MELK (IC50 = 0.41 nM)

OTSSP167 blocks MELK-overexpressed cancer cells A549, T47D, DU4475, and 22Rv1 with IC50 values of 6.7, 4.3, 2.3, and 6.0 nM, respectively. OTSSP167 prevented the phosphorylation of two novel MELK substrates, PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which are crucial for stem cell properties and invasiveness. Through the suppression of PSMA1 phosphorylation, OTSSP167 prevents breast cancer cells from forming mammospheres. [1]

OTSSP167 exhibits significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. OTSSP167 is administered intravenously to the MDA-MB-231 model at a dose of 20 mg/kg once every two days, with a 73% TGI. TGI is 72% when administered orally at a dose of 10 mg/kg once per day. OTSSP167 for various cancer types in a dose- and MELK-dependent manner with little to no body weight loss. [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. Prior to SDS-PAGE, the reaction is stopped by adding SDS sample buffer and boiling for 5 minutes. At room temperature, the gel is dried and autoradiographed with intensifying screens. Before the incubation, DMSO-dissolved OTSSP167 (10 nM final concentration) is added to the kinase buffer.

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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.

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°C. At a wavelength of 450 nm, a spectrophotometer reads the plates. There are three copies of each assay performed.

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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.

Injections of MDA-MB-231 cells are made into NOD's mammary fat pads.Mice CB17-Prkdcscid/J. Female BALB/cSLC-nu/nu mice are subcutaneously injected with 1 105 A549), MIAPaCa-2, and PC-14 cells. Male BALB/cSLC-nu/nu mice receive a subcutaneous injection of DU145 cells in the left flank. Animals are randomized into groups of 6 mice (apart from PC-14, for which groups of 3 mice are used) when MDA-MB-231, A549, DU145, MIAPaCa-2, and PC-14 xenografts have reached average volumes of 100, 210, 110, 250, and 250 mm3, respectively. OTSSP167 and other substances are prepared for oral administration in a vehicle containing 0.5% methylcellulose and administered orally according to the prescribed dose and schedule. Compounds are prepared in 5% glucose for intravenous administration and injected into the tail vein. For both administration routes, a volume of 10 mL per kg of body weight is used. Every other day, tumor volumes are measured using a caliper.

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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]

[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.

4-[7-acetyl-8-[[4-[(dimethylamino)methyl]cyclohexyl]amino]-1H-1,5-naphthyridin-2-ylidene]-2,6-dichloro-1-cyclohexa-2,5-dienone is a naphthyridine derivative.
MELK Inhibitor OTS167 is an orally available inhibitor of maternal embryonic leucine zipper kinase (MELK) with potential antineoplastic activity. Upon administration, OTS167 binds to MELK, which prevents both MELK phosphorylation and activation; thus inhibiting the phosphorylation of downstream MELK substrates. This may lead to an inhibition of both cell proliferation and survival in MELK-expressing tumor cells. MELK, a serine/threonine kinase, is involved in cancer cell survival, invasiveness and cancer-stem cell formation and maintenance; it is highly upregulated in various types of cancer cells and absent in normal, healthy cells.

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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. [1]

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Maternal embryonic leucine zipper kinase (MELK) is upregulated in a variety of human tumors, and is considered an attractive molecular target for cancer treatment. We characterized the expression of MELK in gastric cancer (GC) and measured the effects of reducing MELK mRNA levels and protein activity on GC growth. MELK was frequently overexpressed in primary GCs, and higher MELK levels correlated with worse clinical outcomes. Reducing MELK expression or inhibiting kinase activity resulted in growth inhibition, G2/M arrest, apoptosis and suppression of invasive capability of GC cells in vitro and in vivo. MELK knockdown led to alteration of epithelial mesenchymal transition (EMT)-associated proteins. Furthermore, targeting treatment with OTSSP167 in GC patient-derived xenograft (PDX) models had anticancer effects. Thus, MELK promotes cell growth and invasiveness by inhibiting apoptosis and promoting G2/M transition and EMT in GC. These results suggest that MELK may be a promising target for GC treatment.[2]

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OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays.[3]

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Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (MELK), has been associated with various human cancers and plays an important role in the formation of cancer stem cells. OTSSP167, a MELK selective inhibitor, exhibits a strong in vitro activity, conferring an IC50 of 0.41nM and in vivo effect on various human cancer xenograft models. Here, we report the crystal structure of MPK38 (T167E), an active mutant, in complex with OTSSP167 and describe its detailed protein-inhibitor interactions. Comparison with the previous determined structure of MELK bound to the nanomolar inhibitors shows that OTSSP167 effectively fits into the active site, thus offering an opportunity for structure-based development and optimization of MELK inhibitors.[4]

C25H28CL2N4O2

487.42

486.158

C, 61.60; H, 5.79; Cl, 14.55; N, 11.49; O, 6.56

1431697-89-0

OTSSP167 hydrochloride;1431698-10-0

135398499

Light yellow to yellow solid powder

1.3±0.1 g/cm3

619.0±55.0 °C at 760 mmHg

328.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.644

6.41

2

6

6

33

648

0

ClC1=CC(C2=CC=C(N=CC(C(C)=O)=C3N[C@H]4CC[C@H](CN(C)C)CC4)C3=N2)=CC(Cl)=C1O

DKZYXHCYPUVGAF-UHFFFAOYSA-N

InChI=1S/C25H28Cl2N4O2/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)

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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