Kinase experiment:

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℃. 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℃ 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.

Animal experiment:

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). Mice injected with 1% FBS/PBS solution serve as a sham control for each experiment. Beginning at 4 wk posttransplantation, mice are monitored for engraftment of human cells by flow cytometry. At 6 wk after transplantation, engrafted mice are treated with vehicle alone, dasatinib (5 mg/kg/d) by gavage, or CGP57380 (40 mg/kg/d) intraperitoneally for 3 wk (n=5 mice per group). At the end of treatment, mice are euthanized, and CD45+ cells are isolated from BM and spleen by using anti-human CD45-specific immunomagnetic microbeads. An aliquot of 1×105 human CD45+ cells is seeded into methylcellulose for the colony forming cell (CFC) assay, and colonies are enumerated after 2 wk. All of the remaining human cells from each primary transplant recipient are then transplanted by intrafemoral injection into secondary recipients, and human engraftment is monitored at 2-wk intervals beginning at 4 wk. At the end of 16 wk, all mice are euthanized. Engraftment in BM and blood is assessed by flow cytometry, and BCR-ABL1 transcripts are detected by RT-PCR.

CGP 57380 is a specific and selective inhibitor of MNK1 with IC50 value of 2.2 μM [1].

Mitogen-activated protein (MAP) kinase interacting kinases 1 (MNK1) is a serine/threonine kinase and is able to integrate signal from MAP kinase pathway and phosphorylate eIF4E [1].

CGP 57380 is a specific and selective MNK1 inhibitor. In 293 cells, CGP 57380 (10 μM) inhibited eIF4E phosphorylation in response to fetal calf serum (FCS), arsenite, anisomycin, PMA or tumor necrosis factor alpha. Also, CGP 57380 increased the cap-dependent reporter rluc. In cellular assays, CGP 57380 inhibited eIF4E phosphorylation with IC50 value of 3 μM [1]. In rat vascular smooth muscle cells, CGP 57380 inhibited eIF4E phosphorylation, protein synthesis and VSMC hypertrophy induced by angiotensin II in a dose dependent way [2]. In mouse macrophages, CGP 57380 concentration-dependently inhibited TNFα production stimulated by Escherichia coli lipopolysaccharide (LPS) through posttranscriptional regulation. Also, CGP 57380 inhibited eIF4E phosphorylation. These results suggested that adenine/uridine-rich elements (ARE)-containing TNFα mRNA required eIF4E phosphorylation for initiation of translation [3]. In bone marrow-derived macrophages, CGP 57380 significantly inhibited the production of proinflammatory cytokines monocyte chemoattractant protein-1, TNF and IL-6 [4].

References:
[1].  Knauf U, Tschopp C, Gram H. Negative regulation of protein translation by mitogen-activated protein kinase-interacting kinases 1 and 2. Mol Cell Biol, 2001, 21(16): 5500-5511.
[2].  Ishida M, Ishida T, Nakashima H, et al. Mnk1 is required for angiotensin II-induced protein synthesis in vascular smooth muscle cells. Circ Res, 2003, 93(12): 1218-1224.
[3].  Andersson K, Sundler R. Posttranscriptional regulation of TNFalpha expression via eukaryotic initiation factor 4E (eIF4E) phosphorylation in mouse macrophages. Cytokine, 2006, 33(1): 52-57.
[4].  Rowlett RM, Chrestensen CA, Nyce M, et al. MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages. Am J Physiol Gastrointest Liver Physiol, 2008, 294(2): G452-459.