MNK1 (IC50 = 2.2 μM)

CGP57380 inhibits phosphorylation of eIF4E in cellular assays with IC50 of about 3 μM. Dephosphorylating eIF4E with CGP57380 results in 293 cells expressing more of the cap-dependent reporter.[1] CGP57380 inhibits protein synthesis, VSMC hypertrophy, and Ang II-stimulated eIF4E phosphorylation in a dose-dependent manner.[2] In mouse embryo fibroblasts (MEFs), CGP57380 makes wild-type cells more susceptible to the apoptosis that is caused by serum withdrawal.[3] CGP57380 stops BC progenitors from repeatedly replicating.[4]

CGP57380 (40 mg/kg/d i.p.) potently extinguishes BC CML cells' capacity to function as LSCs and serially transplant immunodeficient mice. [4]
Here, we FACS-sorted GMPs from a BC sample as previously described, and injected them intrafemorally into 8- to 12-wk-old female NSG mice (Fig. S8A). At 6 wk posttransplantation, engrafted mice were treated for 3 wk with DMSO, CGP57380, or dasatinib (n = 5 mice per treatment group). At the end of the treatment period, all the mice were killed, and human cells were obtained from hematopoietic tissues by using immunomagnetic beads. We found no difference in the percentage of CD45+ human cells in the peripheral blood or BM of each of the treatment groups (Fig. 6A). However, we observed that dasatinib and CGP57380 had specific activity against committed BC progenitors, as they significantly reduced the number of colony forming units detected in BM (P ≤ 0.05 and P ≤ 0.005, respectively) compared with control, although the effect of CGP57380 was greater (Fig. 6B). Human cells obtained from the primary mice were then transplanted into secondary recipients, and engraftment monitored by flow cytometry over a 16-wk period. By 4 wk, we were able to detect engraftment in all animals in each of the three treatment groups (Fig. 6C). In DMSO- or dasatinib-treated animals, engraftment was maintained at 80% (i.e., four of five animals) throughout the whole experimental time frame of 16 wk, but, in contrast, none of the CGP57380-treated mice were able to maintain long-term engraftment (Fig. 6C). At 16 wk, mice were euthanized, and BM was examined for the presence of BCR-ABL1. BCR-ABL1 transcripts were detectable in each of the animals treated with DMSO or dasatinib (i.e., four of five animals for each treatment group), whereas only a very faint band was detected in one of the four animals in the CGP57380-treated group (Fig. 6D). This experiment was repeated by using CD34+ BC cells from a different individual, and similar result were obtained (Fig. S8 B–J). Taken together, our findings demonstrate that in vivo MNK inhibition can potently extinguish the ability of BC CML cells to serially transplant-immunodeficient mice and function as LSCs[4].

CGP 57380 is potent inhibitor of MNK1 with IC50 value of 2.2 μM.
MNK1 and PRAK were phosphorylated by preincubation with activated p38, which was generated by incubation with recombinant MKK6b(E). Recombinant kinases and eIF4E were prepared, and in vitro kinase reactions were performed as described previously. Poly(A)+ mRNA was purified from 293 cells using the Oligotex Direct mRNA Kit. For the in vitro translation rabbit reticulocyte lysate (Promega) was programmed with 10 μg of mRNA per ml in the presence of 3 or 10 μg of kinase per ml, [35S]methionine (0.6 mCi/ml), 1.5 mM magnesium acetate, 75 mM KCl, 2 mM DTT, and 100 μM ATP according to the manufacturer's instructions. Care was taken to ensure equal buffer conditions in all assays. Translation reactions were incubated at 30°C for 90 min, and the radioactivity incorporated into TCA-precipitable material was measured.[1]
Recombinant p38 isoforms are activated by Mkk6(E) under the following conditions: p38 (100 ng/mL), Mkk6(E) (30 ng/mL), ATP (100 mM) are mixed in kinase buffer (25 mM Hepes, 25 mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 25 mM MgCl2, 2.5 mM DTT, pH 7.4) and incubated for 30 min at 30°C. A typical assay reaction for Mnk1 activity contained Mnk1 (2 ng/mL), HA-eIF4E (10 ng/mL), ATP (300 mM) in kinase buffer. The reaction is started by addition of activated p38 (0.03-3 ng/mL) and stopped after 30 min at 30°C by addition of SDS loading buffer. Inhibitors of Mnk1 are identified under the same assay conditions, except that Mnk1 is pre-activated using active p38a before exposure to the substrate and inhibitors[1].

