Versatile small molecule kinase assay through real-time, ratiometric fluorescence changes based on a pyrene-DPA-Zn2+complex

Article abstract of DOI:10.1039/d1ra01547h

A real-time kinase assay method based on a ratiometric fluorescence probe that can be applied to various small-molecule kinases is described herein. The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence change. This property facilitates the monitoring of phosphorylation and dephosphorylation in small-molecule kinases, whereas most of the existing methods focus on one of these reactions. To prove the applicability of this method for small-molecule kinase assays, hexokinase and creatine kinase, which phosphorylate and dephosphorylate substrates, respectively, were analyzed. The ratiometric fluorescence change was correlated with the enzyme activity, and the inhibition efficiencies of the well-known inhibitors,N-benzoyl-d-glucosamine and iodoacetamide, were also monitored. Notably, the change in fluorescence can be observed with a simple light source by the naked eye.

Full text of DOI:10.1039/d1ra01547h

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Versatile small molecule kinase assay through real-
time, ratiometric fluorescence changes based on
a pyrene-DPA-Zn2+ complex†
Cite this: RSC Adv., 2021, 11, 10375
*
Jihoon Kim,‡ Jinyoung Oh‡ and Min Su Han
A real-time kinase assay method based on a ratiometric fluorescence probe that can be applied to various
small-molecule kinases is described herein. The probe can trace the reversible interchange of ATP and ADP,
which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence
change. This property facilitates the monitoring of phosphorylation and dephosphorylation in small-
molecule kinases, whereas most of the existing methods focus on one of these reactions. To prove the
applicability of this method for small-molecule kinase assays, hexokinase and creatine kinase, which
phosphorylate and dephosphorylate substrates, respectively, were analyzed. The ratiometric
fluorescence change was correlated with the enzyme activity, and the inhibition efficiencies of the well-
known inhibitors, N-benzoyl-D-glucosamine and iodoacetamide, were also monitored. Notably, the
change in fluorescence can be observed with a simple light source by the naked eye.
Received 26th February 2021
Accepted 4th March 2021
DOI: 10.1039/d1ra01547h
rsc.li/rsc-advances
crucial kinases.15–19 Thus, it remains necessary to develop
a small molecule kinase assay method that can be applied in
Introduction
real time and is based on a property shared by most small
molecule kinases, regardless of their substrate diversity.
In many small molecule kinases, a decrease in ATP, accom-
panied by an increase in ADP, commonly occurs, and this aspect
has received attention as a target for monitoring kinase
activity.20–25 However, some small molecule kinases have dis-
played an increase in ATP and a decrease in ADP for dephosphor-
ylation of the substrate, unlike those kinases using ATP as
a phosphate group donor. For example, creatine kinase (CK), which
plays an important role in cellular energy transformation, is an
example of a kinase that can perform reversible reactions. Mito-
chondrial CK catalyzes the transformation of creatine and ATP to
phosphocreatine and ADP, whereas the production of creatine and
ATP in the cytosol is caused by cytosolic CK to maintain energy
homeostasis.26,27 Therefore, to understand small molecule kinases
more precisely, it is necessary to monitor the reversible changes,
rather than the mono-directional transformation. For this reason, it
is proposed that real-time assay to trace the reversibly changed ATP
and ADP ratio, which is a common feature of small molecule
kinases, can be adopted as a general assay method that will be
applicable to various small molecule kinases.
Kinases mediate the transfer of a phosphate group to an
acceptor for phosphorylation or dephosphorylation of various
substrates. This enzymatic reaction is related to a number of
biological processes, including metabolism, transcription, cell
cycle regulation, and apoptosis.1–4 Because the dysregulation of
kinase enzymes can cause various human diseases, the devel-
opment of kinase assay methods and inhibitors for each kinase
plays a critical role in the diagnosis and therapy of these
diseases.5–10 Although many kinase assay methods have been
reported for the rapid detection of enzyme malfunctions and
inhibitor screening, most of them have focused on protein
kinases, despite the importance of small molecule kinases,
which utilize small molecules as their substrates. Many protein
kinase assay methods have been established by using the
characteristic properties of the protein substrates, and the
dynamic range of the substrates was also suitable for
proteins.11–14 For specic small molecule kinase assays, exam-
ples have been developed by using nanoparticles, and coupled
enzymes. Unfortunately, the enzyme assays showed obvious
drawbacks in their end-point measurement and complex
cascade enzyme reaction. Most of them also attempted to target
unique structures of substrates that were unsuited for other
Ratiometric uorescence probes have received attention
because they allow for ratio change between emission bands at
distinct wavelength and built-in correction to reduce environ-
mental interference and improve sensitivity and accuracy,
permitting the development of detection methods for various
targets.28–30 In particular, certain ratiometric uorescence
probes have been introduced as a method for tracing reversibly
changed analytes based on the signicant property that the change
Department of Chemistry, Gwangju Institute of Science and Technology (GIST), 123
Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Republic of Korea. E-mail: happyhan@
gist.ac.kr
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra01547h
‡ Both authors contributed equally.
