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Issue 20, 2019
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A simple, robust, universal assay for real-time enzyme monitoring by signalling changes in nucleoside phosphate anion concentration using a europium(iii)-based anion receptor

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Abstract

Enzymes that consume and produce nucleoside polyphosphate (NPP) anions represent major targets in drug discovery. For example, protein kinases are one of the largest classes of drug targets in the fight against cancer. The accurate determination of enzyme kinetics and mechanisms is a critical aspect of drug discovery research. To increase confidence in the selection of lead drug compounds it is crucial that pharmaceutical researchers have robust, affordable assays to measure enzyme activity accurately. We present a simple, sensitive microplate assay for real-time monitoring of a range of pharmaceutically important enzyme reactions that generate NPP anions, including kinases and glycosyltransferases. Our assay utilises a single, stable europium(III) complex that binds reversibly to NPP anions, signalling the dynamic changes in NPP product/substrate ratio during an enzyme reaction using time-resolved luminescence. This supramolecular approach to enzyme monitoring overcomes significant limitations in existing assays, obviating the need for expensive antibodies or equipment, chemically labelled substrates or products and isolation or purification steps. Our label and antibody-free method enables rapid and quantitative analysis of enzyme activities and inhibition, offering a potentially powerful tool for use in drug discovery, suitable for high-throughput screening of inhibitors and accurate measurements of enzyme kinetic parameters.

Graphical abstract: A simple, robust, universal assay for real-time enzyme monitoring by signalling changes in nucleoside phosphate anion concentration using a europium(iii)-based anion receptor

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Supplementary files

Article information


Submitted
29 Mar 2019
Accepted
24 Apr 2019
First published
01 May 2019

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2019,10, 5373-5381
Article type
Edge Article

A simple, robust, universal assay for real-time enzyme monitoring by signalling changes in nucleoside phosphate anion concentration using a europium(III)-based anion receptor

S. H. Hewitt, R. Ali, R. Mailhot, C. R. Antonen, C. A. Dodson and S. J. Butler, Chem. Sci., 2019, 10, 5373
DOI: 10.1039/C9SC01552C

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    [Original citation] - Published by The Royal Society of Chemistry.

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