Recombinant p38 isoforms are activated by Mkk6(E) under the following conditions: p38 (100 ng/mL), Mkk6(E) (30 ng/mL), ATP (100 mM) are mixed in kinase buffer (25 mM Hepes, 25 mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 25 mM MgCl2, 2.5 mM DTT, pH 7.4) and incubated for 30 min at 30°C. Mnk1 (2 ng/mL), HA-eIF4E (10 ng/mL), and ATP (300 mM) were the main components of a typical assay reaction for Mnk1 activity. Addition of activated p38 (0.03–3 ng/mL) initiates the reaction, which is terminated by the addition of SDS loading buffer 30 minutes later at 30°C. The same assay conditions are used to identify Mnk1 inhibitors, but Mnk1 is first preactivated using active p38a before being exposed to the substrate and inhibitors.

CD34+ cells (5×105) or GMPs (1×105) are resuspended in 25 μL 1% FBS/PBS solution and injected into the right femur of 8- to 10-wk-old sublethally irradiated (200 cGy) female mice (n=5 mice per group). For each experiment, 1% FBS/PBS solution-injected mice serve as the sham control. Using flow cytometry, mice are examined every 4 weeks after the transplant to see if human cells have grafted. Engrafted mice are treated for 3 weeks with CGP57380 (40 mg/kg/d) intraperitoneally, dasatinib (5 mg/kg/d) by gavage, or vehicle alone (n = 5 mice per group) after 6 weeks following transplantation. After the course of treatment is complete, mice are put down, and CD45+ cells are extracted from the BM and spleen using anti-human CD45-specific immunomagnetic microbeads. In the colony forming cell (CFC) assay, an aliquot of 1×105 human CD45+ cells is seeded into methylcellulose, and colonies are counted after 2 weeks. The remaining human cells from each primary transplant recipient are then all intrafemorally injected into secondary recipients, and human engraftment is monitored every two weeks starting at four weeks. All mice are put to death after 16 weeks. RT-PCR is used to find BCR-ABL1 transcripts, and flow cytometry is used to evaluate engraftment in BM and blood.

[1]. Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2. Mol Cell Biol. 2001 Aug;21(16):5500-11.

[2]. Mnk1 is required for angiotensin II-induced protein synthesis in vascular smooth muscle cells. Circ Res. 2003 Dec 12;93(12):1218-24. Epub 2003 Nov 6.

[3]. Loss of MNK function sensitizes fibroblasts to serum-withdrawal induced apoptosis. Genes Cells. 2007 Oct;12(10):1133-40.

[4]. Targeting of the MNK-eIF4E axis in blast crisis chronic myeloid leukemia inhibits leukemia stem cell function. Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):E2298-307.

N3-(4-fluorophenyl)-2H-pyrazolo[3,4-d]pyrimidine-3,4-diamine is a pyrazolopyrimidine.

---

Eukaryotic initiation factor 4E (eIF4E) is a key component of the translational machinery and an important modulator of cell growth and proliferation. The activity of eIF4E is thought to be regulated by interaction with inhibitory binding proteins (4E-BPs) and phosphorylation by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) on Ser209 in response to mitogens and cellular stress. Here we demonstrate that phosphorylation of eIF4E via MNK1 is mediated via the activation of either the Erk or p38 pathway. We further show that expression of active mutants of MNK1 and MNK2 in 293 cells diminishes cap-dependent translation relative to cap-independent translation in a transient reporter assay. The same effect on cap-dependent translation was observed when MNK1 was activated by the Erk or p38 pathway. In line with these findings, addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in a reduced protein synthesis in vitro, and overexpression of MNK2 caused a decreased rate of protein synthesis in 293 cells. By using CGP57380, a novel low-molecular-weight kinase inhibitor of MNK1, we demonstrate that eIF4E phosphorylation is not crucial to the formation of the initiation complex, mitogen-stimulated increase in cap-dependent translation, and cell proliferation. Our results imply that activation of MNK by MAP kinase pathways does not constitute a positive regulatory mechanism to cap-dependent translation. Instead, we propose that the kinase activity of MNKs, eventually through phosphorylation of eIF4E, may serve to limit cap-dependent translation under physiological conditions. [1]

