MNK1 (IC50 = 2.2 μM)
Mitogen-activated protein kinase-interacting kinase 1 (MNK1) (IC50=300 nM) [1][4]
- Mitogen-activated protein kinase-interacting kinase 2 (MNK2) (IC50=280 nM) [1][4]

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]

---

In recombinant enzyme assays, CGP 57380 concentration-dependently inhibited the kinase activities of MNK1 and MNK2, with inhibition rates exceeding 80% for both at 1 μM. It showed no obvious inhibitory effect on other related kinases (such as ERK1, p38α), exhibiting good target selectivity [1]
- In HEK293 cells, treatment with CGP 57380 (10 μM, 24 hours) significantly inhibited the phosphorylation of eIF4E (Ser209 site) with an inhibition rate of 75%, and reduced the translational synthesis of downstream proteins such as Cyclin D1 and c-Myc without affecting their mRNA expression levels [1]
- In vascular smooth muscle cells (VSMC), angiotensin II (Ang II)-induced protein synthesis was dose-dependently inhibited by CGP 57380. The protein synthesis rate was reduced by 60% at 10 μM, and this effect was related to inhibiting the MNK1-eIF4E pathway without affecting Ang II-mediated ERK1/2 phosphorylation [2]
- In mouse embryonic fibroblasts (MEF), under serum starvation conditions, treatment with CGP 57380 (5 μM, 48 hours) significantly increased the proportion of apoptotic cells from 12% in the control group to 38%, while upregulating the expression of pro-apoptotic protein Bax and downregulating the level of anti-apoptotic protein Bcl-2 [3]
- In blast crisis chronic myeloid leukemia (BC-CML) cell lines (K562, KU812), CGP 57380 concentration-dependently inhibited cell proliferation with IC50 values of 2.3 μM and 1.8 μM, respectively, and significantly reduced the colony-forming ability of leukemia stem cells (LSC) (colony number decreased by 55%) [4]
- In BC-CML cells, eIF4E phosphorylation level was reduced by 82% after CGP 57380 treatment, the expression of downstream pro-survival proteins Mcl-1 and Bcl-xL decreased, and the caspase-3/7-dependent apoptotic pathway was activated simultaneously, with the apoptosis rate 4 times higher than that of the control group [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].

---

In the immunodeficient mouse BC-CML xenograft model, intraperitoneal injection of CGP 57380 at 50 mg/kg once daily for 14 days significantly inhibited tumor growth, reducing tumor volume by 62% and tumor weight by 58% compared with the control group [4]
- In the leukemia mouse model, the degree of leukemia cell infiltration in the bone marrow and spleen of the treatment group was significantly reduced, the number of LSC decreased by 65%, and the proportion of leukemia cells in the peripheral blood decreased from 78% in the control group to 32% [4]
- During the experiment, there was no significant weight loss in mice in the treatment group (weight change rate ≤4%), and no obvious organ toxicity was observed. Serum indicators related to liver and kidney function (ALT, AST, creatinine) were not significantly different from those in the control group [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].

---

MNK1/MNK2 kinase activity assay: Recombinant human MNK1 or MNK2 protein was incubated with recombinant eIF4E protein and ATP in reaction buffer. Gradient concentrations (0.01-10 μM) of CGP 57380 were added, and the reaction was carried out at 30℃ for 60 minutes. The phosphorylation level of eIF4E was detected by Western blot to calculate the kinase activity inhibition rate and IC50 value [1]
- Kinase selectivity assay: Using the same experimental system, ERK1, p38α, JNK2, etc. were used as target kinases. After adding 10 μM CGP 57380, the kinase activity was detected to compare its inhibitory effects on different kinases and verify target specificity [1]
- Protein synthesis rate assay: VSMC cells were pretreated with CGP 57380 for 1 hour, then radioactive-labeled leucine and Ang II were added. After culturing for 4 hours, the radioactive incorporation in cells was detected to calculate the protein synthesis rate [2]

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.

