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

Sarah Hewitt, Rozee Ali, Romain Mailhot, Chloe Antonen, Charlotte Dodson, Stephen Butler

Research output: Contribution to journalArticle

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.
LanguageEnglish
Pages5373-5381
Number of pages9
JournalChemical Science
Volume10
Issue number20
Early online date1 May 2019
DOIs
StatusPublished - 28 May 2019

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

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. / Hewitt, Sarah; Ali, Rozee; Mailhot, Romain; Antonen, Chloe; Dodson, Charlotte; Butler, Stephen.

In: Chemical Science, Vol. 10, No. 20, 28.05.2019, p. 5373-5381.

Research output: Contribution to journalArticle

@article{4e9baf1667bc48dcaa4bac80f0aa98be,
title = "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",
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 monitoringovercomes 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.",
author = "Sarah Hewitt and Rozee Ali and Romain Mailhot and Chloe Antonen and Charlotte Dodson and Stephen Butler",
year = "2019",
month = "5",
day = "28",
doi = "10.1039/C9SC01552C",
language = "English",
volume = "10",
pages = "5373--5381",
journal = "Chemical Science",
issn = "2041-6520",
publisher = "Royal Society of Chemistry",
number = "20",

}

TY - JOUR

T1 - 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

AU - Hewitt, Sarah

AU - Ali, Rozee

AU - Mailhot, Romain

AU - Antonen, Chloe

AU - Dodson, Charlotte

AU - Butler, Stephen

PY - 2019/5/28

Y1 - 2019/5/28

N2 - 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 monitoringovercomes 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.

AB - 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 monitoringovercomes 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.

UR - http://www.scopus.com/inward/record.url?scp=85066090269&partnerID=8YFLogxK

U2 - 10.1039/C9SC01552C

DO - 10.1039/C9SC01552C

M3 - Article

VL - 10

SP - 5373

EP - 5381

JO - Chemical Science

T2 - Chemical Science

JF - Chemical Science

SN - 2041-6520

IS - 20

ER -