At some point in drug development of kinase inhibitors, we should know if the drug can bind (at similar affinities) to the both phosphorylated and un-phosphorylated forms of kinase proteins .
Hi Luiz,
That is very important quisteion in many studies. In my previous study we had similar question we solved it through point mutation of the phosphor-site(s) and also constructed DNA which contained a mutation (to Glu or Asp)that mimicked the phosphorylated form of the protein. Then transfect the cells with your Plasmid and treat them with the drug.
Good luck
Hi Luiz,
That is very important quisteion in many studies. In my previous study we had similar question we solved it through point mutation of the phosphor-site(s) and also constructed DNA which contained a mutation (to Glu or Asp)that mimicked the phosphorylated form of the protein. Then transfect the cells with your Plasmid and treat them with the drug.
Good luck
Isothermal calorimetry would be the gold standard.
A less tedious experiment could be performed using a biotinylated variant of the drug. It's binding to the kinase (regardless of the phosphorylation status) could be monitored by AlphaLISA. A competition with the free drug would then allow to measure its affinity to either form of the protein.
That's a very important and interesting question, as we know that phosphorylation status or at different sites may bring completely different or reversed function for the target protein. Thus I will apply at least two independent assay to validate the protein-compound binding. Fortunately, we have different approches, such as ITC, AlphaLISA, Fluorescence polarization assay, NMR or even crystallization... I guess it really dependent on your goal and resource to choose the proper assays.
We had good results with the kinase binding assay ( Lanthascreen - from Life technologies) when we compared binding to an active (phosphorylated) and a non active ( non phosphorylated ) kinase. With the restriction of course that binding is detected as displacement of a labelled propriety - unknown- molecule. I agree with Mathieu Arcand that isothermal calorimetry would be the golden standard.
Dear all, thank you very much for your comments. I can now say that I am going ahead on isothermal calorimetry. Cheers. M.
Good luck on the experiments. Should you get compounds that have a preference for phosphorylated versus non-phosphorylated protein, then a potential follow up is to determine co-crystal structures of protein bound to compound in order to explain the findings structurally. It's high risk but also high reward.
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