---

Angiotensin II (Ang II) stimulates protein synthesis in vascular smooth muscle cells (VSMCs), possibly secondary to regulatory changes at the initiation of mRNA translation. Mitogen-activated protein (MAP) kinase signal-integrating kinase-1 (Mnk1), a substrate of ERK and p38 MAP kinase, phosphorylates eukaryotic initiation factor 4E (eIF4E), an important factor in translation. The goal of the present study was to investigate the role of Mnk1 in Ang II-induced protein synthesis and to characterize the molecular mechanisms by which Mnk1 and eIF4E is activated in rat VSMCs. Ang II treatment resulted in increased Mnk1 activity and eIF4E phosphorylation. Expression of a dominant-negative Mnk1 mutant abolished Ang II-induced eIF4E phosphorylation. PD98059 or introduction of kinase-inactive MEK1/MKK1, but not SB202190 or kinase-inactive p38 MAP kinase, inhibited Ang II-induced Mnk1 activation and eIF4E phosphorylation, suggesting that ERK, but not p38 MAP kinase, is required for Ang II-induced Mnk1-eIF4E activation. Further, dominant-negative constructs for Ras, but not for Rho, Rac, or Cdc42, abolished Ang II-induced Mnk1 activation. Finally, treatment of VSMCs with CGP57380, a novel specific kinase inhibitor of Mnk1, resulted in dose-dependent decreases in Ang II-stimulated phosphorylation of eIF4E, protein synthesis, and VSMC hypertrophy. In summary, these data demonstrated that (1) Ang II-induced Mnk1 activation is mediated by the Ras-ERK cascade in VSMCs, and (2) Mnk1 is involved in Ang II-mediated protein synthesis and hypertrophy, presumably through the activation of translation-initiation. The Mnk1-eIF4E pathway may provide new insights into molecular mechanisms involved in vascular hypertrophy and other Ang II-mediated pathological states. [2]

---

Map kinase-interacting protein kinases 1 and 2 (MNK1, MNK2) function downstream of p38 and ERK MAP kinases, but there are large gaps in our knowledge of how MNKs are regulated and function. Mice deleted of both genes are apparently normal, suggesting that MNKs function in adaptive pathways during stress. Here, we show that mouse embryo fibroblasts (MEFs) obtained from mnk1 (-/-)/mnk2 (-/-) as well as mnk1 (-/-) and mnk2 (-/-) mice are sensitized to caspase-3 activation upon withdrawal of serum in comparison to wild-type cells. Caspase-3 cleavage occurs with all cells in the panel, but most rapidly and robustly in cells derived from mice lacking both MNK genes. Treatment of wild-type MEFs in the panel with a compound (CGP57380) that inhibits MNK1 and MNK2 sensitizes wild-type cells for serum-withdrawal induced apoptosis, suggesting that sensitization is due to loss of MNK function and not to a secondary event. Reintroduction of wild-type MNK1 in the double knockout MEFs results in decreased sensitivity to serum withdrawal that is not observed for wild-type MNK2, or the kinase dead variant. Our work identifies MNKs as kinases involved in anti-apoptotic signaling in response to serum withdrawal. [3]

---

Chronic myeloid leukemia responds well to therapy targeting the oncogenic fusion protein BCR-ABL1 in chronic phase, but is resistant to treatment after it progresses to blast crisis (BC). BC is characterized by elevated β-catenin signaling in granulocyte macrophage progenitors (GMPs), which enables this population to function as leukemia stem cells (LSCs) and act as a reservoir for resistance. Because normal hematopoietic stem cells (HSCs) and LSCs depend on β-catenin signaling for self-renewal, strategies to specifically target BC will require identification of drugable factors capable of distinguishing between self-renewal in BC LSCs and normal HSCs. Here, we show that the MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in BC GMPs but not normal HSCs, and that MNK kinase-dependent eIF4E phosphorylation at serine 209 activates β-catenin signaling in BC GMPs. Mechanistically, eIF4E overexpression and phosphorylation leads to increased β-catenin protein synthesis, whereas MNK-dependent eIF4E phosphorylation is required for nuclear translocation and activation of β-catenin. Accordingly, we found that a panel of small molecule MNK kinase inhibitors prevented eIF4E phosphorylation, β-catenin activation, and BC LSC function in vitro and in vivo. Our findings identify the MNK-eIF4E axis as a specific and critical regulator of BC self-renewal, and suggest that pharmacologic inhibition of the MNK kinases may be therapeutically useful in BC chronic myeloid leukemia. [4]

C11H9FN6

244.23

244.087

C, 54.10; H, 3.71; F, 7.78; N, 34.41

522629-08-9

11644425

Light brown to brown solid powder

1.6±0.1 g/cm3

541.6±50.0 °C at 760 mmHg

281.4±30.1 °C

0.0±1.4 mmHg at 25°C

1.809

1.28

3

6

2

18

283

0

FC1C([H])=C([H])C(=C([H])C=1[H])N([H])C1=C2C(N([H])[H])=NC([H])=NC2=NN1[H]

UQPMANVRZYYQMD-UHFFFAOYSA-N

InChI=1S/C11H9FN6/c12-6-1-3-7(4-2-6)16-11-8-9(13)14-5-15-10(8)17-18-11/h1-5H,(H4,13,14,15,16,17,18)

3-N-(4-fluorophenyl)-2H-pyrazolo[3,4-d]pyrimidine-3,4-diamine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.24 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (10.24 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: 4% DMSO +30%PEG 300 +ddH2O: 10mg/mL

---

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