MELK (IC50 = 0.41 nM)

OTSSP167 inhibits the growth of A549 (lung), T47D (breast), DU4475 (breast), 22Rv1 (prostate) and HT1197 (bladder) cancer cells with IC50 values of 6.7, 4.3, 2.3, 6.0 and 97 nM, respectively[1].
OTSSP167 can abrogate the mitotic checkpoint, disrupt MCC and MCC-APC/C interaction in MCF7 cells. OTSSP167 causes GFP-MELK localization to cell cortex in prometaphase cells[2].
OTSSP167 is a MELK selective inhibitor, exhibits a strong in vitro activity, conferring an IC50 of 0.41 nM[3].

OTSSP167 (20 mg/kg, i.v.) results in tumor growth inhibition (TGI) of 73% in xenograft mouse model; OTSSP167 (1, 5, and 10 mg/kg, p.o.) reveals TGI of 51, 91, and 108%, respectively. OTSSP167 (20 mg/kg, p.o.) shows no tumor growth suppressive effect on PC-14 xenografts[1].

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Growth suppressive effect of OTSSP167 in xenograft mouse model [1]
We subsequently investigated in vivo anti-tumor effect of OTSSP167 by a xenograft model using MDA-MB-231 cells (MELK-positive, triple-negative breast cancer cells). The compound was administered to mice bearing xenografts for 14 days after the tumor size reached about 100 mm3. The tumor size was measured as a surrogate marker of drug response (tumor growth inhibition (TGI)). Intravenous administration of OTSSP167 at 20 mg/kg once every two days resulted in TGI of 73% (Fig 3A). Since the bioavailability of this compound was expected to be very high (data not shown), we attempted oral administration of this compound. The oral administration at 10 mg/kg once a day revealed TGI of 72% (Fig 3B). Due to the strong growth-suppressive effect on various cancer cell lines, we further investigated in vivo growth-suppressive effect using cancer cell lines of other types and found significant tumor growth suppression by OTSSP167 for multiple cancer types in dose-dependent manners with no or a little body-weight loss (Fig 3 and Supplementary Fig. S1). For example, mice carrying A549 (lung cancer) xenografts that were treated with 1, 5, and 10 mg/kg once a day of OTSSP167 by intravenous administration revealed TGI of 51, 91, and 108%, respectively (Fig 3C) and those by oral administration of 5 and 10 mg/kg once a day revealed TGI of 95 and 124%, respectively (Fig 3D). In addition, we examined DU145 (prostate cancer) and MIAPaCa-2 (pancreatic cancer) xenograft models by oral administration of 10 mg/kg once a day, and observed TGI of 106 and 87%, respectively (Fig 3E and F). To further validate the MELK-specific in vivo tumor suppressive effect, we examined PC-14 lung cancer cells in which MELK expression was hardly detectable (Fig 3G). Oral administration of 10 mg/kg OTSSP167 once a day for 14 days showed no tumor growth suppressive effect on PC-14 xenografts (Fig 3H), further supporting the MELK-dependent antitumor activity of OTSSP167.

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Efficacy of OTSSP16 treatment in preclinical GC patient-derived xenograft (PDX) mouse models [2]
Two MELK-positive, and one MELK-negative, GC-PDX models were chosen from our established banks to evaluate whether MELK is an effective therapeutic target for GC in vivo (Fig. 6A, 6B, and 6C). Third generation PDX mice were used in this experiment. When the TumorGraft volume reached 100-200 mm3, the PDX mice were intravenously treated with OTSSP167 (15 mg/kg) or vehicle once every other day for two weeks. The reaction to OTSSP167 was quantified by tumor growth inhibition (TGI). In the two MELK-positive models, TGI values were 106% and 112% at the end of drug administration (Fig.6D and 6E, right panel). In the MELK-negative model, the TGI value was only 19% (Fig. 6F, right panel). MELK expression levels in the TumorGraft tissues were subsequently evaluated by IHC. In both MELK-positive cases, MELK expression was eliminated in the TumorGraft after OTSSP167 treatment but not after vehicle treatment (Fig. 6D and 6E, middle panel). These data strongly suggest that MELK might be an effective molecular target for the treatment of gastric cancer.

