Hi! I am trying to inhibit Chk1 kinase phosporilation upon DNA damage. I was thinking on using the inhibitor AZD-7762. What is the best way to evaluate that the AZD is working? Should a western blot against pS296-Chk1 work? Thanks!
Dear Ariel
For the DNA damage analysis you should check the genotoxicity of the inhibitor using COMET assay.
But as per my knowledge this inhibitor is not passed in phase-I clinical stage due to toxicity. Kindly go through literature regarding it for proper research. Chk1 is very important target.
best of luck
I would suggest doing flow cytometry for cell cycle first. It's quicker and cheaper. Synchronize your cells, release them and then treat with a DNA damaging agent (irradiation or drugs) when they entered S phase (typically 18-22 h post-release) +/- your inhibitor. Collect samples every 4-6 h during the entire experiment to monitor how cell cycle phases change. If chk1 signalling is intact they will arrest at G2/M, if inhibited they will go through mitosis (and likely die). This is a functional test. To validate that the effect is actually mediated by chk1 inhibition you'll need western blot for chk1 + a few more targets of chk1 for verification.
Thanks to both of you for your answers! I'll look into those experiements and in parallel anayze p-ChK1 345 and 296 by western blot upon DNA damage + / - AZD.
Thanks again!
Ari
- Badges
- Science method