M443 is an irreversible and specific inhibitor of MRK, with an IC50<125 nM.
MRK depletion decreases cell viability after IR by 33% of control at 3 Gy. Similarly, the clonogenic assay shows a significant decrease in survival with a dose enhancement factor (DEF) of 1.6 at 10% viability. MRK activation by IR is maximal at 30 minutes after exposure to radiation. Therefore, for subsequent analysis, this time point is used. In both cell cultures, the IR-stimulated activation of MRK, Chk2, and p38 is greatly inhibited by 500 nM M443. Cells are seeded on coverslips, pretreated with 500 nM M443 or vehicle, exposed to 6 Gy of IR, fixed at different times after IR and processed for immunofluorescence with the MPM2 phospho-specific antibody that specifically stains mitotic cells. In contrast to control cells, the M443-treated cells fail to arrest after IR and maintained a similar mitotic index as the nonirradiated cells. Thus, inhibition of MRK leads to inhibition of Chk2 and failure to arrest in the cell cycle in response to IR-induced DNA damage[1].
Control mice survive with a median of 32 days after tumor cell implantation. Treatment with M443 alone adds 5.5 days to this survival, whereas the chosen low dose of radiation does not significantly increase survival. In contrast, the combination of M443 and IR extend survival with a median of 16 days longer than control. Treatment with M443 does not affect the animal weight, as the weight loss observed is observed in all groups just a few days before the animals became moribund. It is showed that the tumor-containing fraction has elevated levels of both total and active MRK (lane RB in the vehicle-treated brain). In contrast, the tumor-containing fraction from the M443-treated brain shows total loss of MRK activity. Interestingly, the fractions containing normal brain, which in the control brain show some level of MRK protein, in the treated brain also has lost MRK, suggesting that diffusion of M443 across the whole cerebellum inhibit normal MRK as well[1].
[1]. Markowitz D, et al. Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma. Mol Cancer Ther. 2016 Aug;15(8):1799-808.