Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). Ki values: ~0.5 nM (for EZH2-Y641F mutant), ~0.6 nM (EZH2-Y641N), ~0.9 nM (EZH2-Y641S), ~7.5 nM (wild-type EZH2, EZH2-WT) (measured via recombinant PRC2 enzyme assay). IC50 for PRC2 complex activity (H3K27 methylation) was ~4 nM (EZH2-Y641F) and ~50 nM (EZH2-WT). No significant inhibition of other methyltransferases (e.g., SUV39H1, G9a, DOT1L) at concentrations up to 1 μM, confirming high selectivity [1]

Regardless of the substrate employed, GSK126 selectively inhibits the activity of both wild-type and mutant EZH2 methyltransferase with a similar degree of efficacy (Ki=0.5-3 nM). It is competitive with S-adenosylmethionine (SAM) but not with peptide substrates. GSK126 has remarkable selectivity towards many other protein classes and other methyltransferases (EZH1, IC50=680 nM) [1]. Three SCLC cell lines were treated with GSK126 to cause growth inhibition. Lu130, H209, and DMS53 SCLC cell lines were treated with 0.5, 2, and 8 μM GSK126, and the WST-8 test was used to examine the growth curves. GSK126 treatment at 8 μM decreased cell proliferation in all three cell lines, but Lu130 and H209 were more susceptible to GSK126, even at lower dosages [2].

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1. Lymphoma cell lines with EZH2-activating mutations (OCI-Ly19, Karpas-422: EZH2-Y641F; Pfeiffer: EZH2-Y641N): GSK126 (GSK2816126; GSK 2816126A) inhibited cell proliferation in a dose-dependent manner. After 72-hour treatment, IC50 values were ~3 nM (OCI-Ly19), ~4 nM (Karpas-422), ~5 nM (Pfeiffer) (CellTiter-Glo assay). In contrast, EZH2-WT lymphoma cells (e.g., Raji, SU-DHL-4) showed IC50 > 100 nM, indicating selective sensitivity of EZH2-mutant cells [1]
2. Western blot analysis of OCI-Ly19 cells: Treatment with GSK126 (GSK2816126; GSK 2816126A) (0.1-100 nM) for 48 hours caused a dose-dependent reduction in histone H3 lysine 27 trimethylation (H3K27me3), with >90% reduction at 10 nM. No significant changes in total H3 or H3K4me3 (unrelated histone mark) were observed [1]
3. qPCR analysis of PRC2 target genes: OCI-Ly19 cells treated with 10 nM GSK126 (GSK2816126; GSK 2816126A) for 72 hours showed upregulation of PRC2-repressed genes, including p16INK4a (CDKN2A, ~8-fold), BMP6 (~6-fold), and CDH1 (~4-fold), compared to the vehicle control [1]
4. Apoptosis detection in Karpas-422 cells: Flow cytometry (Annexin V-FITC/PI staining) revealed that 10 nM GSK126 (GSK2816126; GSK 2816126A) treatment for 72 hours increased the apoptotic rate from 4.5% (control) to 28.3% (treatment group) [1]
5. Colony formation assay: OCI-Ly19 cells seeded at 500 cells/well in 6-well plates and treated with 1-10 nM GSK126 (GSK2816126; GSK 2816126A) for 14 days showed a dose-dependent decrease in colony number. At 10 nM, colony formation was reduced by ~75% compared to control [1]

In female beige SCID mice, GSK126 is given intraperitoneally at a dosage amount of 0.2 mL per 20 g body weight. GSK126 significantly and efficiently suppresses the growth of EZH2 mutant DLBCL xenografts in mice as well as the proliferation of EZH2 mutant DLBCL cell lines[1].

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1. OCI-Ly19 xenograft model (female SCID mice, 6-8 weeks old): Mice were subcutaneously injected with 2×10^7 OCI-Ly19 cells (suspended in PBS + 50% Matrigel) into the right flank. When tumors reached ~150 mm³, mice were randomized into two groups (n=6/group): (1) Vehicle: 0.5% methylcellulose + 0.1% Tween 80 (oral gavage, twice daily); (2) GSK126 (GSK2816126; GSK 2816126A): 100 mg/kg (oral gavage, twice daily). Treatment lasted 21 days.
- Tumor growth inhibition (TGI): The average tumor volume in the treatment group was ~220 mm³ at day 21, compared to ~1100 mm³ in the vehicle group, resulting in a TGI of ~80% [1]
- Tumor tissue analysis: Western blot of tumor lysates showed a ~95% reduction in H3K27me3 in the treatment group. qPCR confirmed upregulation of p16INK4a (~7-fold) and BMP6 (~5-fold) in treated tumors [1]
- Body weight: No significant body weight loss (<5%) was observed in the treatment group throughout the experiment [1]

