Semi-automated high-throughput substrate screening assay for nucleoside kinases

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.