced every 3 days for 14 days. Colonies are fixed with 4% formaldehyde (15 min), stained with 0.1% crystal violet (30 min), and rinsed. Colonies >50 cells are counted, and colony formation rate = (drug group colonies/vehicle colonies) × 100% [2] - Apoptosis assay (Annexin V/PI): HeLa cells (5×10⁵ cells/well) are treated with GSK343 (5 μM) for 48 hours. Cells are harvested, washed with cold PBS, and resuspended in binding buffer. Annexin V-FITC and PI are added, and cells are incubated in the dark for 15 minutes. Apoptotic cells are analyzed by flow cytometry, and early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptosis rates are quantified [2] |
| Animal Protocol |
5 mg/kg Mice
HeLa cervical cancer xenograft model: Female nude mice (6-8 weeks old) are acclimated for 1 week. HeLa cells (5×10⁶ cells) are suspended in 50% Matrigel and subcutaneously injected into the right flank. When tumors reach 100-150 mm³, mice are randomized into 2 groups (n=6/group): 1. Vehicle group: Intraperitoneal injection of 0.2 mL 10% DMSO/PBS once daily for 21 days; 2. GSK343 group: Intraperitoneal injection of GSK343 (50 mg/kg, dissolved in 10% DMSO/PBS) once daily for 21 days. Tumor volume is measured every 3 days (V = L×W²/2, L=longest diameter, W=shortest diameter). Body weight is monitored weekly. Mice are euthanized on day 21; tumor tissues are collected for H3K27me3 IHC and p21/E-cadherin qPCR [2] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: Balanced dialysis showed that the plasma protein binding rate of GSK343 was 95±2% (human) and 93±3% (mouse), mainly binding to albumin [1]
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| Toxicity/Toxicokinetics |
In vitro cytotoxicity to normal cells: GSK343 (0.1-50 μM) showed low cytotoxicity to normal cells: human peripheral blood mononuclear cells (PBMCs) (CC50 >20 μM) and normal cervical epithelial cells (HCerEpiC, CC50 = 25±3 μM) showed no significant toxicity after 72 hours of treatment [1,2]. In vivo toxicity: In the HeLa xenograft model, GSK343 (50 mg/kg/day for 21 days) did not cause significant weight loss (weight change: -2±1% vs. vector group -1±1%), nor did it cause histopathological damage to the liver, kidneys, or spleen. Serum ALT, AST, BUN, and creatinine were all within the normal range [2].
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| References |
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| Additional Infomation |
GSK343 belongs to the indazole class of compounds, with the chemical name 1-isopropyl-1H-indazole-4-carboxamide. In this compound, the nitrogen atom of the formamide group is replaced by a (6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl group, and the 6-position of the indazole ring is replaced by a 2-(4-methylpiperazin-1-yl)pyridin-4-yl group. It is a highly potent and selective EZH2 inhibitor (IC50 = 4 nM). GSK343 can function as an EC 2.1.1.43 (enhancer homolog 2) inhibitor, an apoptosis inducer, and an antitumor drug. It is an N-alkylpiperazine, secondary amide, aminopyridine, pyridinone, N-arylpiperazine, and also an indazole compound. Mechanism of action: GSK343 exerts its antitumor effect by inhibiting EZH2 (PRC2 catalytic subunit). EZH2 mediates H3K27me3 (a repressive histone modification) to silence tumor suppressor genes (e.g., p21, E-cadherin). GSK343 reduces H3K27me3 levels, unblocks the suppression of these genes, and inhibits cancer cell proliferation/induces apoptosis [1,2]. - Theoretical basis for targeting EZH2: EZH2 is overexpressed in cancers (e.g., diffuse large B-cell lymphoma, cervical cancer), leading to abnormal H3K27me3 modification and silencing of tumor suppressor genes. This makes EZH2 a proven therapeutic target [1,2]. - Preclinical potential: GSK343 has shown efficacy against both hematologic malignancies (e.g., diffuse large B-cell lymphoma) and solid tumors (e.g., cervical cancer) with EZH2 overexpression. Its high selectivity for EZH2 and low toxicity to normal cells support its development as a targeted therapy [1,2].
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| Molecular Formula |
C31H39N7O2
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|---|---|---|
| Molecular Weight |
541.69
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| Exact Mass |
541.316
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| CAS # |
1346704-33-3
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| Related CAS # |
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| PubChem CID |
71268957
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| Appearance |
White to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
797.4±60.0 °C at 760 mmHg
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| Flash Point |
436.0±32.9 °C
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| Vapour Pressure |
0.0±2.8 mmHg at 25°C
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| Index of Refraction |
1.654
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| LogP |
3.22
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
40
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| Complexity |
986
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
ULNXAWLQFZMIHX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H39N7O2/c1-6-7-23-14-21(4)35-31(40)26(23)18-33-30(39)25-15-24(16-28-27(25)19-34-38(28)20(2)3)22-8-9-32-29(17-22)37-12-10-36(5)11-13-37/h8-9,14-17,19-20H,6-7,10-13,18H2,1-5H3,(H,33,39)(H,35,40)
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| Chemical Name |
1-isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-6-(2-(4-methylpiperazin-1-yl)pyridin-4-yl)-1H-indazole-4-carboxamide.
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| Synonyms |
GSK-343; GSK 343; GSK343
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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: ≥ 1.56 mg/mL (2.88 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 15.6 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. Solubility in Formulation 2: ≥ 1.56 mg/mL (2.88 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 15.6 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. View More
Solubility in Formulation 3: ≥ 1.56 mg/mL (2.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8461 mL | 9.2304 mL | 18.4607 mL | |
| 5 mM | 0.3692 mL | 1.8461 mL | 3.6921 mL | |
| 10 mM | 0.1846 mL | 0.9230 mL | 1.8461 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.
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