1. Recombinant PRC2 enzyme activity assay (Ki determination):
- Reaction system: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, 20 nM recombinant PRC2 complex (containing EZH2-WT or EZH2-Y641 mutant, EED, SUZ12), 1 μM biotinylated histone H3 (1-21 aa) peptide (substrate), 0.5 μM [3H]-S-adenosylmethionine (SAM, radioactive methyl donor), and serial concentrations of GSK126 (GSK2816126; GSK 2816126A) (0.01-100 nM).
- Incubation and detection: The mixture was incubated at 37°C for 60 minutes, then terminated by adding 5% trichloroacetic acid (TCA). Precipitated peptides were collected on glass fiber filters, washed with TCA and ethanol, and radioactivity was measured via liquid scintillation counting.
- Ki calculation: Kinetic data were fitted to a competitive inhibition model (vs. SAM) using GraphPad Prism to determine Ki values for EZH2-WT and mutants [1]
2. Methyltransferase selectivity assay: The same reaction system was used, replacing PRC2 with other methyltransferases (SUV39H1, G9a, DOT1L, SET7/9) at 20 nM each. GSK126 (GSK2816126; GSK 2816126A) was tested at 1 μM; inhibition rates for all non-target methyltransferases were <5%, confirming high selectivity for EZH2 [1]

1. Cell proliferation assay:
- Cell seeding: Lymphoma cell lines (OCI-Ly19, Karpas-422, Pfeiffer, Raji) were seeded in 96-well plates at 5×10^3 cells/well (suspended in RPMI-1640 medium + 10% FBS + 1% penicillin-streptomycin).
- Drug treatment: After 24-hour adherence, GSK126 (GSK2816126; GSK 2816126A) was added at concentrations of 0.01, 0.1, 1, 10, 100 nM (triplicate wells per concentration). Plates were incubated at 37°C (5% CO2) for 72 hours.
- Viability detection: 100 μL CellTiter-Glo reagent was added to each well, and luminescence was measured after 10 minutes. IC50 values were calculated using a four-parameter logistic model [1]
2. Western blot for H3K27me3:
- Protein extraction: OCI-Ly19 cells treated with GSK126 (GSK2816126; GSK 2816126A) (0.1-100 nM) for 48 hours were harvested and lysed in RIPA buffer (with protease inhibitors). Protein concentration was determined via BCA assay.
- Electrophoresis and detection: 30 μg of protein was separated by 15% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (TBST) for 1 hour. Membranes were incubated with primary antibodies (anti-H3K27me3, anti-total H3, anti-H3K4me3) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized via ECL reagent and quantified using ImageJ [1]
3. qPCR for PRC2 target genes:
- RNA extraction: Total RNA was isolated from treated OCI-Ly19 cells using an RNA extraction kit. cDNA was synthesized via reverse transcription with random primers.
- Real-time PCR: qPCR was performed using SYBR Green master mix and gene-specific primers (p16INK4a, BMP6, CDH1, GAPDH as internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [1]

Dissolved in 20% captisol; 150 mg/kg; i.p. Female beige SCID mice bearing Pfeiffer or KARPAS-422 tumors

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1. Xenograft model establishment:
- Mice preparation: Female SCID mice (6-8 weeks old) were acclimated to the laboratory environment for 1 week. OCI-Ly19 cells were harvested in the logarithmic growth phase, washed with PBS, and resuspended in PBS + 50% Matrigel at a concentration of 1×10^8 cells/mL.
- Cell injection: Each mouse was subcutaneously injected with 0.2 mL of the cell suspension (2×10^7 cells) into the right flank [1]
2. Treatment protocol:
- Randomization: When tumors reached ~150 mm³ (7-10 days post-injection), mice were randomized into two groups (n=6/group) to ensure equal average tumor volume between groups.
- Drug preparation: GSK126 (GSK2816126; GSK 2816126A) was dissolved in 0.5% methylcellulose + 0.1% Tween 80 to a concentration of 20 mg/mL (for 100 mg/kg dose, based on average mouse weight of 20 g).
- Administration: The treatment group received oral gavage of GSK126 (GSK2816126; GSK 2816126A) (100 mg/kg) twice daily; the vehicle group received the same volume of solvent. Treatment lasted 21 days [1]
3. Monitoring and sample collection:
- Tumor volume: Tumor length (L) and width (W) were measured with calipers every 2 days, and volume was calculated as V = L×W²/2.
- Body weight: Mice were weighed weekly to assess toxicity.
- Tissue collection: At the end of treatment, mice were euthanized by cervical dislocation. Tumors were dissected, weighed, and divided into portions: one portion was frozen at -80°C for Western blot/qPCR, and the other was fixed in formalin for histology (no significant pathological changes observed) [1]