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of analytes can be monitored directly by the change of uorescence
emissions.31,32 For instance, the real-time interchange of bisulfate
and hydrogen peroxide in a biological redox cycle was monitored
by a ratiometric change of uorescence emission.32 Considering
the properties of ratiometric uorescence probes, they could be
effective for detecting bidirectional changes between ATP and ADP
in a kinase reaction, and thus, they could become an appropriate
real-time assay platform for various small molecule kinases asso-
ciated with phosphorylation or dephosphorylation.
In this study, a general method for a real-time small mole-
cule kinase uorescence assay was proposed. To develop the assay,
a pyrene-dipicolylamine (DPA)-Zn2+ complex was prepared because
this probe allowed discrimination between ATP and ADP with
distinct ratiometric uorescence emissions.33,34 It was speculated
that a ratiometric uorescence change in the pyrene-DPA-Zn2+
complex would occur in conjunction with the interchange of ATP
and ADP, representing the real-time progression of the small
molecule kinase reaction (Scheme 1). In addition, the reversible
uorescence change of the pyrene-DPA-Zn2+ complex would facil-
itate monitoring of the dephosphorylation as well as phosphory-
lation of the substrate by various small molecule kinases. As proof
of this concept, the activity of two small molecule kinases, namely
hexokinase (HK) to phosphorylate glucose and CK to dephos-
phorylate phosphocreatine, were analyzed, and inhibitor analyses
for the enzymes were also demonstrated.
Synthesis of 1-pyrenebutanol (1). 1-Pyrenebutyric acid
(1.00 g, 3.45 mmol) in anhydrous THF (22 mL) was stirred at
0 C. To the solution lithium aluminum hydride (1.25 g, 32.5
ꢀ
mmol) dissolved in anhydrous THF (32.5 mL) was added
dropwise slowly and stirred for 30 min. Aer then, the solution
was reuxed for 23 h and quenched through Fieser method. The
residue was evaporated and puried via silica gel column
chromatography (MeOH : CHCl3 ¼ 1 : 99) to give a pale yellow
1
solid (0.64 g, 68%). H-NMR (400 MHz, CDCl3) d 8.27 (d, J ¼
9.2 Hz, 1H), 8.16 (d, J ¼ 2.1 Hz, 1H), 8.14 (d, J ¼ 2.4 Hz, 1H),
8.11–8.08 (m, 2H), 8.02–7.96 (m, 3H), 7.86 (d, J ¼ 7.9 Hz, 1H),
3.72–3.68 (m, 2H), 3.37 (t, J ¼ 7.8 Hz, 2H), 1.97–1.90 (m, 2H),
1.77–1.70 (m, 2H) 1.25 (s, 1H).
Synthesis of 1-(4-bromobutyl)pyrene (2). To 4-pyrenyl-1-
butanol (0.500 mg, 1.82 mmol) solution in anhydrous DCM
(30 mL) was added CBr4 (1.212 g, 3.64 mmol), and K2CO3
(0.504 g, 2.73 mmol) at 0 ꢀC. PPh3 (0.716 mg, 2.73 mmol) in
anhydrous DCM (20 mL) was added dropwise to the solution
and stirred at room temperature for 4 h. Aer the reaction
mixture was concentrated under reduced pressure, the residue
was dissolved with EA and washed with water, brine, and dried
on anhydrous Na2SO4, and concentrated under reduced pres-
sure. The residue was puried by silica gel column chroma-
tography (Hex : DCM ¼ 7 : 1) to give a white solid (0.506 g, 82%).
1H-NMR (400 MHz, CDCl3) d 8.27 (d, J ¼ 9.2 Hz, 1H), 8.18–8.11
(m, 4H), 8.05–7.97 (m, 3H), 7.86 (d, J ¼ 7.9 Hz, 1H), 3.49–3.46
(m, 2H), 3.40–3.36 (m, 2H), 2.05–2.02 (m, 4H).
