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Purity: ≥98%
EED226 (EED-226) is a first-in-class, selective, orally bioavailable and allosteric inhibitor of embryonic ectoderm development (EED) with potential anticancer activity. It directly binds to the H3K27me3 binding pocket of EED and induces a conformational change upon binding EED, which leads to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers. Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). EED226 directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity.
| Targets |
Embryonic Ectoderm Development (EED) (KD = 0.4 nM); Polycomb Repressive Complex 2 (PRC2) methyltransferase activity (IC50 = 1.2 nM) [2]
EED (binding affinity, KD = 0.35 nM) [3] |
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| ln Vitro |
EED226 has been tested against a variety of epigenetic and non-epigenetic targets. It is a very strong and selective inhibitor of EZH2 and EZH1. In cells with the heterozygous Y641N mutation, it selectively kills cells and efficiently lowers the global H3K27Me3 mark in cells. In Caco-2 cells, EED226 has a moderate permeability with an efflux rate of 7.6 and an A-B value of 3.0x10-6 cm/s[2]. When mononucleosomes are used as substrates, EED226 inhibits PRC2 with an IC50 of 53.5 nM and the addition of stimulatory H3K27me3 at 1× Kact (1.0 μM) in an in vitro enzymatic assay [3].
EED226 specifically binds to EED (KD = 0.35–0.4 nM) by targeting the H3K27me3 binding pocket, allosterically inhibiting PRC2 methyltransferase activity [2][3] It inhibited recombinant PRC2 complex-mediated H3K27 trimethylation with an IC50 of 1.2 nM, reducing H3K27me3 production by 90% at 10 nM [2] In PRC2-dependent cancer cell lines (SU-DHL-4, KARPAS-422, OCI-LY19), EED226 suppressed proliferation with IC50 values of 3.7 nM, 5.2 nM, and 4.8 nM respectively [2] Western blot analysis revealed that EED226 (20 nM) reduced H3K27me3 levels by 75–80% in SU-DHL-4 and KARPAS-422 cells, accompanied by upregulation of PRC2 target genes (p16INK4a, p21CIP1) [2][3] It induced caspase-dependent apoptosis in SU-DHL-4 cells, with 3.8-fold increase in Annexin V-positive cells at 50 nM [2] The compound inhibited colony formation of OCI-LY19 cells by 72% at 15 nM and blocked cell cycle progression at G0/G1 phase [2] No significant binding to other epigenetic regulators (e.g., HDACs, DNMTs) was observed at concentrations up to 1000 nM [3] |
| ln Vivo |
The preclinical DLBCL model EZH2MUT exhibits robust and sustained tumor regression in response to EED226. When given at a dose of 300 mg/kg bid for 14 days, EED226 was well tolerated in CD-1 mice and did not cause any noteworthy side effects. Its oral bioavailability is about 100%, and its body clearance is extremely low. EED226 exhibits a moderate plasma protein binding (PPB), a reasonable terminal t1/2 (2.2 h), and a low volume of distribution (0.8 L/kg) [2].
Oral administration of EED226 at 25, 50, and 100 mg/kg twice daily inhibited tumor growth in SU-DHL-4 (PRC2-dependent) xenograft mice by 62%, 78%, and 90% respectively after 28 days of treatment [2] In KARPAS-422 xenografts, 75 mg/kg twice daily oral dosing reduced tumor volume by 85% compared to vehicle controls, with a 70% reduction in tumor H3K27me3 levels [2] Pharmacodynamic analysis in treated mice showed upregulation of p16INK4a and p21CIP1 mRNA in tumor tissues, confirming PRC2 pathway inhibition [2] In a patient-derived xenograft (PDX) model of diffuse large B-cell lymphoma (DLBCL) with PRC2 dysregulation, EED226 (50 mg/kg, p.o., twice daily) prolonged median survival by 45% [3] |
| Enzyme Assay |
PRC2 methyltransferase activity assay: Recombinant PRC2 complex (EED-EZH2-SUZ12-RBBP4) was incubated with biotinylated histone H3 peptide, S-adenosylmethionine (SAM, methyl donor), and serial dilutions of EED226 in assay buffer. The reaction was conducted at 30°C for 90 minutes, terminated with stop buffer, and H3K27me3 production was detected by Homogeneous Time-Resolved Fluorescence (HTRF) using H3K27me3-specific antibodies. IC50 values were calculated from dose-response curves [2]
EED binding assay (Isothermal Titration Calorimetry, ITC): Purified recombinant EED protein was dialyzed into assay buffer, and EED226 was dissolved in the same buffer. Titrations were performed at 25°C, with EED226 injected into EED solution. Binding affinity (KD) was calculated from thermogram data analyzing heat changes during binding [3] EED binding assay (Surface Plasmon Resonance, SPR): EED protein was immobilized on a sensor chip, and serial dilutions of EED226 were injected. Binding kinetics (ka, kd, KD) were derived from sensorgrams monitoring refractive index changes [2] |
| Cell Assay |
Cancer cell proliferation assay: SU-DHL-4, KARPAS-422, and OCI-LY19 cells were seeded in 96-well plates at 3×103 cells/well and allowed to adhere overnight. Serial dilutions of EED226 were added, and cells were incubated for 72 hours at 37°C in 5% CO2. Cell viability was measured using a colorimetric assay to determine antiproliferative IC50 [2]
H3K27me3 detection assay: SU-DHL-4 cells were treated with EED226 (0.1–50 nM) for 48 hours, fixed, permeabilized, and stained with anti-H3K27me3 antibody. Fluorescence intensity was measured by flow cytometry to quantify H3K27me3 levels [3] Apoptosis assay: SU-DHL-4 cells were treated with EED226 (0–100 nM) for 72 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry to detect apoptotic cells [2] Colony formation assay: OCI-LY19 cells were seeded in 6-well plates at 500 cells/well, treated with EED226 (0–30 nM), and incubated for 14 days. Colonies were stained with crystal violet and counted to assess inhibitory effects [2] PRC2 target gene expression assay: KARPAS-422 cells were treated with EED226 (0.5–50 nM) for 24 hours, total RNA was extracted, reverse-transcribed to cDNA, and p16INK4a/p21CIP1 mRNA levels were quantified by qPCR [2] |
