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

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Nucleoside kinases (NKs) are crucial enzymes for phosphorylating nucleoside analogues used in cancer and antiviral therapies.
• Characterizing NK specificities is essential for optimizing drug design and in vitro nucleotide analogue production.
• A rapid and dependable method for screening NK substrates is highly needed.

Purpose of the Study:

• To validate a luciferase-based assay for detecting nucleoside kinase activity in a 96-well plate format.
• To establish a semi-automated, high-throughput screening method for NK enzymes.
• To compare the substrate specificities of different NKs and identify novel substrates.

Main Methods:

• Validation of a luciferase-based assay for NK activity detection.
• Semi-automation of the assay using a liquid handling robot for high-throughput screening.
• Comparison of assay results with High-Performance Liquid Chromatography (HPLC) analysis.

Main Results:

• The assay demonstrated good linearity (r² > 0.98) and accepted alternative phosphate donors (dATP, CTP).
• The high-throughput assay yielded results comparable to HPLC analysis.
• Screening of four NKs against 20 substrates identified previously unknown substrates for three enzymes, correlating well with literature data.

Conclusions:

• The developed semi-automated, high-throughput assay is a suitable tool for identifying optimal nucleoside kinases for various substrates.
• This method facilitates efficient characterization of NK enzyme specificities, crucial for drug development.
• The assay enables the discovery of novel NK substrates, advancing nucleotide analogue research.