Synthesis of [(2,20-dipicolylamino)methyl]pyrene (3). To 1-(4-
bromobutyl)pyrene (0.100 g, 0.297 mmol) solution in ACN, 2,20-
dipicolylamine (0.071 g, 0.36 mmol), and K2CO3 (0.147 g, 1.07
mmol) was added and reuxed for 28 h. Aer evaporating
solvent under reduced pressure, the residue was diluted with
water and extracted with DCM. The collected organic layer was
concentrated and puried by silica gel column chromatography
(EA) to give a pale yellow oil (0.093 g, 69%). 1H-NMR (400 MHz,
CDCl3) d 8.49 (d, J ¼ 4.6 Hz, 2H), 8.22–8.14 (m, 3H), 8.08–7.95
(m, 5H), 7.78 (d, J ¼ 7.6 Hz, 1H), 7.55 (td, J ¼ 7.6, 1.8 Hz, 2H),
7.46 (d, J ¼ 7.6 Hz, 2H), 7.08 (ddd, J ¼ 7.2, 5.0, 1.1 Hz, 2H), 3.79
(s, 4H), 3.26 (t, J ¼ 7.6 Hz, 2H), 2.63 (t, J ¼ 7.2 Hz, 2H), 1.88–1.80
(m, 2H), 1.74–1.67 (m, 2H).
Experimental
Materials and instruments
Chemical reagents were purchased from commercial sources
and, unless otherwise stated, were used without further puri-
cation. For enzyme assay, hexokinase (HK, EC 2.7.1.1) and
creatine kinase (CK, EC 2.7.3.2) were purchased from Sigma
Aldrich. Florescence spectra were recorded on an Agilent Cary
Eclipse uorescence spectrometer using a 1 cm path length
1
quartz cell. H NMR spectrums were recorded on a JEOL 400
MHz NMR spectrometer.
Synthesis
In situ preparation of pyrene-DPA-Zn2+ complex. Pyrene-
DPA-Zn2+ complex was generated in situ by addition of
Zn(ClO4)2 to the buffer solution (HEPES, 20 mM, pH 7.4)
containing compound 3. Without further incubation of the
solution, other components were also added to the solution,
The pyrene-DPA-Zn2+ complex was prepared following as
Scheme 2.
Scheme 1 Schematic illustration of the real-time assay method for
various small molecule kinases based on a ratiometric fluorescence Scheme
2
Synthetic route for preparation of pyrene-DPA-Zn2+
probe.
complex.
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and uorescence spectra were recorded with excitation at
341 nm.
Fluorescence measurement
Model study for real-time uorescence kinase assay. To
buffer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-
Zn2+ complex (20 mM), different ratio of ATP and ADP (total 10
mM) were in 1 mL of volume. Emission spectra (lex ¼ 341 nm)
were recorded using uorescence spectrometer.
Monitoring phosphorylation in a kinase reaction. To buffer
solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-Zn2+
complex (20 mM), ATP (10 mM), MgCl2 (80 mM), and glucose (1
mM), various concentrations (0 to 1 unit) of HK were added. The
uorescence intensity was measured at 1 min interval for
30 min with excitation at 341 nm. The observed rate (kobs) for
HK was estimated using initial change of uorescence ratio of
F376 and F476 from 0 to 5 min.
Kinetic study of hexokinase for glucose. Various concentra-
tions of glucose (from 0 to 1.4 mM) was added to the buffer
solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-Zn2+
complex (20 mM), ATP (10 mM), MgCl2 (80 mM). Aer addition of
HK (1 unit), the uorescence intensity was measured at 1 min
interval for 10 min with excitation at 341 nm. The Michaelis–
Menten constant (Km), maximum initial velocity (Vmax) as the
enzymatic parameters were estimated from Lineweaver–Burk
plots and the values were compared with reported results.
Monitoring dephosphorylation in a kinase reaction. To
buffer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-
Zn2+ complex (20 mM), ADP (10 mM), MgCl2 (80 mM), and
phosphocreatine (0.75 mM), various concentrations (0 to 1 unit)
of CK were added. The uorescence intensity was measured at
1 min interval for 15 min with excitation at 341 nm. The
observed rate (kobs) for HK was estimated using initial change of
uorescence ratio of F476 and F376 from 0 to 4 min.
