A new fluorescence method for monitoring PNK activity in vitro, natural compounds screening and intracellular imaging.

• A new method has been proposed for monitoring PNK activity in vitro , natural compound screening and intracellular imaging. • NS17 isolated from Kadsura heteroclita screened as PNK inhibitor, which effect confirmed in vitro and in cell level. • NS17 can scavenge intracellular ROS, inhibit inflammatory cells proliferation and down-regulate COX-2 expression. Polynucleotide kinase (PNK) is critical for nucleic acid metabolism, as its overexpression can increase ROS production and activate the inflammation pathway, leading to chronic inflammation. In this study, we proposed a new fluorescence method for monitoring PNK in vitro , natural compounds screening and intracellular imaging. Quantitative detection depicted that the limit of detection (LOD) of PNK is 0.0021 U/mL in the linear range of 0.01∼10 U/mL. Methodological evaluation showed that both of the selectivity and the inhibitory effect of ADP and EDTA on PNK activity with satisfactory results. Fluorescence imaging of inflammatory cells demonstrated the reliability of platform for capturing PNK change in situ regulated by inhibitor of natural compound NS17 isolated from Kadsura heterolita. Mechanism study showed that the inhibitor NS17 can reduce inflammation by scavenging intracellular ROS, killing inflammatory cells and down-regulating COX-2 expression, the processes of which are related with PNK activity. In summary, above results suggest that this new platform could monitor PNK activity in situ and screen inflammation inhibitors targeted on PNK. [ABSTRACT FROM AUTHOR]