Kinase experiment:

For in vitro kinase assay, MELK recombinant protein (0.4 μg) is mixed with 5 μg of each substrate in 20 μL of kinase buffer containing 30 mM Tris-HCl (pH), 10 mM DTT, 40 mM NaF, 10 mM MgCl2, 0.1 mM EGTA with 50 μM cold-ATP and 10 Ci of [γ-32P]ATP for 30 min at 30℃. The reaction Is terminated by addition of SDS sample buffer and boiled for 5 min prior to SDS-PAGE. The gel is dried and autoradiographed with intensifying screens at room temperature. OTSSP167 (final concentration of 10 nM) is dissolved in DMSO and added to kinase buffer before the incubation.

Cell experiment:

In vitro cell viability is measured by the colorimetric assay using Cell Counting Kit-8. Cells are plated in 100 μL in 96-well plates at a density that generates continual linear growth (A549, 1×103 cells; T47D, 3×103 cells; DU4475, 4×103 cells; 22Rv1, 6×103 cells; and HT1197, 2×103 cells, in 100 μL per well). The cells are allowed to adhere overnight before exposure to OTSSP167 for 72 hours at 37℃. Plates are read using a spectrophotometer at a wavelength of 450 nm. All assays are carried out in triplicate.

Animal experiment:

MDA-MB-231 cells are injected into the mammary fat pads of NOD.CB17-Prkdcscid/J mice. A549, MIAPaCa-2 and PC-14 cells (1×105 cells) are injected subcutaneously in the left flank of female BALB/cSLC-nu/nu mice. DU145 cells are injected subcutaneously in the left flank of male BALB/cSLC-nu/nu mice. When MDA-MB-231, A549, DU145, MIAPaCa-2, and PC-14 xenografts has reached an average volume of 100, 210, 110, 250, and 250 mm3, respectively, animals are randomized into groups of 6 mice (except for PC-14, for which groups of 3 mice are used). For oral administration, OTSSP167 and other compounds are prepared in a vehicle of 0.5% methylcellulose and given by oral garbage at the indicated dose and schedule. For intravenous administration, compounds are formulated in 5% glucose and injected into the tail vein. An administration volume of 10 mL per kg of body weight is used for both administration routes. Tumor volumes are determined every other day using a caliper.

OTSSP167 is an inhibitor of maternal embryonic leucine zipper kinase(MELK) with IC50 value of 0.41nM [1].

MELK belongs to the AMPK serine/threonine kinase family and involves in the mammalian embryonic development. It overexpresses in various types of human cancer. OTSSP167 is synthetized from a compound which is a hit of a high-throughput screening. In vitro anti-proliferative assay shows that OTSSP167 suppresses cell growth of various cancer cell lines, such as A549(IC50=6.7nM), T47D(IC50=4.3nM), DU4475(IC50=2.3nM) and 22Rv1(IC50= 6.0nM). In vivo assay also shows OTSSP167 can significantly suppress tumor growth in the xenograft model using various cancer cell lines. The effects are observed by both intravenous administration and oral administration. When investigating the substrates of MELK, OTSSP167 is found to inhibit the phosphorylation of DBNL and PSMA1 in vitro. Since PSMA1 is essential for survival of cancer cells, the reduction of phosphorylated PSMA1 caused by OTSSP167 can subsequently suppress mammosphere formation of cancer stem cells [1].

References:
[1] Suyoun Chung, Hanae Suzuki, Takashi Miyamoto et al. Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer. Oncotarget. 2012, 3: 1629-1640.