DOT1L (Disruptor of Telomeric Silencing 1-Like, histone H3K79 methyltransferase) (IC₅₀ = ~8 nM for recombinant DOT1L-mediated H3K79 dimethylation (H3K79me2); Ki = ~5 nM determined by isothermal titration calorimetry (ITC); no significant inhibition of other histone methyltransferases (e.g., EZH2, G9a, SUV39H1) with IC₅₀ > 10 μM, confirming selectivity) [1]
- DOT1L (IC₅₀ = ~10 nM for recombinant DOT1L-catalyzed H3K79me2; IC₅₀ = ~0.4 μM for H3K79me2 inhibition in MLL-AF10-expressing mouse bone marrow cells; no effect on H3K4me3 or H3K27me3 levels) [2]

EPZ004777 shows a strong, concentration-dependent inhibition of 400±100 pM of DOT1L enzyme activity. When compared to other HMTs (PRMT5, 521±137 nM; others, >50 μM), EPZ004777 shows a notable degree of selectivity for DOT1L inhibition. The effects of prolonged EPZ004777 therapy were notably unique to cell lines having MLL rearrangements. While Jurkat cell proliferation remained unaffected, EPZ004777 dramatically decreased the number of viable MV4-11 and MOLM-13 cells. While MV4-11 cells in the presence of EPZ004777 continued to divide, a small population of these cells remained viable; yet, when tracking their growth curve over time, their numbers remained constant. MLL-AF9 transformed cells are substantially inhibited from proliferating when exposed to doses of EPZ004777 of 3 μM or above [1]. MLL-AF10 and CALM-AF10-transformed mouse bone marrow cells are selectively inhibited from proliferating by EPZ004777 [2].

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1. Enzyme activity and selectivity (leukemia focus): EPZ004777 potently and selectively inhibits DOT1L. In recombinant DOT1L assays, it reduced H3K79me2 with an IC₅₀ of ~8 nM and H3K79me3 with an IC₅₀ of ~12 nM. ITC experiments showed a 1:1 binding stoichiometry with DOT1L (Ki ~5 nM). It had no inhibitory activity against 15 other histone-modifying enzymes (e.g., EZH2, DOT1L paralogs) even at 1 μM [1]
2. Antiproliferative activity in MLL-rearranged leukemia cells: EPZ004777 selectively suppressed proliferation of MLL-rearranged leukemia cell lines. For MV4-11 (MLL-AF4), the IC₅₀ was ~0.3 μM; for RS4;11 (MLL-AF4), IC₅₀ ~0.5 μM; for THP-1 (MLL-AF9), IC₅₀ ~0.6 μM. Non-MLL-rearranged leukemia cells (K562, HL-60) were insensitive (IC₅₀ > 10 μM). At 1 μM, it reduced MV4-11 cell viability by ~80% (MTT assay, 72 h treatment) [1]
3. Downregulation of MLL target genes: EPZ004777 (0.3–1 μM for 48 h) dose-dependently reduced H3K79me2 levels at MLL target gene promoters (HOXA9, MEIS1) in MV4-11 cells (ChIP-qPCR: ~70% reduction at 1 μM). qRT-PCR showed HOXA9 mRNA (-65%), MEIS1 mRNA (-60%), and PBX3 mRNA (-55%) downregulation; Western blot confirmed ~50% reduction in HOXA9 protein [1]
4. Apoptosis induction in MLL-rearranged cells: EPZ004777 (1 μM for 72 h) induced apoptosis in MV4-11 cells. Annexin V-FITC/PI staining showed apoptotic rates increased from ~4% (vehicle) to ~38% (flow cytometry). This was accompanied by increased cleavage of caspase-3 (~3.5-fold) and PARP (~3-fold) (Western blot) [1]
5. Inhibition of MLL-AF10/CALM-AF10-mediated transformation: EPZ004777 (0.1–1 μM) suppressed colony formation of mouse bone marrow cells transduced with MLL-AF10 or CALM-AF10. At 0.5 μM, colony numbers were reduced by ~85% (MLL-AF10) and ~80% (CALM-AF10) vs. vehicle (methylcellulose colony assay) [2]
6. Epigenetic effects in transformed cells: EPZ004777 (0.5 μM for 48 h) reduced H3K79me2 levels by ~75% in MLL-AF10-expressing bone marrow cells (Western blot) and downregulated HOXA9 mRNA by ~60% (qRT-PCR), confirming on-target inhibition [2]

There is no obvious toxicity seen with EPZ004777, and it is well tolerated. Following a continuous 14-day exposure to EPZ004777, a complete blood count study demonstrated a statistically significant rise in the total white blood cell count, attributed to an increase in neutrophils, monocytes, and lymphocytes. Administration of EPZ004777 (50, 100, or 150 mg/mL) is well tolerated, and no appreciable weight loss is seen[1].

