CGP 57380 (Synonyms: MNK1 Inhibitor)

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°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.

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.

References:

[1]. Knauf U, et al. 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]. Ishida M, et al. 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]. Chrestensen CA, et al. Loss of MNK function sensitizes fibroblasts to serum-withdrawal induced apoptosis. Genes Cells. 2007 Oct;12(10):1133-40.
[4]. Lim S, et al. 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