JNJ-47117096 hydrochloride (MELK-T1 hydrochloride)

Kinase experiment:

Inhibition of MELK kinase activity is measured using a radioactive filter binding assay. Briefly, each assay well contains 1.25 nM MELK (human, residues 1-340) 10 μM ATP, 6.7 uCi/mL γ33P-ATP, 3 μM biotinylated ZIP-tide peptide (Biotin-KKLNRTLSFAEPG) in 30 μL reaction buffer (25 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 1 mM EGTA, 0.1% Triton X100). Kinase reactions are performed for 25 minutes at room temperature before stopping with 40 μL 2% orthophosphoric acid. Unbound radioactivity is removed by filtering the reaction through a MAPH filter plate. The trapped 33P labelled peptide is then washed twice with 200 μL 0.5% orthophosphoric acid, 20 μL Microscint-20 added per well and the amount of radioactivity determined by scintillation counting using a Topcount. To calculate compound IC50, semi-log serial dilutions are used to produce 8-point dose-response curves in duplicate. IC50 values are then derived using the four parameter logistic fit method in GraphPad Prism 5.0[1].

Cell experiment:

Compounds (JNJ-47117096) dissolved in DMSO are sprayed into 384-well plates (100 nL/well). A suspension of Ba/F3-Flt3 cells is added (20,000 cell/well), followed by the addition of 10 ng/mL IL3. The cells are incubated for 24 h at 37°C and 5% CO2. Alamar Blue solution is added, and after 4 h incubation at 37°C, the fluorescent intensity is measured on a Fluorescence plate reader (540 nm excitation and 590 nm emission). The control experiment in the absence of IL3 is performed in the same way. To calculate compound IC50, semi-log serial dilutions are used to produce 8- point dose-response curves in duplicate. A best-fit curve is fitted by a minimum sum of squares method to the plot of %Control vs. compound concentration. From this an IC50 value is calculated[1].

References:

[1]. Johnson CN, et al. Fragment-based discovery of type I inhibitors of maternal embryonic leucine zipper kinase. ACS Med Chem Lett. 2014 May 23;6(1):25-30.
[2]. Beke L, et al. MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells. Biosci Rep. 2015 Oct 2;35(6). pii: e00267.