EPZ004777

EPZ004777 wird in DMSO 3-fach seriell verdünnt, für insgesamt zehn Konzentrationen, beginnend bei 1 mM. Ein 1 μL Aliquot jeder Inhibitorverdünnung wird in eine 384-Well-Mikrotiterplatte gegeben. Die 100%ige Hemmkontrolle bestand aus einer Endkonzentration von 2,5 mM des Produkthemmers S-Adenosyl-L-Homocystein (SAH). Diese Verbindung wird 30 Minuten lang mit 40 ml pro Well 0,25 nM DOT1L(1-416) in Assaypuffer (20 mM TRIS [pH 8.0] 10 mM NaCl, 0.002% Tween 20, 0.005% Bovine Skin Gelatin, 100 mM KCl und 0.5 mM DTT) inkubiert. 10 ml pro Well Substratmischung, bestehend aus Assaypuffer mit 200 nM ³H-SAM (American Radiolabeled Chemicals: 80 Ci/mmol), 600 nM unmarkiertem SAM und 20 nM Nukleosomen, werden hinzugefügt, um die Reaktion zu initiieren (beide Substrate sind in der endgültigen Reaktionsmischung zu ihren jeweiligen KM-Werten vorhanden). Die Reaktionen werden 120 Minuten lang inkubiert und mit 10 ml pro Well 800 mM SAM gequencht. Der Einbau von Radioaktivität in das Nukleosomsubstrat wird in einer Flashplate gemessen. IC50-Werte für Enzyme im Histone Methyltransferase-Panel werden unter ähnlichen ausgewogenen Assaybedingungen bestimmt, wobei sowohl SAM als auch Protein-/Peptidsubstrat in Konzentrationen vorliegen, die ihren jeweiligen KM-Werten entsprechen.

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