Hello. I'm trying to do FRET-based in vitro kinase assay these days with MAPK and MAPK inhibitor. I expected kinase activity to decrease in dose-dependent manner when treated with kinase inhibitor but I constantly gain result that there's no difference. (And inhibition percentage is a negative number)
I already put quite high concentration of inhibitor so I don't think the inhibitor concentration is the cause...
(and I put 100uM ATP)
Can someone give me advice??
Have you checked that your experimental system is working as expected by demonstrating that a well-known and potent MAPK inhibitor is actually resulting in the expected potency of inhibition?
A negative percent inhibition indicates either that the compound enhances the activity of the enzyme or, more likely for a novel compound of low potency, that it is causing an interference in the detection method of the assay.
It is not entirely clear what type of assay you are doing. Is this in vitro but in living cells with a genetically encoded FRET reporter? Or is it in vitro with purified components?
FRET reporters tend to work well in cells, where their phosphorylation is counteracted by phosphatases: a 50% reduction in kinase activity changes the fraction of FRET sensor molecules that is phosphorylated in steady state. It is generally a good idea to test inhibitors first in a radioactive phosphorylation type assay using purified kinase and substrate with gamma-P-32-ATP.
The next question is, whether the inhibitor penetrates the cells. You might be able to verify this by Western Blot and phospho-specific antibodies for the substrates of your MAPK.
When you say there is no difference, have you tried to distinguish whether the FRET probe is in a high-FRET or low-FRET state? You might be able to get an impression of this by bleaching donor or acceptor, or by forcing phosphorylation with an unspecific phosphatase inhibitor like okadaic acid.
If you are doing these assays fully in vitro, there will be no phosphatase, and ultimately all sensor molecules will be phosphorylated. Only if you manage to 100% kill the kinase before adding it to the FRET sensor substrate will you see a difference, but this is generally not how these (often ATP-competitive) inhibitors work.
You could consider using our assays, www.pamgene.com.
We can do in vivo Kinome activity profiling and also ex-vivo spike in experiments of your inhibitors. This way you can also find interresting off target effects in a unbiassed way. If you want we can also exchange thoughts on email: [email protected] best of luck!