Fig. 1 (a) Fluorescence spectra of pyrene-DPA-Zn2+ complex with
various ratios of ADP and ATP in buffer solution (HEPES, 20 mM, pH
7.4); (b) fluorescence intensity ratio of monomer (F376) and excimer
(F476) emission against the ratio of ADP and ATP. [pyrene-DPA-Zn2+
complex] ¼ 20 mM, [ADP] + [ATP] ¼ 10 mM, lex ¼ 341 nm.
added concentration, whereas the excimer emission at 476 nm
increased gradually as the concentration of ATP increased without
a signicant change in the monomer emission (Fig. S1†). As
a result, ATP and ADP clearly had different effects on the pyrene-
DPA-Zn2+ complex and induced two distinct pyrene complexes, as
represented by the diverse emission spectra. In fact, it is postulated
that the kinase reaction induces an interchange of ATP and ADP,
not the uctuation of each molecule, which may be the result of an
increase in the ADP/ATP ratio for phosphorylation or decrease in
the ratio for dephosphorylation. Therefore, the uorescence
change following a change in the ADP/ATP ratio was evaluated as
a model study for monitoring kinase reactions. As shown in Fig. 1,
a sequential change of the ADP/ATP ratio transformed the overall
spectra, with an increase or decrease in the excimer emission
accompanied by the opposite change in the monomer emission.
This means that the interchange of ADP and ATP as a progression
of the kinase reaction can be monitored in real time from the
ratiometric uorescence change.
Monitoring phosphorylation in a kinase reaction and its
kinetic study
Inhibitor assay for the kinases. To buffer solution (HEPES,
20 mM, pH 7.4) containing pyrene-DPA-Zn2+ complex (20 mM),
ATP (10 mM), MgCl2 (80 mM), and glucose (1 mM), a various
concentration of N-benzoyl-D-glucosamine (0 to 4 mM), and
without incubation HK (1 unit) was added. Inhibition efficiency
was obtained by the change of excimer and monomer emission
ratio at 20 min aer the measurement.
To apply the system for assaying a biological kinase that phos-
phorylates its substrates, HK was analyzed as a model enzyme.
HK, which plays an important role in the rst step of glycolysis
and has been well studied because of its relationship with many
diseases, transfers a phosphate group from ATP to the substrate
To buffer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-
Zn2+ complex (20 mM), ADP (10 mM), MgCl2 (80 mM), and phospho-
creatine (0.75 mM), mixture with various concentrations of iodoace-
tamide (0 to 100 mM) and CK (1 unit per mL) incubated for 10 min was
added. Inhibition efficiency was obtained by the change of excimer
and monomer emission ratio at 15 min aer the measurement.
Results and discussion
Model study for real-time uorescence kinase assay
Fig. 2 (a) Enzymatic reaction formula of HK associated with phos-
phorylation; (b) fluorescence intensity ratios of monomer (F376) and
excimer (F476) emissions over time for the pyrene-DPA-Zn2+ complex
with various concentrations of HK (0 to 1 unit) in buffer solution
(HEPES, 20 mM, pH 7.4); (c) plot of the logarithm of kobs against
concentration of HK. [pyrene-DPA-Zn2+] ¼ 20 mM, [ATP] ¼ 10 mM,
To evaluate the inuence on the pyrene-DPA-Zn2+ complex of ATP
and ADP, which are common components for general kinase
reactions, the uorescence changes with ATP and ADP were
measured. Addition of ADP to the pyrene-DPA-Zn2+ complex
increased the monomer emission at 376 nm in proportion to the [Mg2+] ¼ 80 mM, [glucose] ¼ 1 mM, lex ¼ 341 nm.
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glucose, thereby increasing the ADP/ATP ratio (Fig. 2a).35–37 As
Most typical kinase assays except for recently reported
the HK reaction proceeded and the ADP/ATP ratio increased research, which assayed both hexokinase and pyruvate kinase
gradually, the excimer emission at 476 nm gradually decreased as a sequential monitoring,31 have focused on the analysis of
with a concomitant increase in monomer emission at 376 nm, only mono-directional changes, either phosphorylation or
in conjunction with an increase in the concentration of HK dephosphorylation, although the methods were based on
from 0 to 1 unit (Fig. S2†); the monomer/excimer emission ratio changes in ATP and ADP and then extended for several
changed in proportion to the concentration of HK during the kinases.17,20 However, considering the reversibility of reactions
reaction time (Fig. 2b). In addition, the logarithm of observed exhibited by kinases, it is necessary for a general kinase assay to
rate constant (kobs) for the enzyme reaction measured by using analyze the bidirectional changes between ATP and ADP that
the ratiometric uorescence change showed a linear correlation indicate both phosphorylation and dephosphorylation. The
with the concentration of HK (Fig. 2c) and enzymatic parame- developed assay method in this study provided evidence for the
ters (Km, Vmax) obtained from the method showed little differ- effective utilization of bidirectional changes in kinase reactions,
ence with reported value (Fig. S3, Table S1†), allowing and consequently, the results are suggested as a general method
quantication and kinetic study of the enzyme from the uo- for versatile small molecule kinase assays.
