Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer.

We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC₅₀ of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.

Kinase assay for compound screening. Kinase activity was measured in the presence or absence of compounds using FAM-Glycogen Synthase-derived peptide (Molecular Devices Corporation) as a substrate. The extent of FAM-GS-derived peptide phosphorylation was measured by immobilized metal ion affinity-based fluorescence polarization (IMAP) technology [17] using IMAP FP Progressive Binding System (Molecular Devices Corporation). Test compounds were dissolved in DMSO at 12.5 mM and then serially diluted as the DMSO concentration in the assays to be 1%. The serially diluted compounds, 0.3 ng/μL MELK and 100 nM FAM-Glycogen Synthase-derived peptide were reacted in a reaction buffer (20 mM HEPES, 0.01% Tween-20, 0.3 mM MgCl 2 , 2 mM dithiothreitol, 20 μM ATP, pH 7.4) at room temperature for 3 hours. The reaction was terminated by addition of three-fold assay volume of IMAP binding solution (Molecular Devices Corporation). Following 30 min incubation at room temperature, fluorescence polarization was measured by Wallac EnVision 2103 multilabel reader (PerkinElmer). IC 50 values were calculated by nonlinear four parameter fit using SigmaPlot, version 10.0 (Systat Software, Inc.).
Cell-based assay. In vitro cell viability was measured by the colorimetric assay using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.). Cells were plated in 100 μL in 96-well plates at a density that generated continual linear growth (A549, 1x10 3 cells; T47D, 3x10 3 cells; DU4475, 4x10 3 cells; 22Rv1, 6x10 3 cells; and HT1197, 2x10 3 cells, in 100 μL per well). The cells were allowed to adhere overnight before exposure to compounds for 72 hours at 37 °C. Plates were read using a spectrophotometer at a wavelength of 450 nm. All assays were carried out in triplicate.
High-throughput screening. Liquid handling operations were performed using several instruments. Small volume transfers of 100 nL were performed using a Genomic Solutions Hummingbird 384-well non-contact nanoliter dispenser. Assays were initiated by sequential addition of reagents using an Aurora Discovery's BioRapTR FRD non-contact dispenser. Addition of IMAP binding reagent was performed using a Titertek Multidrop 384 liquid dispenser. A Beckman Coulter BioMek FX equipped with Span-8 was used for hit picking, and BioMek FX equipped with a 384-well head was used to prepare dose response source places. The fluorescent polarization was read using an Analyst GT multimode plate reader from Molecular Devices.The MELK kinase assay protocol was optimized for a low volume 384-well format for high-throughput screening. All assay plates had test compounds in columns 3-22, and controls in columns 1-2 and 23-24. The MAX (high signal or no inhibition) control wells received all assay components, including enzyme. The MIN (low signal) control wells received substrate and enzyme buffer but no enzyme. Based on preliminary concentration tests, the samples were tested at 30 μM against MELK. The standard operating procedure is outlined below: (1) Transfer library compounds resuspended in 100% DMSO into a dry 384-well low volume non-binding surface black polystyrene assay plate using the Hummingbird. Control wells receive equal volumes of 100% DMSO. (2) Dispense substrate followed by enzyme to the assay plate in a final volume of 5.0 μL using the BioRapTR. MIN control wells receive substrate followed by enzyme buffer without enzyme. (3) Shake plate on a plate shaker at high setting for 2 min. (4) Cover and incubate at room temperature for 120 min. (5) Dispense 10.0 μL IMAP binding reagent to all wells of the plate and incubate at room temperature for an additional 90 min. (6) Read plate on an Analyst GT with the following settings: Excitation, 485 nm; Emission, 530 nm; Dichroic, 505 nm; G Factor, 1; Z Height, 1 mm; Integration time, 0.1 s; and Attenuator, Out.
Synthesis of compound OTSSP167. The scheme of the synthesis of OTSSP167 is as shown below: Pd ( d pp f ) Cl 2

Ethyl 2-(ethoxymethylene)-3-oxobutanoate.
To a 1 L round bottom flask equipped with a distillation apparatus was added ethyl 3-oxobutanoate (100 g, 0.77 mol), triethyl orthoformate (130 g, 0.92 mol), and acetic anhydride (150 g, 1.5 mol), and the reaction mixture was stirred with heating at 135 °C until the desired amount of ethanol was collected (~ 35 mL). The reaction was cooled, concentrated to remove all volatiles and the residue was distilled under high vacuum to obtain the desired product (100 g, 70%) as a pale yellow oil: ESI MS m/z 187 [M + H] + .