In vitro activity: Prexasertib (also know LY2606368) is a novel, potent, selective and ATP competitive inhibitor of the CHK1 (checkpoint kinase 1) protein kinase with IC50 values of<1 nM and 8 nM for CHK1 and CHK2, respectively. CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Prexasertib as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of Prexasertib is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with Prexasertib results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Prexasertib shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. In summary, Prexasertib is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.

Kinase Assay: Prexasertib (LY2606368) potently and selectively inhibits CHK1 with an IC50 of<1 nM, and also inhibits CHK2, with an IC50 of 8 nM. LY2606368 has an EC50 of 1 nM for CHK1 activity through autophosphorylation of serine 296 and <31 nM for HT-29 CHK2 autophosphorylation (S516). LY2606368 potently abrogates the G2-M checkpoint activated by doxorubicin in p53-deficient HeLa cells with an EC50 of 9 nM. However, 100 nM LY2606368 does not inhibit PMA-stimulated RSK but instead weakly stimulates phosphorylation of S6 on serines 235/236. LY2606368 is broadly antiproliferative with IC50s of 3 nM, 3 nM, 10 nM, 37 nM, and 68 nM against U-2 OS, Calu-6, HT-29, HeLa, and NCI-H460 cell lines, respectively. LY2606368 (4 nM) results in a large shift in cell-cycle populations from G1 and G2-M to S-phase with an accompanied induction of H2AX phosphorylation in U-2 OS cells. LY2606368 (25 μM) exhibits inhibitory activities against proliferation of AGS and MKN1 cells. LY2606368 (20 nM) inhibits HR repair capacity DR-GFP cells. LY2606368 (5 nM) in combination with PARP inhibitor BMN673, displays synergistic anticancer effects in gastric cancer cells.

Cell Assay: Proliferation inhibition effect of CHK1 ablation, IR sensitivity, anticancer effect of BMN673 and LY2606368 are detected by MTS Cell Proliferation Colorimetric Assay Kit. Cells are seeded into 96 wells cell culture plate, then treated with indicated experiment conditions, then added 20 μL MTS reagent to each well subsequently, after incubated for 2 hours, cell viability of each well is detected on microplate reader at a wavelength of 490 nM.