Structure
GSK126, inhibitor
GSK126
Purity
99.59%
Synonyms
GSK126; inhibitor
Purity/Purification
99.59%
Form/Format
POWDER
CAS
1346574-57-9
Formula
C31H38N6O2
Solubility
DMSO: 3 mg/ml (5.69 mM)
Ethanol: < 1 mg/ml
Ethanol: < 1 mg/ml
Biological Activity
GSK126 is a excellently specific EZH2 methyltransferase inhibitor (IC50=9.9 nM).
Smiles
CC[C@H](C)n1cc(C)c2c(cc(cc12)-c1ccc(nc1)N1CCNCC1)C(=O)NCc1c(C)cc(C)[nH]c1=O
Receptor (IC50)
EZH2: 9.9nM
Animal Experiment
Animal Model: Febeige SCID mice bearing Pfeiffer or KARPAS-422 tumors.
Preparation and Storage
2 years -80°C in solvent; 3 years -20°C powder
Structure
Structure
Related Product Information for GSK126, inhibitor
Kinase Assay
EZH2 assay: The five-member PRC2 complex (DYKDDDDK-EZH2, EED, SUZI2, AEBP2, RbAp48) containing either wild-type or mutant EZH2 is prepared. GSK126 is dissolved in DMSO and tested at concentrations of 0.6 nM to 300 nM with a final DMSO concentration of 2.5%. In contrast to wild-type EZH2 which prefers H3K27meO as a substrate in vitro, EZH2 Y641 mutants prefer H3K27me2 and have little activity with H3K27meO or H3K27mel. The A677G mutant is distinct from both the wild-type and Y641 mutant forms of EZH2 in that it efficiently methylates H3K27meO, H3K27mel, and H3K27me2; therefore, histone H3 peptides (residues 21-44; 10 uM final) with either K27meO (wild type, A677G EZH2), K27mel (A677G EZH2), or K27me2 (A677G, Y641N, Y641C, Y641H, Y641S and Y641F EZH2) are used as methyltransferase substrates. GSK126 is added to plates followed by addition qf 6 nM EZH2 complex and peptide. As the potency of GSK126 is at or near the tight binding limit of an assay run at [SAM] = Km, IC50 values are measured at a high concentration of the competitive substrate SAM relative to its Km (7.5 uM SAM where the SAM Km is 0.3 uM). Under these conditions, the contribution from the enzyme concentration becomes relatively small and accurate estimates of Ki can be calculated. Reactions are initiated with [3H]-SAM, incubated for 30 min, quenched with the addition of 500-fold excess unlabelled SAM, and the methylated product peptide is captured on phosphocellulose filters according to the vendor supplied protocol for MSPH Multiscreen plates. Plates are read on a TopCount after adding 20 III of Microscint-20 cocktail. Apparent Ki values are calculated using the ChengPrus off relationship for a competitive inhibitor. IC50=Ki (1+[S]/Km)+[E]/2, where E is the enzyme and S is the substrate.
Cell Assay:
The optimal cell seeding is determined empirically for all cell lines by examining the growth of a wide range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells are then plated at the optimal seeding density 24 h before treatment (in duplicate) with a 20-point two fold dilution series of GSK126 or 0.15% DMSO. Plates are incubated for 6 days at 3JOC in 5% CO2. Cells are then lysed with CeliTiter-Glo (CTG) and chemiluminescent signal is detected with a TECAN Safire2 microplate reader. In addition, an untreated plate of cells is harvested at the time of compound addition (TO) to quantify the starting number of cells. CTG values obtained after the 6 day treatment are expressed as a percent of the TO value and plotted against compound concentration. Data are fit with a four-parameter equation to generate a concentration response curve and the concentration of GSK126 required to inhibit 50% of growth (growth IC50) is determined.(Only for Reference)
EZH2 assay: The five-member PRC2 complex (DYKDDDDK-EZH2, EED, SUZI2, AEBP2, RbAp48) containing either wild-type or mutant EZH2 is prepared. GSK126 is dissolved in DMSO and tested at concentrations of 0.6 nM to 300 nM with a final DMSO concentration of 2.5%. In contrast to wild-type EZH2 which prefers H3K27meO as a substrate in vitro, EZH2 Y641 mutants prefer H3K27me2 and have little activity with H3K27meO or H3K27mel. The A677G mutant is distinct from both the wild-type and Y641 mutant forms of EZH2 in that it efficiently methylates H3K27meO, H3K27mel, and H3K27me2; therefore, histone H3 peptides (residues 21-44; 10 uM final) with either K27meO (wild type, A677G EZH2), K27mel (A677G EZH2), or K27me2 (A677G, Y641N, Y641C, Y641H, Y641S and Y641F EZH2) are used as methyltransferase substrates. GSK126 is added to plates followed by addition qf 6 nM EZH2 complex and peptide. As the potency of GSK126 is at or near the tight binding limit of an assay run at [SAM] = Km, IC50 values are measured at a high concentration of the competitive substrate SAM relative to its Km (7.5 uM SAM where the SAM Km is 0.3 uM). Under these conditions, the contribution from the enzyme concentration becomes relatively small and accurate estimates of Ki can be calculated. Reactions are initiated with [3H]-SAM, incubated for 30 min, quenched with the addition of 500-fold excess unlabelled SAM, and the methylated product peptide is captured on phosphocellulose filters according to the vendor supplied protocol for MSPH Multiscreen plates. Plates are read on a TopCount after adding 20 III of Microscint-20 cocktail. Apparent Ki values are calculated using the ChengPrus off relationship for a competitive inhibitor. IC50=Ki (1+[S]/Km)+[E]/2, where E is the enzyme and S is the substrate.
Cell Assay:
The optimal cell seeding is determined empirically for all cell lines by examining the growth of a wide range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6 days. Cells are then plated at the optimal seeding density 24 h before treatment (in duplicate) with a 20-point two fold dilution series of GSK126 or 0.15% DMSO. Plates are incubated for 6 days at 3JOC in 5% CO2. Cells are then lysed with CeliTiter-Glo (CTG) and chemiluminescent signal is detected with a TECAN Safire2 microplate reader. In addition, an untreated plate of cells is harvested at the time of compound addition (TO) to quantify the starting number of cells. CTG values obtained after the 6 day treatment are expressed as a percent of the TO value and plotted against compound concentration. Data are fit with a four-parameter equation to generate a concentration response curve and the concentration of GSK126 required to inhibit 50% of growth (growth IC50) is determined.(Only for Reference)
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Product Notes
The GSK126 (Catalog #AAA577697) is an Inhibitor and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "GSK126, Inhibitor" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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