In the presence of DNA damage, Chk1 (checkpoint kinase 1) can be activated through phosphorylation by ATR and then lead to the phosphorylation and degradation of CDC25A, the nuclear exclusion of CDC25B and CDC25C. Through Chk1-mediated loss of CDC25A activity, CDK2 in an inactive phosphorylated state blocking initiation of DNA replication origins can be suspended. Cytosolic sequestration of CDC25B and CDC25C prevents the activation of CDK1 resulting in cell-cycle arrest at the G2–M boundary. Thus, Chk1 acts as a key regulator to maintain functional intra-S and G2–M checkpoints. LY2606368 (Prexasertib) is a selective Chk1 inhibitor with IC50 value <1nM, modest to Chk2 and Rsk with IC50 values of 8nM and <10nM (measured by enzymatic assay). LY2606368 potently abrogated the G2–M checkpoint activated by doxorubicin in p53-deficient HeLa cells with an EC50 of 9 nM. Treatment with 33-100nM LY2606368 for 7h resulted in DNA damage, revealed by TUNEL or increased pH2AX-S139, in HeLa cells during S-phase. When U-2 OS cells, depleted of CDC25A or CDK2 through siRNA, were treated for 6 hours with 4nM LY2606368, H2AX phosphorylation was greatly reduced relative to LY2606368 alone. This indicated that the DNA damage effects of inactivation of Chk1 by LY2606368 were dependent upon CDC25A and CDK2. Treatment with 33nM LY2606368 for a total of 12 hours caused replication catastrophe in HeLa cells. Subcutaneous injection of 1, 3.3, or 10 mg/kg of LY2606368 twice daily for 3 days followed by 4 days of rest for three cycles caused statistically significant tumor growth inhibition (up to 72.3%) in mice bearing Calu-6 xenograft tumors[1]. Several phase 2 studies of LY2606368 treatment for small cell lung cancer, ovarian cancer, breast cancer and prostate cancer are under recruiting now (see https://www.clinicaltrials.gov/).
作用机制
LY2606368 is an ATP-competitive protein kinase inhibitor with a Ki of 0.9nM against purified Chk1.
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