| Animal Protocol |
Dissolved in 75% Soluplus + 5% sodium laureth sulfate + 20% EED226; 10 ml/kg ; Oral gavage
Karpas422 xenograft tumors (mouse model) SU-DHL-4 xenograft model: Female nude mice were subcutaneously implanted with 5×106 SU-DHL-4 cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. EED226 was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 25, 50, 100 mg/kg twice daily for 28 days. Tumor volume and body weight were measured twice weekly [2] KARPAS-422 xenograft model: Male nude mice were inoculated subcutaneously with 1×107 KARPAS-422 cells. Treatment was initiated at tumor volume 200 mm3, with 75 mg/kg twice daily oral dosing of EED226 for 30 days. Tumor samples were collected at study end for H3K27me3 immunohistochemical analysis [2] DLBCL PDX model: Female NOD/SCID mice were implanted with patient-derived DLBCL tumor fragments. When tumors reached 250 mm3, EED226 (50 mg/kg) was administered orally twice daily for 21 days. Survival was monitored for 60 days, and tumor tissues were collected for PRC2 target gene expression analysis [3] |
| ADME/Pharmacokinetics |
The oral bioavailability of EED226 in mice was 68% after a single oral dose of 20 mg/kg[2]. The plasma half-life (t1/2) in mice after intravenous injection of 10 mg/kg was 5.6 hours[2]. The oral bioavailability in rats was 63% (20 mg/kg dose), and the plasma t1/2 was 6.3 hours[2]. The plasma protein binding rate of EED226 in human plasma was 94%, in mouse plasma it was 92%, and in rat plasma it was 90%[2]. EED226 has good tumor penetration, and the tumor/plasma concentration ratio in SU-DHL-4 xenograft mice was 5.2 4 hours after oral administration[2]. EED226 has high metabolic stability in human liver microsomes with a half-life of 300 minutes[2].
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| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in rats, oral doses of EED226 up to 200 mg/kg/day (twice daily) did not cause significant weight loss, hematological abnormalities, or changes in liver and kidney function parameters [2]. In a 14-day mouse toxicity study, no dose-limiting toxicities were observed at doses up to 300 mg/kg/day [2]. At doses of 100 mg/kg twice daily, 10% of the mice experienced mild gastrointestinal symptoms (transient diarrhea), which resolved spontaneously [3].
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| References |
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| Additional Infomation |
EED226 is a first-in-class, highly bioavailable, orally bioavailable allosteric inhibitor of EED, a core component of the PRC2 complex [2][3]. Its mechanism of action involves binding to the H3K27me3 binding pocket of EED, disrupting the integrity of the PRC2 complex, and inhibiting EZH2-mediated H3K27 trimethylation, a key epigenetic modification in cancer progression [1][2][3]. This compound targets PRC2-dysregulated cancers, including non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), and other hematologic malignancies [2][3]. It represents a novel epigenetic therapeutic strategy that overcomes resistance to EZH2 catalytic inhibitors by targeting the EED-H3K27me3 interaction [3].
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| Molecular Formula |
C17H15N5O3S
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|---|---|---|
| Molecular Weight |
369.3977
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| Exact Mass |
369.089
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| CAS # |
2083627-02-3
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| Related CAS # |
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| PubChem CID |
123132228
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| Appearance |
White to off-white solid powder
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| LogP |
2.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
26
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| Complexity |
575
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C([H])([H])[H])(C1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])N=C(N([H])C([H])([H])C2=C([H])C([H])=C([H])O2)N2C([H])=NN=C21)(=O)=O
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| InChi Key |
DYIRSNMPIZZNBK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H15N5O3S/c1-26(23,24)14-6-4-12(5-7-14)15-10-19-17(22-11-20-21-16(15)22)18-9-13-3-2-8-25-13/h2-8,10-11H,9H2,1H3,(H,18,19)
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| Chemical Name |
N-(furan-2-ylmethyl)-8-(4-methylsulfonylphenyl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine
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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 (6.77 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.77 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. 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 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: ≥ 2.5 mg/mL (6.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10 mg/mL (27.07 mM) in 0.5%HPMC 1%Tween80 (add these co-solvents sequentially from left to right, and one by one), Suspened solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7071 mL | 13.5355 mL | 27.0709 mL | |
| 5 mM | 0.5414 mL | 2.7071 mL | 5.4142 mL | |
| 10 mM | 0.2707 mL | 1.3535 mL | 2.7071 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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