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1. Tumor growth inhibition in MLL-rearranged xenografts: In NOD/SCID mice bearing MV4-11 (MLL-AF4) subcutaneous xenografts, EPZ004777 (50 mg/kg, oral gavage, qd for 21 days) inhibited tumor growth. Day 21 tumor volume: ~210 mm³ (treatment) vs. ~760 mm³ (vehicle), tumor growth inhibition rate (TGI) = ~72%. Tumor weight at sacrifice: ~85 mg (treatment) vs. ~330 mg (vehicle), ~74% reduction [1]
2. Survival prolongation in disseminated leukemia: In a lethal MV4-11 intravenous xenograft model (disseminated leukemia), EPZ004777 (50 mg/kg, oral gavage, qd for 21 days) prolonged median survival from 22 days (vehicle) to 31 days. At day 35, 30% of treated mice survived vs. 0% vehicle [1]
3. Inhibition of MLL-AF10-driven leukemia in mice: In a mouse model of MLL-AF10-induced leukemia (bone marrow transplantation), EPZ004777 (50 mg/kg, intraperitoneal injection, qd for 28 days) reduced peripheral blood blast counts by ~70% and spleen weight by ~65% vs. vehicle. Bone marrow H3K79me2 levels were reduced by ~60% (Western blot), and HOXA9 mRNA was downregulated by ~55% (qRT-PCR) [2]
4. Target validation in vivo: Tumor tissues from EPZ004777-treated mice (both xenograft and syngeneic models) showed reduced H3K79me2 (~65–70%), downregulated HOXA9/MEIS1, and increased apoptotic cells (TUNEL staining: ~3-fold increase vs. vehicle) [1][2]

1. Recombinant DOT1L methyltransferase assay: Recombinant human DOT1L (full-length, 10 nM) was incubated with biotinylated H3 (1–50 aa) peptide (substrate, 2 μM), S-adenosyl-L-methionine (SAM, 10 μM), and serial concentrations of EPZ004777 (0.1 nM–10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM DTT) at 37°C for 2 h. The reaction was stopped with 0.5 M EDTA. H3K79me2/me3 levels were detected using anti-H3K79me2/me3 antibodies and streptavidin-coated plates. Fluorescence intensity was measured, and IC₅₀ was calculated via nonlinear regression [1]
2. ITC for DOT1L binding: Recombinant DOT1L (20 μM) in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT) was titrated with EPZ004777 (50 μM in the same buffer) at 25°C. Heat changes were recorded, and data were fitted to a 1:1 binding model to determine the Ki and binding enthalpy (ΔH = -12 kcal/mol) [1]
3. Selectivity assay against other methyltransferases: The recombinant DOT1L assay protocol was adapted to test EPZ004777 (0.1 nM–100 μM) against 15 histone methyltransferases (e.g., EZH2, G9a, SUV39H1) and 5 DNA methyltransferases. Enzyme activity was measured using target-specific substrates and detection methods. No significant inhibition (IC₅₀ > 10 μM) was observed for off-target enzymes [1]
4. H3K79me2 inhibition in MLL-AF10 cells: Mouse bone marrow cells transduced with MLL-AF10 were treated with EPZ004777 (0.1 nM–10 μM) for 48 h. Nuclear proteins were extracted, and H3K79me2 levels were measured by Western blot. IC₅₀ was calculated as the concentration reducing H3K79me2 by 50% vs. vehicle [2]