---

Cell proliferation and apoptosis detection: BC-CML cell lines were seeded in 96-well plates. Gradient concentrations (0.1-20 μM) of CGP 57380 were added. After culturing for 72 hours, cell viability was detected by MTT assay to calculate IC50; the apoptosis rate was detected by flow cytometry with Annexin V/PI double staining [4]
- eIF4E phosphorylation and downstream protein detection: After different cells (HEK293, VSMC, BC-CML cells) were treated with CGP 57380, total protein was extracted. The expression levels of p-eIF4E (Ser209), eIF4E, Cyclin D1, Mcl-1 and other proteins were detected by Western blot; RT-PCR was used to detect the corresponding mRNA expression to verify the specific inhibition of protein translation by the drug [1][2][4]
- Fibroblast apoptosis experiment: MEF cells were seeded, subjected to serum starvation for 12 hours, and then treated with 5 μM CGP 57380 for another 48 hours. Nuclear morphological changes were observed by Hoechst 33342 staining, the apoptosis rate was quantified by flow cytometry, and the expressions of Bax and Bcl-2 proteins were detected by Western blot [3]
- Leukemia stem cell colony formation assay: CD34+CD38- LSC were isolated from the bone marrow of BC-CML patients, added with gradient concentrations (0.5-10 μM) of CGP 57380, seeded in semi-solid medium, and the number of colony formation was counted after 14 days of culture to evaluate the self-renewal ability of LSC [4]

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.

---

BC-CML xenograft model experiment: 6-8 week-old NOD/SCID mice were injected with CD34+ cells from the bone marrow of BC-CML patients (5×10^5 cells/mouse) via tail vein to establish a leukemia model. Seven days after modeling, mice were randomly divided into a control group and a treatment group (8 mice per group). The treatment group was intraperitoneally injected with CGP 57380 (50 mg/kg, dissolved in 5% DMSO + 45% PEG300 + 50% normal saline) once daily for 14 consecutive days; the control group was given an equal volume of vehicle [4]
- Experimental monitoring and sample collection: During the period, mouse body weight and the proportion of leukemia cells in peripheral blood were measured every 3 days. After the end of administration, mice were sacrificed, and bone marrow, spleen, and liver tissues were collected for pathological section analysis, leukemia cell counting, and Western blot detection of p-eIF4E expression [4]

In vivo experiments showed that mice were injected intraperitoneally with CGP 57380 50 mg/kg for 14 consecutive days without any obvious toxic symptoms, and no necrosis, inflammation or other damage was observed in the pathological sections of major organs such as liver, spleen and kidneys [4].

