A bioorthogonal turn-on fluorescent strategy for the detection of lysine acetyltransferase activity

Article abstract of DOI:10.1039/c8cc02987c

Lysine acetylation plays vital roles in the regulation of fundamental cellular processes, which is mediated by lysine acetyltransferases (KATs). Developing chemical biology probes for KAT activity detection is of important value in providing improved understanding of their biological functions. We reported a panel of "turn-on" fluorescent probes for sensitive and selective detection of KAT enzymatic activity through a simple mix-and-read format. Combined with bioorthogonal substrate labelling and click chemistry, these probes produced strong "turn-on" fluorescent signals in response to KAT-mediated acylation process. This chemical biology strategy diversifies the assay toolboxes to investigate functions and mechanisms of acetyltransferase enzymes.

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A Bioorthogonal Turn-On Fluorescent Strategy for the Detection
of Lysine Acetyltransferase Activity
Maomao He,a Zhen Han,a Jing Qiao,a Liza Ngo,a May P. Xiong,a Y. George Zheng*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Lysine acetylation plays vital roles in the regulation of assays suffer from issues such as high cost of radioactive
fundamental cellular processes, which is mediated by lysine materials, radioactive hazard concerns, and experimental
acetyltransferases (KATs). Developing chemical biology probes for inflexibility (i.e. discontinuous endpoint measurement). Non-
KAT activity detection is of important value in providing improved radioactive spectroscopic methods such as fluorescent assays,
understanding of their biological functions. We reported a panel have been explored as alternative strategies.7-9 However,
of “turn-on” fluorescent probes for sensitive and selective almost all fluorescent methods detect KAT activity by
detection of KAT enzymatic activity through a simple mix-and-read examining the formation of byproduct CoA through either
format. Combined with bioorthogonal substrate labelling and click enzyme or chemical reagent coupling. Drawbacks such as
chemistry, these probes produced strong “turn-on” fluorescent background fluorescence, inner filter effect, and incapability
signals in response to KAT-mediated acylation process. This for in vivo detection limit their biological applications.
chemical biology strategy diversifies the assay toolboxes to We recently explored the bioorthogonal strategy that utilizes
investigate functions and mechanisms of acetyltransferase synthetic acetyl-CoA analogs and engineered KAT enzymes to
enzymes.
provide a chemically maneuverable technology to interrogate
substrate profiles of KAT enzymes.10-12 As an extension of the
bioorthogonal labeling strategy, here we created a set of small
molecule nitrobenzoxadiazole (NBD) probes to achieve mix-
and-read, “turn-on” fluorescence detection of KAT activities.
The design takes advantage of the principles that: 1)
nucleophilic amino acid residues (lysine, arginine) abundantly
exist in histone and non-histone proteins; 2) acetyl-CoA
surrogates such as 3-azidopropanoyl CoA (3AZ-CoA) can be
excellent bioorthogonal cofactors of specific wild-type or
engineered KAT enzymes; and 3) oxygen-linked NBD unit (O-
NBD) is non-fluorescent, while nitrogen-linked NBD unit (N-
NBD) exhibits strong fluorescence in the wavelength range of
520-550 nm. As a potential fluorophore precursor, the NBD
unit has little interference on the ligand-target interaction due
to its small size and planar structure. The unique fluorescence-
switchable property of NBD makes it a good candidate for
probe design,13-15 which was recently applied to study KDAC
activities.14,15 We report here the first application of NBD-
based probes to detect KAT activity through integral alignment
of bioorthogonal acylation labeling, alkyne-azide click
chemistry, and “turn-on” fluorescence. As illustrated in
Scheme 1, upon KAT-catalyzed bioorthogonal acylation, 3-
azidopropanoyl group is transferred from 3AZ-CoA to the
substrate, which is further conjugated to an alkynyl-O-NBD
probe through click chemistry. This event would subsequently
Acetylation of lysine residues in proteins represents a pivotal
post-translational modification (PTM) conserved from
prokaryotes to yeasts and higher organisms. Lysine
acetyltransferases (KATs) and lysine deacetylases (KDACs) are
two types of enzymes with opposing activities that fine tune
the dynamic homeostasis of lysine acetylation.1 Since their
genetic identification in the mid-1990s, KATs have been
extensively studied in aspects of structural characterization,
biochemical properties, and functions in physiology and
pathology. Mounting evidence reveals that dysregulation of
KATs is causative to the pathogenesis of many human diseased
phenotypes,
inflammation, diabetes, and cancer.2,3
including
neurodegenerative
disorders,
Chemical biology probes that detect and quantify KAT
activities are essential tools for understanding the scope and
depth of acetyltransferase biology.4,5 Because of the
chemically inert nature of the acetyl group, direct detection of
KAT-medicated protein acetylation has been technically
challenging, which typically relies on either radioisotope
labeling or antibody recognition. The radiometric assays have
satisfying sensitivity and reliability.6 However, radiometric
trigger
a
proximity-promoted intramolecular nucleophilic
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substitution of O-NBD by free amines of adjacent lysine contrast, fluorescence signals of the control group without the