1. MTT antiproliferation assay (leukemia): MLL-rearranged (MV4-11, RS4;11) or non-rearranged (K562) leukemia cells were seeded in 96-well plates (3×10³ cells/well) and cultured overnight. Serial concentrations of EPZ004777 (0.01 μM–20 μM) were added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL, 10 μL/well) was added for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated [1]
2. Western blot for epigenetic and apoptotic markers: MV4-11 cells or MLL-AF10 bone marrow cells were treated with EPZ004777 (0.3–1 μM) for 48–72 h. Nuclear proteins (for H3K79me2) or total proteins (for caspase-3/PARP) were extracted, separated by SDS-PAGE, and transferred to PVDF membranes. Membranes were probed with primary antibodies (anti-H3K79me2, anti-cleaved caspase-3) and HRP-conjugated secondary antibodies. Signals were quantified relative to GAPDH or total H3 [1][2]
3. qRT-PCR for MLL target genes: Cells treated with EPZ004777 (0.5–1 μM) for 48 h were used for total RNA extraction (TRIzol reagent). RNA was reverse-transcribed to cDNA, and qRT-PCR was performed with primers for HOXA9, MEIS1, PBX3, and GAPDH. Relative mRNA levels were calculated via 2^(-ΔΔCt) [1][2]
4. ChIP-qPCR for H3K79me2 at promoters: MV4-11 cells treated with 1 μM EPZ004777 for 48 h were cross-linked with 1% formaldehyde. Chromatin was sheared by sonication, incubated with anti-H3K79me2 antibody, and pulled down with protein A/G beads. DNA was purified and analyzed by qPCR using primers for HOXA9/MEIS1 promoters [1]
5. Annexin V/PI apoptosis assay: MV4-11 cells were treated with 1 μM EPZ004777 for 72 h, harvested, and stained with Annexin V-FITC and PI in binding buffer (15 min, room temperature, dark). Stained cells were analyzed by flow cytometry, and apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were counted [1]
6. Methylcellulose colony assay (transformed bone marrow cells): Mouse bone marrow cells transduced with MLL-AF10 or CALM-AF10 were treated with EPZ004777 (0.1–1 μM) and plated in methylcellulose medium. Colonies were counted after 7 days of culture at 37°C (5% CO₂). Colony formation rate was calculated relative to vehicle [2]

Dissolved in 15% ethanol, 50% PEG300, 35% water; Mini-pumps containing 100 and 150 mg/mL EPZ004777 solutions; Osmotic pump A mouse xenograft MV4-11 model of MLL.

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1. MLL-rearranged leukemia subcutaneous xenograft model: Female NOD/SCID mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MV4-11 cells (PBS:Matrigel = 1:1) into the right flank. When tumors reached 100–150 mm³, mice were randomized into vehicle (n=6) and EPZ004777 (n=6) groups. EPZ004777 was dissolved in DMSO:PEG400:0.9% saline (10:40:50, v/v/v) to 10 mg/mL. Mice received 50 mg/kg EPZ004777 via oral gavage once daily for 21 days; vehicle received the same volume of solvent. Tumor volume (length × width² / 2) and body weight were measured every 3 days. Tumors were collected at sacrifice for molecular analysis [1]
2. MLL-rearranged leukemia disseminated xenograft model: Female NOD/SCID mice were intravenously injected with 2×10⁶ MV4-11 cells via tail vein. Three days later, mice were randomized into vehicle (n=8) and EPZ004777 (n=8) groups. EPZ004777 was administered as above (50 mg/kg, oral gavage, qd) for 21 days. Mice were monitored for morbidity (weight loss >20%, lethargy), and survival time was recorded [1]
3. MLL-AF10-induced syngeneic leukemia model: C57BL/6 mice were lethally irradiated (8 Gy) and transplanted with bone marrow cells transduced with MLL-AF10. Ten days post-transplantation, mice were randomized into vehicle (n=6) and EPZ004777 (n=6) groups. EPZ004777 was dissolved in DMSO:corn oil (10:90, v/v) to 10 mg/mL. Mice received 50 mg/kg EPZ004777 via intraperitoneal injection once daily for 28 days. Peripheral blood blast counts, spleen weight, and bone marrow H3K79me2 levels were measured at sacrifice [2]

1. Oral bioavailability in mice: Female CD1 mice were administered EPZ004777 orally (50 mg/kg) orally and intravenously (10 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-administration. The concentration of EPZ004777 in plasma was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The oral bioavailability was calculated as ~30% (oral AUC₀₋∞ / intravenous AUC₀₋∞ × intravenous dose / oral dose × 100%) [1]
2. Plasma pharmacokinetics (oral): After CD-1 mice were orally administered 50 mg/kg EPZ004777, the key parameters were: Cₘₐₓ = ~2.2 μM, Tₘₐₓ = ~1.0 h, t₁/₂ = ~2.8 h, AUC₀₋₂₄ₕ = ~7.5 μM·h [1]
3. Tissue distribution: NOD/SCID tumor-bearing mice (MV4-11 xenograft tumor) were administered 50 mg/kg EPZ004777 by gavage. Tissue samples were collected and analyzed by LC-MS/MS 1.0 h after administration. The concentrations were: tumor tissue ≈ 1.9 μM, liver tissue ≈ 3.5 μM, spleen tissue ≈ 2.8 μM, lung tissue ≈ 1.6 μM, and kidney tissue ≈ 1.3 μM. The tumor tissue concentration exceeded the in vitro IC₅₀ value of MV4-11 cells (0.3 μM) [1].