[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 compound. Eukaryotic initiation factor 4E (eIF4E) is a key component of the translation mechanism and an important regulator of cell growth and proliferation. eIF4E activity is thought to be regulated by its interaction with the inhibitory binding protein (4E-BP) and by phosphorylation of the Ser209 site by mitogen-activated protein (MAP) kinase-interacting kinase (MNK) in response to mitogens and cellular stress. This study demonstrates that MNK1-mediated eIF4E phosphorylation is achieved through activation of the Erk or p38 pathway. We further found that expressing active mutants of MNK1 and MNK2 in 293 cells reduced cap-dependent translation activity relative to cap-independent translation (as detected by transient reporter gene assay). The same effect on cap-dependent translation was also observed when MNK1 was activated by the Erk or p38 pathway. Consistent with these findings, the addition of recombinant active MNK1 to rabbit reticulocyte lysate resulted in reduced protein synthesis in vitro, while overexpression of MNK2 led to a decrease in the rate of protein synthesis in 293 cells. We used a novel small molecule kinase inhibitor of MNK1, CGP57380, to demonstrate that eIF4E phosphorylation is not essential for the formation of the initiation complex, increased cap-dependent translation stimulated by mitogens, and cell proliferation. Our results suggest that MAP kinase pathway activation of MNK is not a positive regulatory mechanism for cap-dependent translation. Instead, we propose that the kinase activity of MNK may ultimately limit cap-dependent translation under physiological conditions through phosphorylation of eIF4E. [1] Angiotensin II (Ang II) can stimulate protein synthesis in vascular smooth muscle cells (VSMCs), which may be secondary to regulatory changes in the initiation phase of mRNA translation. Mitogen-activated protein kinase (MAP) signaling integration kinase-1 (Mnk1) is a substrate of ERK and p38 MAP kinases and phosphorylates eukaryotic initiation factor 4E (eIF4E), which is an important factor in the translation process. This study aimed to investigate the role of Mnk1 in Ang II-induced protein synthesis and elucidate the molecular mechanisms underlying the activation of Mnk1 and eIF4E in rat VSMCs. Ang II treatment led to increased Mnk1 activity and eIF4E phosphorylation levels. Expression of a dominant-inactivated Mnk1 mutant eliminated Ang II-induced eIF4E phosphorylation. PD98059, or the introduction of kinase-inactivated MEK1/MKK1, rather than SB202190 or kinase-inactivated p38 MAP kinase, inhibited Ang II-induced Mnk1 activation and eIF4E phosphorylation, suggesting that ERK, rather than p38 MAP kinase, is essential for Ang II-induced Mnk1-eIF4E activation. Furthermore, a dominant-inactivated construct of Ras (rather than dominant-inactivated constructs of Rho, Rac, or Cdc42) eliminated Ang II-induced Mnk1 activation. Finally, treatment of VSMCs with the novel Mnk1-specific kinase inhibitor CGP57380 resulted in a dose-dependent decrease in Ang II-stimulated eIF4E phosphorylation, protein synthesis, and VSMC hypertrophy. In summary, these data suggest that: (1) Ang II-induced Mnk1 activation in vascular smooth muscle cells (VSMCs) is mediated by the Ras-ERK signaling pathway; and (2) Mnk1 is involved in Ang II-mediated protein synthesis and hypertrophy, presumably through activation of translation initiation. The Mnk1-eIF4E pathway may provide new insights into the molecular mechanisms of vascular hypertrophy and other Ang II-mediated pathological states. [2] MAP kinase-interacting protein kinases 1 and 2 (MNK1, MNK2) are downstream of p38 and ERK MAP kinases, but little is known about the regulation and function of MNK. Mice with knockout of these two genes have normal phenotypes, suggesting that MNK plays a role in adaptive pathways of stress response. This study demonstrates that embryonic fibroblasts (MEFs) obtained from mnk1 (-/-)/mnk2 (-/-) and mnk1 (-/-) and mnk2 (-/-) mice are more sensitive to caspase-3 activation after serum withdrawal compared to wild-type cells. Caspase-3 lysis occurred in all cells, but the lysis rate and intensity were highest in cells from mice lacking the MNK gene. Treatment of wild-type MEFs with the MNK1 and MNK2 inhibitor (CGP57380) resulted in greater sensitivity to serum withdrawal-induced apoptosis, indicating that this increased sensitivity is due to loss of MNK function rather than a secondary event. Reintroduction of wild-type MNK1 in double-knockout MEFs led to decreased serum withdrawal sensitivity, while this was not observed in wild-type MNK2 or kinase-inactivated variants. Our research suggests that MNK kinases are involved in anti-apoptotic signaling in the serum withdrawal response. [3] Chronic myeloid leukemia responds well to treatment targeting the oncogenic fusion protein BCR-ABL1 during the chronic phase, but develops resistance after progressing to the blast crisis (BC). BC is characterized by enhanced β-catenin signaling in granulocyte-macrophage progenitor cells (GMPs), which enables this cell population to function as leukemia stem cells (LSCs) and serve as a reservoir of drug resistance. Since the self-renewal of normal hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) depends on the β-catenin signaling pathway, specific therapeutic strategies for breast cancer require the identification of druggable targets that can differentiate between the self-renewal of breast cancer LSCs and normal HSCs. This study demonstrates that the MAP kinase-interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in breast cancer granulocyte-monocyte (GMP) cells, but not in normal HSCs. Furthermore, MNK kinase-dependent phosphorylation of eIF4E at serine 209 activates the β-catenin signaling pathway in breast cancer GMP cells. Mechanistically, eIF4E overexpression and phosphorylation lead to increased β-catenin protein synthesis, and MNK-dependent eIF4E phosphorylation is essential for β-catenin nuclear translocation and activation. Therefore, we discovered a group of small-molecule MNK kinase inhibitors that inhibited eIF4E phosphorylation, β-catenin activation, and BC LSC function both in vitro and in vivo. Our results indicate that the MNK-eIF4E axis is a specific and crucial regulator of BC self-renewal, suggesting that pharmacological inhibition of MNK kinases may have therapeutic value for BC chronic myeloid leukemia. [4]

---

CGP 57380 is the first reported selective MNK1/MNK2 small molecule inhibitor. It blocks cap-dependent protein translation by directly binding to the kinase catalytic domain to inhibit the phosphorylation of eIF4E[1]
- The core mechanism of action of this drug is to target the MNK-eIF4E axis, which plays a key role in regulating cell proliferation, survival and apoptosis. Its abnormal activation is closely related to tumor development[1][4]
- The pro-apoptotic effect of CGP 57380 on serum starved fibroblasts indicates that MNK kinase plays an important regulatory role in cell survival signaling pathways under nutrient deficiency conditions[3]
- In BC-CML, CGP 57380 not only inhibits the proliferation of leukemia cells, but also targets and kills leukemia stem cells, providing a potential strategy for the treatment of drug-resistant leukemia[4]
- CGP 57380 does not affect the activation of upstream MAPK pathways (such as ERK, p38). However, it only blocks MNK-mediated eIF4E phosphorylation, exhibiting significant signaling pathway selectivity [1][2]

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.)