EPZ-5676

Biological Activity

EPZ-5676 is a novel small molecule inhibitor of DOT1L. EPZ-5676 has an inhibition constant value of 80 pM, and demonstrates 37000-fold selectivity over all other methyltransferases tested. EPZ-5676 inhibits proliferation of MLL-AF4 rearranged cell line MV4-11 with an IC50 of 9 nM. EPZ-5676 reduces H3K79 dimethylation with a cellular IC50 of 2.6 nM in MV4-11 cells. EPZ-5676 blocks the activity of the histone lysine-methyltransferase DOT1L, thereby inhibiting H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

Customer Product Validations & Biological Datas

	Source	Clin Epigenetics (2017). Figure 5. EPZ-5676
	Method	immunoblotting
	Cell Lines	H358 cells
	Concentrations	1 μM
	Incubation Time	48 h
	Results	First, in order to evaluate the involvement of H3K79me3 in TGF-β1-induced EMT, A549 and H358 cells were treated with DOT1L inhibitors, EPZ5676 and SGC0946, simultaneously with TGF-β1

Protocol

Cell Experiment
Cell lines	MV4-11 cells
Preparation method	Quantitative real-time polymerase chain reaction (qRT-PCR). To assess inhibition of HOXA9 and MEIS1 messenger RNA (mRNA) expression by EPZ-5676, exponentially growing MV4-11 cells were seeded in 75-cm2 culture flasks at 2 × 105 cells/mL and incubated in the presence of 0.2% DMSO or increasing concentrations of EPZ-5676. On day 4, cells were maintained in log phase culture by reseeding at 5 × 105 cells/mL and compound was replenished. On day 6, cells were harvested, total RNA extracted, and HOXA9 and MEIS1 mRNA levels assessed and normalized to the β2-microglobulin by qRT-PCR as previously described.
Concentrations	0~1µM
Incubation time	3 days

Animal Experiment
Animal models	Rat MV4-11 xenograft models
Formulation	5% hydroxypropyl-β-cyclodextrin in saline
Dosages	70 mg/kg
Administration	IV infusion into the femoral vein continuously (24 hours/day), or intermittently (8 hours/day).

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Chemical Information

References

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia.
Daigle SR, et al. Blood. 2013 Aug 8;122(6):1017-25. PMID: 23801631.