Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases

International Journal of Molecular Sciences

Katja F. Hellendahl¹  Maryke Fehlau¹  Anke Kurreck¹  Sebastian Hans¹  Peter Neubauer¹ 

[1] Chair of Bioprocess Engineering, Faculty III Process Sciences, Institute of Biotechnology, Technische Universität Berlin, Ackerstraße 76, 13355 Berlin, Germany;

关键词: nucleoside kinase;    nucleoside analogues;    nucleotide;    screening;    high throughput;    luminescent assay;   

DOI  :  10.3390/ijms222111558

来源: DOAJ

【 摘 要 】

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 nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0–500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 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.

【 授权许可】

Unknown