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].

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.

[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.

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]

1431697-89-0; 1431698-10-0

Typically exists as solid at room temperature

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)