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 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.

Fig. 1. (a) Structure of Eu(iii)-based phosphoanion receptor [Eu.1]⁺. (b) Cartoon illustrating real-time monitoring of the kinase-catalyzed conversion of ATP to ADP using [Eu.1]⁺. (c) Emission spectra of [Eu.1]⁺ (8 μM) in the presence of different phosphoanions (1 mM) and MgCl₂ (5 mM) in 10 mM HEPES, pH 7.0, λ_exc = 330 nm. (d) and (e) Titration of ADP (1 mM) into ATP (1 mM) in the presence of [Eu.1]⁺ (8 μM) and MgCl₂ (5 mM) showing the change in emission spectra (d) and the change in the emission intensity ratio at 616.5/599.5 nm as the mole fraction of ADP increases (e) in 10 mM HEPES, pH 7.0, λ_exc = 330 nm.

Fig. 2. Microplate-based real-time monitoring of a kinase reaction. (a) Cartoon depicting the use of [Eu.1]⁺ to monitor hexokinase. (b) Kinase simulation in standard assay conditions (1 mM ATP + ADP, 5 mM MgCl₂, 8 μM [Eu.1]⁺, 10 mM HEPES, pH 7.0), measuring the time-resolved luminescence intensity (λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs) of differing ratios of ADP/(ATP + ADP) (% ADP). (c) and (d) Real-time monitoring of different concentrations of hexokinase using the time-resolved luminescence intensity of [Eu.1]⁺ and calculation of initial rates (d). Conditions: 1 mM ATP, 5 mM MgCl₂, 10 mM glucose, 8 μM [Eu.1]⁺, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs.

Fig. 3. Real-time monitoring of Aurora A kinase reactions. (a) Enzyme reaction at different concentrations of Aurora A. (b) Initial AurA kinase (1 μM) reaction rate at different concentrations of the peptide substrate, kemptide, fitted to a Michaelis–Menten equation. (c) and (d) Inhibition of AurA (1 μM) by a range of known inhibitors measured in real-time (c), each showing a decrease in initial reaction rate (d, shown as %activity of the enzyme without inhibitor). (e) Titration of staurosporine into Aurora A (50 nM) reaction to derive an IC₅₀. Conditions: 1 mM ATP, 0.5 mM kemptide (except kemptide titration), 0.25 mM DTT, 5 mM MgCl₂, 8 μM [Eu.1]⁺, 2.5% glycerol, 50 mM NaCl, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs), 2.5% DMSO for inhibitor screen, 5% DMSO for staurosporine titration.

Fig. 4. Real-time monitoring of a glycosyl transferase reaction. (a) Cartoon depicting the monitoring of glycosyl transferase activity using [Eu.1]⁺ (b) LgtC simulation in standard assay conditions (1 mM UDP-galactose + UDP, 10 mM lactose, 2 mM MgCl₂, 8 μM [Eu.1]⁺, 0.02% Triton X-100, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm,, λ_em = 615–625 nm, integration time = 60–400 μs), measuring the time-resolved luminescence intensity of differing ratios of UDP/(UDP-galactose + UDP) (% UDP). (c) and (d) Real-time monitoring of the LgtC catalysed transfer of galactose from UDP-galactose to lactose, generating UDP at different concentrations of LgtC using the time-resolved luminescence intensity of [Eu.1]⁺ (c) and the calculation of the initial enzyme reaction rate (d). Conditions: 1 mM UDP-galactose, 2 mM MgCl₂, 10 mM lactose, 8 μM [Eu.1]⁺, 0.02% Triton X-100, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs.

Fig. 5. Real-time monitoring of phosphodiesterase activity. (a) Cartoon depicting the monitoring of cyclic nucleotide phosphodiesterase using [Eu.1]⁺, (b) and (c) Simulations of the phosphodiesterase reactions, cGMP to GMP (b) and cAMP to AMP (c), measuring the time-resolved luminescence intensity of [Eu.1]⁺ with increasing NMP/(cNMP + NMP) ratio (% NMP). Conditions: 1 mM cNMP + NMP, 5 mM MgCl₂, 8 μM [Eu.1]⁺, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs. (d)–(g) Real-time monitoring of the PDE-catalysed conversion of cGMP to GMP (d) and cAMP to AMP with calmodulin (CaM) activation (e), using the time-resolved luminescence intensity of [Eu.1]⁺, and comparing the change in initial rate on changing concentration of enzyme (f) and addition of calmodulin (g). Conditions: 1 mM cNMP, 5 mM MgCl₂, 0.03 mM CaCl₂ (d and e), 8 μM [Eu.1]⁺, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs.

Fig. 6. Real-time monitoring of sequential enzyme reactions, involving hexokinase (HK, ATP to ADP) and pyruvate kinase (PK, ADP to ATP). (a) Cartoon depicting sequence of enzyme monitoring using [Eu.1]⁺. (b) Real-time monitoring of hexokinase followed by pyruvate kinase using the time-resolved emission of [Eu.1]⁺. Conditions: 1 mM ATP, 1 mM glucose, 1 mM PEP, 5 mM MgCl₂, 50 mM KCl, 8 μM [Eu.1]⁺, 10 mM HEPES, pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs).