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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
journal contribution
posted on 2019-06-14, 12:31 authored by Sarah Hewitt, Rozee Ali, Romain Mailhot, Chloe R. Antonen, Charlotte A. Dodson, Stephen ButlerStephen Butler© 2019 The Royal Society of Chemistry. 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.
Funding
This work was supported by a Wellcome Trust Seed Award (204500/Z/16/Z) and a Royal Society Research Grant.
History
School
- Science
Department
- Chemistry
Published in
Chemical ScienceVolume
10Issue
20Pages
5373 - 5381Citation
HEWITT, S.H. ... et al., 2019. 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. Chemical Science, 10(20), pp. 5373 - 5381.Publisher
Royal Society of ChemistryVersion
- VoR (Version of Record)
Publisher statement
This work is made available according to the conditions of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) licence. Full details of this licence are available at: http://creativecommons.org/licenses/by/3.0/Acceptance date
2019-04-24Publication date
2019-05-01Copyright date
2019Notes
This is an Open Access Article. It is published by Royal Society of Chemistry under the Creative Commons Attribution 3.0 Unported Licence (CC BY). Full details of this licence are available at: http://creativecommons.org/licenses/by/3.0/ISSN
2041-6520eISSN
2041-6539Publisher version
Language
- en