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℃ and 5% CO2. Alamar Blue solution is added, and after 4 h incubation at 37℃, 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].

JNJ-47117096 hydrochloride is potent and selective MELK inhibitor, with an IC50 of 23 nM, also effectively inhibits Flt3, with an IC50 of 18 nM.

JNJ-47117096 hydrochloride is potent and selective MELK inhibitor, with an IC50 of 23 nM, also effectively inhibits Flt3, with an IC50 of 18 nM, and slighitly blocks CAMKIIδ, Mnk2, CAMKIIγ, and MLCK (IC50, 810 nM, 760 nM, 1000 nM, 1000 nM). JNJ-47117096 (MELK-T1) suppresses the proliferation of Flt3-driven Ba/F3 cell lines, with an IC50 of 1.5 μM in the absence of IL-3, while no inhibitory activity is observed in the presence of IL-3. JNJ-47117096 does not inhibit the proliferation of Ba/F3 cell lines transfected with either FGFR1, FGFR3, or KDR, either in the presence or absence of IL-3[1]. JNJ-47117096 (MELK-T1, 10 μM) delays the progression of MCF-7 cells through S-phase. JNJ-47117096 inhibits MELK, and then exerts stalled replication forks and DNA double-strand breaks (DSBs). JNJ-47117096 activates the ATM-mediated DNA-damage response (DDR). JNJ-47117096 (3, 10 μM) results in a growth arrest and a senescent phenotype. Moreover, JNJ-47117096 induces a strong phosphorylation of p53, a prolonged up-regulation of p21 and a down-regulation of FOXM1 target genes[2].

[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.