rescence change. As a result, it was conrmed that the HK
activity involved in phosphorylation can be estimated from the
developed assay system in real time by tracking the ADP/ATP
Inhibitor assay for the kinases
The fact that most kinases play an essential role in biological
processes makes the development of inhibitors crucial. To
demonstrate that the suggested kinase assay method can be used
for inhibitor assays, the efficiency of inhibitors for both HK and CK
was measured by using the change in ratiometric uorescence.
The inhibition efficiencies of well-known inhibitors of HK and CK,
N-benzoyl-D-glucosamine (NBG) and iodoacetamide (IAA), respec-
tively, were conrmed against an increased concentration of
inhibitor (Fig. 4). The half maximal inhibitory concentration (IC50)
of the inhibitors estimated with the developed assay system was
compared with the reported values (Table S2†). The results corre-
lated without any large differences, indicating that the system was
applicable as an inhibitor assay.38,39
In addition, rapid and simple screening methods for
multiple inhibitor candidates are required in the pharmaceu-
tical eld. To achieve this purpose, the microwell plates were
photographed upon irradiation with a handheld UV lamp to
record the uorescence changes for the enzyme activity and
inhibitor efficiency. As shown in Fig. 4c, during
ratio change, rather than the enzyme substrate, glucose.
Monitoring dephosphorylation in a kinase reaction
Although most kinases are involved in phosphorylation of
substrates, some are used for dephosphorylation, with transfer
of the phosphate group from the substrate to ADP. To establish
a more versatile kinase assay method, it is also crucial to analyze
the kinases associated with dephosphorylation. Herein, CK, which
transfers a phosphate group from phosphocreatine to ADP, was
exploited as an example enzyme to show the method's application
for the dephosphorylation process (Fig. 3a).26 Progression of the
CK reaction caused a decrease in the ADP/ATP ratio, the opposite
result to that with HK, which could be inferred from the decreased
monomer emission at 376 nm and increased excimer emission at
476 nm (Fig. S4†). The change in ratiometric uorescence was
proportional to the concentration of CK from 0 to 1 unit, and the
rate constant, which was estimated from the uorescence change,
displayed a linear correlation with the concentration of CK (Fig. 3b
and c). Therefore, the developed kinase assay system can be
applied to analyze dephosphorylation as well as phosphorylation
in real time without additional assistance, such as a modied
substrate or cascade enzyme reaction.
Fig.
3
(a) Enzymatic reaction formula of CK associated with Fig.
4 Inhibition efficiency for (a) HK and (b) CK with various
dephosphorylation; (b) fluorescence intensity ratios of excimer (F476
)
concentrations of NBG and IAA, respectively, in buffer solution (HEPES,
and monomer (F376) emissions over time for the pyrene-DPA-Zn2+ 20 mM, pH 7.4) with each substrate; (c) photographs of the pyrene-
complex with various concentrations of CK (0 to 1 unit per mL) in DPA-Zn2+ complex in the presence of kinases and inhibitors. [pyrene-
buffer solution (HEPES, 20 mM, pH 7.4); (c) plot of kobs against DPA-Zn2+ complex] ¼ 20 mM, [ATP] or [ADP] ¼ 10 mM, [Mg2+] ¼ 80 mM,
concentration of CK. [pyrene-DPA-Zn2+ complex] ¼ 20 mM, [ADP] ¼ [glucose] ¼ 1 mM, [phosphocreatine] ¼ 0.75 mM, [HK] ¼ 1 unit, [NBG]
10 mM, [Mg2+] ¼ 80 mM, [phosphocreatine] ¼ 0.75 mM, lex ¼ 341 nm. ¼ 5 mM, [CK] ¼ 1 unit per mL, [IAA] ¼ 100 mM, lex ¼ 341 nm.
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phosphorylation, the excimer emission observed in the pres-
ence of ATP was changed to monomer emission by HK activity,
and the inhibition of enzyme activity by NBG allowed the probe
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