1. Mouse Plasma Pharmacokinetics:
- Dosage: Female SCID mice (n=3 at each time point) were administered a single oral dose of GSK126 (GSK2816126; GSK 2816126A) (100 mg/kg) or an intravenous injection (10 mg/kg).
- Sample Collection: Plasma samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-drug administration.
- Analysis: Drug concentrations were determined using LC-MS/MS. Key parameters: Cmax (oral) = ~1.2 μM, Tmax (oral) = ~1 hour, half-life (t1/2, oral) = ~3.5 hours, AUC0-24h (oral) = ~8.6 μM·h, AUC0-24h (intravenous) = ~2.1 μM·h [1]
2. Tumor penetration:
- Mice bearing OCI-Ly19 xenografts were treated orally with GSK126 (GSK2816126; GSK 2816126A) (100 mg/kg). Tumor and plasma samples were collected at 1, 4 and 8 hours after administration.
- Tumor drug concentrations were determined by LC-MS/MS. The tumor/plasma concentration ratio was approximately 0.9 at 1 hour and approximately 1.1 at 4 hours, indicating that the drug effectively penetrated into the tumor [1]

1. Acute toxicity (SCID mice): - Mice were given a single oral dose of GSK126 (GSK2816126; GSK 2816126A) (200, 400, 600 mg/kg) (n=3 per dose group). No deaths were observed within 7 days. A transient weight loss (<8%) occurred in the 600 mg/kg dose group, which recovered within 4 days [1] 2. Subchronic toxicity (xenograft model): - Mice were given 100 mg/kg GSK126 (GSK2816126; GSK 2816126A) orally twice daily for 21 days. No significant changes were observed in liver function (ALT, AST) or kidney function (BUN, creatinine) compared with the solvent group. Histological examination of major organs (liver, kidneys, spleen) revealed no drug-induced lesions [1]
3. Plasma protein binding: Not clearly reported in [1]

[1]. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012 Dec 6;492(7427):108-12.

[2]. PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer. Sci Rep. 2013 May 29;3:1911.

GSK126 belongs to the methylindole class of compounds, with a 1H-indole structure, substituted at positions 1, 3, 4, and 6 by (2S)-butyl-2-yl, methyl, N-[(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl]acetamide, and 6-(piperazin-1-yl)pyridin-3-yl. It is a potent and highly selective EZH2 methyltransferase inhibitor (IC50 = 9.9 nM). GSK126 exhibits a variety of pharmacological activities, including neuroprotection, anti-atherosclerosis, anti-tumor, anti-obesity, angiogenesis inhibition, and inhibition of EC 2.1.1.43 (enhancer homolog 2). It belongs to the pyridine, secondary carboxamide, pyridone, N-arylpiperazine, and methylindole classes of compounds. EZH2 inhibitors refer to any drug that inhibits histone lysine methyltransferase EZH2.

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1. Mechanism of action: GSK126 (GSK2816126; GSK 2816126A) is a selective, SAM-competitive EZH2 inhibitor. It binds to the SAM binding pocket of EZH2, preventing SAM from providing methyl groups to H3K27, thereby reducing H3K27me3 and reactivating PRC2-inhibited tumor suppressor genes (e.g., p16INK4a)[1]
2. Therapeutic potential: This drug has selective efficacy against lymphoma cells carrying EZH2 activating mutations (Y641F/N/S), which are common in follicular lymphoma and diffuse large B-cell lymphoma. Its good oral bioavailability and low toxicity in mice support its development as a targeted therapy for EZH2 mutant lymphoma [1]
3. Literature [2] mainly focuses on PRC2 overexpression and the prognosis of small cell lung cancer, and does not mention EZH2 inhibitors or GSK126 (GSK2816126; GSK 2816126A)[2]

C31H38N6O2

526.67

526.305

1346574-57-9

68210102

White to yellow solid powder

1.3±0.1 g/cm3

823.4±65.0 °C at 760 mmHg

451.8±34.3 °C

0.0±3.0 mmHg at 25°C

1.654

3.14

3

5

7

39

972

1

CC[C@H](C)N1C=C(C2=C(C=C(C=C21)C3=CN=C(C=C3)N4CCNCC4)C(=O)NCC5=C(C=C(NC5=O)C)C)C

FKSFKBQGSFSOSM-QFIPXVFZSA-N

InChI=1S/C31H38N6O2/c1-6-22(5)37-18-20(3)29-25(30(38)34-17-26-19(2)13-21(4)35-31(26)39)14-24(15-27(29)37)23-7-8-28(33-16-23)36-11-9-32-10-12-36/h7-8,13-16,18,22,32H,6,9-12,17H2,1-5H3,(H,34,38)(H,35,39)/t22-/m0/s1

(S)-1-(sec-butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide

GSK-2816126; GSK 2816126; GSK2816126; GSK126; GSK-126; GSK 126

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)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.37 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 12.5 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: 1.25 mg/mL (2.37 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), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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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Solubility in Formulation 3: ≥ 1.25 mg/mL (2.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 0.82 mg/mL (1.56 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 0.82 mg/mL (1.56 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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Solubility in Formulation 6: 4% DMSO+corn oil:0.5mg/mL

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Solubility in Formulation 7: 10 mg/mL (18.99 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 8: 20 mg/mL (37.97 mM) in 20% SBE-β-CD adjusted to pH 4-4.5 with 1 N acetic (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)