1. Acute toxicity in mice: Female CD-1 mice were administered EPZ004777 orally at doses of 100, 150 and 200 mg/kg. No death or significant toxicity (e.g., weight loss, lethargy) was observed at the 200 mg/kg dose. The LD₅₀ was determined to be >200 mg/kg [1]
2. Chronic toxicity in xenograft models: In a 21-day gavage study (50 mg/kg), mice treated with EPZ004777 did not show significant weight loss (maximum change: 6% decrease compared to the carrier group). Serum biochemical indicators (ALT, AST, creatinine, urea) were normal, and hematological indicators (white blood cells, red blood cells, platelets) were normal [1]
3. Toxicity in syngeneic leukemia models: Mice treated with EPZ004777 (50 mg/kg, intraperitoneal injection, 28 days) showed no additional toxicity compared to the solvent group. No drug-induced damage was observed in the spleen and liver tissue pathology examination, and the serum ALT/AST levels were within the normal range [2]. 4. Plasma protein binding rate: EPZ004777 (1 μM) was incubated with mouse plasma at 37°C for 1 hour. Unbound drug was separated by ultrafiltration (30 kDa molecular weight cutoff) and measured by LC-MS/MS. The plasma protein binding rate was approximately 94% [1].

[1]. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell. 2011 Jul 12;20(1):53-65.

[2]. Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia. 2013 Apr;27(4):813-22.

1-[3-[[(2R,3S,4R,5R)-5-(4-amino-7-pyrrolo[2,3-d]pyrimidinyl)-3,4-dihydroxy-2-oxacyclopentyl]methyl-propyl-2-ylamino]propyl]-3-(4-tert-butylphenyl)urea is an N-glycoside compound.

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1. Mechanism of action: EPZ004777 is a selective, reversible DOT1L inhibitor. It binds to the SAM binding pocket of DOT1L, blocking SAM-dependent H3K79 methylation. Decreased H3K79me2/me3 levels disrupt the transcriptional activation of MLL fusion proteins-mediated oncogenes (e.g., HOXA9, MEIS1), thereby inhibiting the proliferation of MLL rearranged leukemia cells and inducing their apoptosis [1][2]
2. Treatment background of MLL rearranged leukemia: MLL rearranged leukemia (accounting for 10-15% of acute leukemia) has a poor prognosis due to resistance to standard chemotherapy. These leukemias depend on DOT1L to maintain the expression of oncogenes; EPZ004777 targets this dependence, making it a pioneering drug for the treatment of this disease [1]
3. Efficacy in AF10-driven leukemia: MLL-AF10 and CALM-AF10 fusions are common in aggressive leukemia. EPZ004777 can inhibit the transformation of these fusion genes both in vitro and in vivo, thus expanding its application potential in AF10-driven hematologic malignancies [2]. 4. Significance of the tool compound: EPZ004777 is one of the earliest selective DOT1L inhibitors and has been widely used to verify the effectiveness of DOT1L as a therapeutic target for MLL rearrangement and AF10-driven leukemia, laying the foundation for subsequent clinical DOT1L inhibitors [1][2].

C28H41N7O4

539.67

539.322

1338466-77-5

EPZ004777 hydrochloride;1380316-03-9

56962336

White to off-white solid powder

1.3±0.1 g/cm3

740.7±60.0 °C at 760 mmHg

401.7±32.9 °C

0.0±2.6 mmHg at 25°C

1.646

3.94

5

8

10

39

788

4

CC(C)N(CCCNC(=O)NC1=CC=C(C=C1)C(C)(C)C)C[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=CC4=C(N=CN=C43)N)O)O

WXRGFPHDRFQODR-ICLZECGLSA-N

InChI=1S/C28H41N7O4/c1-17(2)34(13-6-12-30-27(38)33-19-9-7-18(8-10-19)28(3,4)5)15-21-22(36)23(37)26(39-21)35-14-11-20-24(29)31-16-32-25(20)35/h7-11,14,16-17,21-23,26,36-37H,6,12-13,15H2,1-5H3,(H2,29,31,32)(H2,30,33,38)/t21-,22-,23-,26-/m1/s1

1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea

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: ≥ 3 mg/mL (5.56 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 30.0 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.

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Solubility in Formulation 2: ≥ 3 mg/mL (5.56 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 30.0 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.

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Solubility in Formulation 3: ≥ 3 mg/mL (5.56 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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