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 °C. 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.

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Recombinant proteins and in vitro kinase assay for substrate screening [1]
MELK recombinant protein was generated as described previously. The full coding sequence of each of MELK substrate candidates was amplified by RT-PCR and cloned into the pGEX6p-1 vector. The GST-tagged recombinant proteins were expressed in BL21 codon-plus RIL competent cells and purified using Glutathione Sepharose 4B beads according to the supplier's instructions. The GST-tag was removed by PreScission protease according to the supplier's instructions. For in vitro kinase assay, MELK recombinant protein (0.4 μg) was 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 °C. The reaction was terminated by addition of SDS sample buffer and boiled for 5 min prior to SDS-PAGE. The gel was dried and autoradiographed with intensifying screens at room temperature. OTSSP167 (final concentration of 10 nM) was dissolved in DMSO and added to kinase buffer before the incubation.

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In vitro kinase assays [3]
Kinases were provided as recombinant proteins purified from E. coli or immunoprecipitates from mitotic cell lysates. For IP-kinase assays, the immunoprecipitates were washed twice with cell lysis buffer (1× PBS, 10% glycerol, 0.5% NP-40) supplemented with protease inhibitors (Protease Inhibitor Cocktail set III, EDTA-Free) and phosphatase inhibitors (100 mM NaF, 1mM Na3VO4, 60 mM β-glycerophosphate) and twice with 1× kinase buffer (25 mM Tris-HCl, pH 7.5, 60mM ß-glycerophosphate, 10mM MgCl2). Myelin basic protein was purchased from Sigma and Histones H3.3 and H10 were purchased from New England Labs as substrates. For kinase reactions, 4μl of 5× kinase buffer was mixed with recombinant or immunoprecipitated kinases, substrates, 5μCi 32P –ATP or cold ATP. H2O was added to make the final volume of 20 μl. The reactions were incubated at 30°C for 30 min and then terminated by adding 20 μl 2×SDS sample buffer. Samples were subjected to SDS-PAGE followed by transferring to PVDF membranes. Phosphorylation of the substrates was visualized by autoradiography or phospho-specific antibodies.

OTSSP167 inhibits cell proliferation of a variety of cancer cell lines including A549, T47D, DU4475 and 22Rv1. The IC50 values are 6.7nM, 4.3nM, 2.3nM and 6nM, respectively.

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Flow cytometric analysis [2]
For cell cycle analysis, cells near 50% confluence were synchronized in the G0/G1 phase by overnight incubation in serum-free medium. Cells were then incubated in the complete medium containing 25 nM or 50 nM OTSSP167. After 24 hours (BGC823) or 18 hours (SGC7901) of incubation, the cells were trypsinized, washed with PBS, and fixed with 70% ethanol for 16 hours at −20°C. The samples were washed with PBS and stained with PI/RNase Staining Buffer for 15 minutes. Cell cycle analysis was performed by fluorescence flow cytometry on a FACScan machine. For apoptotic analysis, cells were stained using an Annexin V/PI double staining kit according to the manufacturer's protocol.

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Matrigel invasion assay and mammosphere formation assay [1]
NIH3T3 cells transfected with plasmids expressing MELK (pCAGGSnHc-MELK), DBNL (pcDNA3.1-Myc-His-DBNL) or both were grown to near confluence in DMEM containing 10% FBS. After the incubation of 24 hours, the cells were harvested by trypsinization, washed in DMEM without addition of serum, and suspended in serum-free DMEM. The cells (1'104 cells) were seeded onto the Matrigel matrix chamber and incubated for 22 hours. The cells invading to Matrigel were stained by Giemsa and counted. For sphere formation assay, 1'103 cells of MCF-7 cells which transiently over-expressed wild-type MELK, kinase-dead MELK, PMSA1, PSMA1 and wild-type MELK, or PMSA and kinase-dead MELK were seeded onto Ultra-Low attachment plate. For knockdown experiments, MDA-MB-231 cells (1'103 cells) which seeded onto Ultra-Low attachment plate were transfected with oligo siRNA for EGFP, MELK or PSMA1 as described above. For examination of sphere formation under treatment of MELK inhibitor OTSSP167, 1'103 MCF-7 cells were seeded with 0.01, 0.02, 0.04, 0.08, or 0.16 μM of OTSSP167, respectively. DMSO alone was used as a control. Following incubation for two weeks, cell viability was measured by using Cell-counting kit-8.

