Application of a titanium-based metal-organic framework to protein kinase activity detection and inhibitor screening

In this work, we used a titanium-based metal-organic framework (MOF, Ti-MIL125-NH₂) as a novel enrichment platform to detect protein kinase A (PKA) activity and to screen relevant kinase inhibitors. This method took advantage of the highly specific recognition of phosphate groups by the Ti-MIL125-NH₂ nanoparticle. In the presence of PKA and adenosine 5'-triphosphate (ATP), the fluorophore-labeled peptide substrate was phosphorylated, and the generated phosphopeptide could then specifically bind to the titanium sites of Ti-MIL125-NH₂. This resulted in fluorescence enrichment, which could be efficiently detected by the system. Under optimal conditions, the method presented a linear relationship in the experimental range of 0.00005-0.01 U μL^-1, and the limit of detection was 0.00003 U μL^-1 (3σ, n = 11). Furthermore, protein kinase Akt1 was tested to verify the universality of this method. The method was also successfully applied in cell lysates for kinase activity analysis and inhibitor screening. Thus, the new, highly sensitive fluorescence method based on MOF for detecting PKA activity is an excellent tool that has potential applications in kinase-related disease and basic research.