EPZ004777

EPZ004777 se diluye en serie 3 veces en DMSO para un total de diez concentraciones, comenzando en 1 mM. Se deposita una alícuota de 1 μL de cada dilución del inhibidor en una placa de micropocillos de 384 pocillos. El control de inhibición del 100% consistió en una concentración final de 2,5 mM del inhibidor del producto S-adenosil-L-homocisteína (SAH). Este compuesto se incuba durante 30 min con 40 ml por pocillo de 0,25 nM DOT1L(1-416) en tampón de ensayo (20 mM TRIS [pH 8.0] 10 mM NaCl, 0.002% Tween 20, 0.005% gelatina de piel bovina, 100 mM KCl y 0.5 mM DTT). Se añaden 10 ml por pocillo de mezcla de sustrato que comprende tampón de ensayo con 200 nM de ³H-SAM (American Radiolabeled Chemicals: 80 Ci/mmol), 600 nM de SAM sin marcar y 20 nM de nucleosomas para iniciar la reacción (ambos sustratos están presentes en la mezcla de reacción final a sus respectivos valores de KM). Las reacciones se incuban durante 120 min y se inactivan con 10 ml por pocillo de 800 mM SAM. La incorporación de radioactividad en el sustrato de nucleosomas se mide en una flashplate. Los valores de IC50 para las enzimas en el panel Histone Methyltransferase se determinan bajo condiciones de ensayo equilibradas similares con SAM y el sustrato de proteína/péptido presentes en concentraciones iguales a sus respectivos valores de KM.

Human leukemia cell lines MV4-11, THP-1, RS4;11, Kasumi-1, HL-60, REH, and Jurkat. SEM, KOPN-8, 697, U937 and MOLM-13 cells.

~50 μM

14-18 days

For assessment of cell proliferation and viability in human cell lines, exponentially growing cells are plated, in triplicate, in 96-well plates in a final volume of 150 ml. Cells are incubated in the presence of 3 μM (proliferation curve), or increasing concentrations (IC50 determination) of EPZ004777 up to 50 μM. Viable cell number is determined every 3–4 days for up to 18 days using the Guava Viacount assay and analyzed on a Guava EasyCyte Plus instrument according to the manufacturer’s protocol. On days of cell counts, growth media and this compound are replaced and cells split back to a density of 5×10⁴ cells/well. Total cell number is expressed as split-adjusted viable cells per well. For each cell line, IC50 values are determined from concentration-dependence curves at each time point using Graphpad Prism software. Experiments to determine IC50 values continues until IC50 values stabilized (day 18 for THP-1 cells, day 14 for all other cell lines). For assessment of the effect of this compound treatment on transformed murine hematopoietic progenitors, cells from two independent transductions for each virus are plated in 24-well plates at a density of 0.5–1×10⁵ cell/well in 1 ml media in 24-well plates and exposed to increasing concentrations of this chemical up to 30 mM. Cells are counted and replated at equal cell numbers in fresh media with fresh compound every 3–4 days. For MTT assays, cells from serial replatings are harvested on day 10 and plated, in triplicate at 2×10⁴ cells/well in 100 ml media with the appropriate concentration of this compound. Cells are incubated for 2.5 days, the exposed to 10 ml MTT reagent for 3 hr, and lysed over night in 100 ml MTT- solubilization buffer (both from Cell Proliferation Kit I [MTT]).

A mouse xenograft MV4-11 model of MLL.

Mini-pumps containing 100 and 150 mg/mL EPZ004777 solutions

Osmotic pump