Biochemical enzyme inhibition assays

The enzyme inhibition constant (Ki) value for EPZ5676 was determined by fitting inhibition data to the Morrison quadratic equation. Residence time for EPZ5676 was calculated as the reciprocal of the enzymatic-ligand dissociation rate, determined by surface plasmon resonance.

Cell lines

MV4-11 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ for several months.

Reaction Conditions

0.0005 ~ 10 μM; 14 days

Applications

EPZ5676 potently inhibited MV4-11 cell proliferation with an IC50 value of 3.5 nM. Antiproliferative activity was realized after 4 days and was most clear after 7 days of EPZ-5676 treatment.

Animal models

Nude rats bearing MV4-11 xenografts

Dosage form

35, 67 or 70 mg/kg/day; i.v.; for 21 days

Applications

In nude rats bearing MV4-11 xenografts, EPZ5676 caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

EPZ5676 is a potent inhibitor of DOT1L histone methyltransferase, according to X-ray crystallographic analysis, that occupies the S-adenosyl methionine (SAM) binding pocket of DOT1L and induces conformational changes in DOT1L resulting in the opening of a hydrophobic pocket beyond the amino acid portion of SAM. EPZ5676 selectively inhibits DOTIL with a value of 50% inhibition concentration IC₅₀of 0.8 nM, which is 37000-fold greater in selectivity than other methyltransferases, including CARM1, EHMT1/2, EZH1/2, PRMT1/2/5/6/8, SETD7, SMYD2/3, and WHSC1/1L1. EPZ5676 has been investigated for the treatment of MLL-rearranged leukemia in multiple studies where results have shown that EPZ5676 inhibits H3K79 methylation and the expression of MLL-fusion target gene and potently kills acute leukemia cell lines bearing MLL translocation.

Reference

[1].Daigle SR, Olhava EJ, Therkelsen CA, Basavapathruni A, Jin L, Boriack-Sjodin PA, Allain CJ, Klaus CR, Raimondi A, Scott MP, Waters NJ, Chesworth R, Moyer MP, Copeland RA, Richon VM, Pollock RM. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood. 2013 Aug 8;122(6):1017-1025.