MDA-MB-231 cells were injected into the mammary fat pads of NOD.CB17-Prkdcscid/J mice. A549, MIAPaCa-2 and PC-14 cells (1 × 107 cells) were injected subcutaneously in the left flank of female BALB/cSLC-nu/nu mice. DU145 cells were 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 had reached an average volume of 100, 210, 110, 250, and 250 mm3, respectively, animals were randomized into groups of 6 mice (except for PC-14, for which groups of 3 mice were used). For oral administration, compounds such as /OTSSP167 were prepared in a vehicle of 0.5% methylcellulose and given by oral garbage at the indicated dose and schedule. For intravenous administration, compounds were formulated in 5% glucose and injected into the tail vein. An administration volume of 10 ml per kg of body weight was used for both administration routes. Concentrations were indicated in main text and Figures. Tumor volumes were determined every other day using a caliper. The results were converted to tumor volume (mm3) by the formula length × width2 × 1/2. The weight of the mice was determined as an indicator of tolerability on the same days. The animal experiments using A549 xenografts were conducted by a CRO company in accordance with their Institutional Guidelines for the Care and Use of Laboratory Animals. The other animal experiments were conducted at a CRO company. in accordance with their Institutional Guidelines for the Care and Use of Laboratory Animals. Tumor growth inhibition (TGI) was calculated according to the formula {1 – (T – T0) / (C – C0)}×100, where T and T0 are the mean tumor volumes at day 14 and day 0, respectively, for the experimental group, and C – C0 are those for the vehicle control group. [1]

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MELK-positive and -negative GC-PDX mouse models were randomly selected according to the results of IHC performed on the original GC and TumorGraft tissues. When tumor volume reached 100-200 mm3, the mice were randomly assigned to treatment and control groups and dosing was initiated. OTSSP167 was administered at 15 mg/kg intravenously to the third-generation mice once every two days for 2 weeks. The control group was treated with vehicle (PBS) in the same way. Tumor size was monitored every two days by caliper measurements. The weight of the mice was also measured as an indicator of treatment toleration. Tumor growth inhibition (TGI) was assessed in accordance with the formula {1–(T–T0) / (C–C0)} × 100, where T and T0 are the mean tumor volumes at the end of the drug administration and day 0, respectively, for the treated group, and C−C0 are those for the vehicle control group.[2]

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Formulated in 0.5% methylcellulose; 1, 5, 10, 20 mg/kg; p.o.

MDA-MB-231, A549, DU145 xenografts

[1]. Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer. Oncotarget. 2012 Dec;3(12):1629-40.

[2]. Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in gastric cancer. Oncotarget. 2016 Feb 2;7(5):6266-80.

[3]. OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases. PLoS One. 2016 Apr 15;11(4):e0153518.

[4]. The crystal structure of MPK38 in complex with OTSSP167, an orally administrative MELK selective inhibitor. Biochem Biophys Res Commun. 2014 Apr 25;447(1):7-11.

[5]. MELK inhibition in Diffuse Intrinsic Pontine Glioma. Clin Cancer Res. 2018 Jul 30. pii: clincanres.0924.2018.

We previously reported that MELK (maternal embryonic leucine zipper kinase) is a novel therapeutic target for breast cancer. MELK has been reported to be highly expressed in various human cancers. It is believed to play an indispensable role in cancer cell survival and is involved in the maintenance of tumor-initiating cells. We performed high-throughput screening of a compound library and conducted structure-activity relationship studies, successfully obtaining a highly potent MELK inhibitor, OTSSP167, with an IC₅₀ value of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome α subunit 1) and DBNL (drebrin-like protein). We found that PSMA1 and DBNL are novel substrates of MELK, crucial for stem cell characteristics and invasiveness. This compound inhibited mammary globulin formation in breast cancer cells and, in a mouse xenograft model, significantly inhibited tumor growth in breast cancer, lung cancer, prostate cancer, and pancreatic cancer cell lines via intravenous and oral administration. This MELK inhibitor shows promise as an effective compound for inhibiting tumor-initiating cell growth and treating various human cancers. [1] Maternal embryonic leucine zipper kinase (MELK) is upregulated in a variety of human tumors and is considered an attractive molecular target for cancer therapy. We characterized the expression of MELK in gastric cancer (GC) and examined the effects of reducing MELK mRNA levels and protein activity on GC growth. MELK is frequently overexpressed in primary GC, and higher MELK levels are associated with poorer clinical prognosis. Reducing MELK expression or inhibiting kinase activity suppressed GC cell growth, leading to G2/M phase arrest, apoptosis, and inhibition of their invasive ability in vitro and in vivo. MELK knockdown resulted in alterations in epithelial-mesenchymal transition (EMT)-related proteins. Furthermore, targeted therapy with OTSSP167 showed anticancer effects in a patient-derived xenograft (PDX) model of gastric cancer. Therefore, MELK promotes the growth and invasion of gastric cancer cells by inhibiting apoptosis, promoting G2/M phase transition, and EMT. These results suggest that MELK may be a promising therapeutic target for gastric cancer. [2]

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OTSSP167 has recently been shown to be a potent inhibitor of maternal embryonic leucine zipper kinase (MELK) and is currently undergoing a phase I clinical trial for solid tumors that have failed other treatments. This article reports that OTSSP167 can block mitotic checkpoints at concentrations that inhibit MELK. This blocking effect could not be reproduced in cells by RNAi-mediated MELK silencing. Although OTSSP167 does inhibit MELK, it exhibits off-target activity against Aurora B kinase both in vitro and in cells. In addition, OTSSP167 also inhibits BUB1 and Haspin kinases and reduces the phosphorylation levels of histones H2AT120 and H3T3, leading to Aurora B and its associated chromosomal passenger complex misalignment from the centromere/kinetosome. These results suggest that OTSSP167 may have other mechanisms of action that kill cancer cells, and therefore OTSSP167 should be used with caution as a MELK-specific kinase inhibitor in biochemical and cellular experiments. [3]

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Mouse protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (MELK), is associated with a variety of human cancers and plays an important role in the formation of cancer stem cells. OTSSP167 is a selective inhibitor of MELK with strong in vitro activity, an IC50 value of 0.41 nM, and in vivo inhibitory effects on various human cancer xenograft models. This article reports the crystal structure of the complex of the active mutant MPK38 (T167E) and OTSSP167 and describes its protein-inhibitor interaction in detail. Compared with the previously resolved structures of MELK binding to nanomolar inhibitors, OTSSP167 can effectively bind to the active site, thus providing an opportunity for the development and optimization of structure-based MELK inhibitors. [4]

C25H29CL3N4O2

523.88

522.135

C, 57.32; H, 5.58; Cl, 20.30; N, 10.69; O, 6.11

1431698-10-0

OTSSP167;1431697-89-0

135565272

Light yellow solid powder

3

6

6

34

648

0

ClC1C(=C(C([H])=C(C=1[H])C1C([H])=C([H])C2C(=C(C(C(C([H])([H])[H])=O)=C([H])N=2)N([H])C2([H])C([H])([H])C([H])([H])C([H])(C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C([H])([H])C2([H])[H])N=1)Cl)O[H].Cl[H]

XDGWHISAOWEFML-BFLZMHAMSA-N

InChI=1S/C25H28Cl2N4O2.ClH/c1-14(32)18-12-28-22-9-8-21(16-10-19(26)25(33)20(27)11-16)30-24(22)23(18)29-17-6-4-15(5-7-17)13-31(2)3;/h8-12,15,17,33H,4-7,13H2,1-3H3,(H,28,29);1H/t15-,17-;

1-(6-(3,5-dichloro-4-hydroxyphenyl)-4-(((1r,4r)-4-((dimethylamino)methyl)cyclohexyl)amino)-1,5-naphthyridin-3-yl)ethanone hydrochloride

OTSSP167; OTSSP-167; OTS-167; OTS 167; OTSSP 167; OTS167; OTS167 HCl; OTSSP167 hydrochlorideOTSSP167 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 3 mg/mL (5.73 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1 mg/mL (1.91 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)