residues in the substrate and thus turns on the fluorescence of treatment of p300 stayed at a very low level. For PN-2, the
the resulting N-NBD motif.
fluorescence increase was slightly higher than that of PN-1
with the addition of p300, while remained unchanged for the
negative control. Likely, the PEG linker in PN-2 has a higher
degree of flexibility and hydrophilicity that helps to reduce the
nonspecific intermolecular N-substitution reactions.13-15
Scheme 1 Illustration of the bioorthogonal “turn-on” fluorescence strategy for KAT
activity detection.
Fig. 1 Synthetic route and click reaction time dependent spectral study of the O-NBD
probes. a) Synthesis of PN-1 and PN-2. b) Fluorescence change of PN-1 (5 μM) and PN-
2 (5 μM) with or without adding p300 as a function of click reaction time. H4(1-21)
As a proof of concept, we chose p300 as the KAT enzyme to
design the bioorthogonal “turn-on” fluorescent probes for KAT
activity detection. p300 was previously shown to utilize bulky
acetyl-CoA surrogates such as 3AZ-CoA to modify histone
substrates.10-12 Giving that the length of the linker in the NBD
probe may affect the efficiency of intramolecular N-
substitution reaction, two propargyl NBD probes (PN-1 and
PN-2) bearing different lengths of linkers were synthesized
peptide:
5 μM, 3AZ-CoA: 10 μM, p300: 0.2 ꢀM. λex = 485 nm. λem = 545nm. c)
Absorption spectra of PN-2 with and without adding p300. Enzymatic reaction time
was 30 min. click reaction time was 2h.
Absorption spectral changes of PN-2 upon KAT acylation
reaction were also measured. As seen in Fig 1c, the spectrum
of the reaction mixture containing PN-2 without the treatment
of p300 at enzymatic acylation stage showed a strong
absorption peak at 380 nm, which corresponded to the
maximum absorption wavelength of the O-NBD group.15 In
contrast, when the enzymatic reaction was performed in the
presence of p300, a new absorption peak appeared at 485 nm,
which corresponded to the maximum absorption peak of N-
NBD group. These experiments together demonstrated that
the fluorescence of NBD can be turned on as a function of click
reaction time only together with the occurrence of
bioorthogonal KAT labeling. We also tested the fluorescence
responses of PN-2 to the acylation of human H3 protein by
p300. As expected, the fluorescence spectra showed a steady
increase as a function of CuAAC/N-substitution reaction time
from NBD fluoride (NBD-F) (Fig. 1a and Schemes S1-
S2).14
Briefly, NBD-F was treated with propargyl alcohol or PTG-1
under basic condition to yield PN-1 and PN-2, respectively.
Before testing our designed assay strategy on KAT activity
detection, we first studied the fluorescent responses of PN-1
with the chemical components present in the KAT reaction and
the subsequent click reaction. As shown in Fig. S1a
-b, The
fluorescence intensity of PN-1 (10 ꢀM) is similar to NBD-F and
remained unchanged upon addition of either HEPES buffer (pH
8.0) or 3AZ-CoA, while addition of H4(1-21) substrate peptide
(N-terminal 21 residues of histone H4 protein, for full
sequence see the SI) brought to a slight increase of the
fluorescence intensity. In contrast, when the H4(1-21) peptide
was first reacted with 3AZ-CoA in the presence of p300, then
treated with PN-1 together with click reaction cocktail, a
dramatic increase of fluorescence signals was observed (Fig.
S1b). The results demonstrated that the fluorescence of PN-1
was selectively turned on only by reacting with the acylated
substrate. We then explored the “turn-on” fluorescent
responses with respect to the click/N-intramolecular
substitution reaction time. In the model study, H4(1-21) was
used as the KAT substrate, 3AZ-CoA as the cofactor, and p300
as the KAT enzyme. The KAT enzymatic reaction was allowed
to proceed under ambient temperature for 30 min and then
the click reaction cocktail in DMSO containing PN-1 was added.
The fluorescence signals were recorded during further
incubation at room temperature. As shown in Fig. 1b, the
fluorescence signals were weak initially, then increased
pronouncedly during incubation and plateaued at 2 h. In
(
Fig. S2).
To further demonstrate that the fluorescence enhancement
was caused by O-NBD to N-NBD transformation through
reacting with the adjacent amine groups in the substrate, we
prepared H4(1-21) peptide with a 3AZ group on its K16 lysine
residue [H4(1-21)K163AZ] and performed click reaction with
PN-2 at pH 2.0 (Fig. S3a). After click reaction (3 h), the pH was
adjusted to 8.0 to allow the intramolecular substitution
reaction to occur. The process was monitored by HPLC (Fig.
S3b). At pH 2.0, no further reaction occurred after the click
cycloaddition because amine residues on the substrate peptide
were protonated so that intramolecular N-substitution was
hindered. After increasing the pH to 8.0, however, amine
groups on the substrate were activated which led to the
nucleophilic attack on the O-NBD group. Indeed, two new
peaks formed on the HPLC chromatogram, which possessed
the same mass value (Fig. S3-S4). We also measured the
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absorption spectra of the reaction solution at pH 2.0 and 8.0. reaction to tag 3AZ-labeled proteins with PN-2. As shown in
Our observation showed that the maximum absorption Fig. 3, treatment of the cell lysate with 3AZ-CoA in the
wavelength shifted from 380 nm to 485 nm after pH presence of p300 and PN-2 resulted in strong fluorescence
adjustment (Fig. S3c), which corresponded to the maximum signals, indicating efficient labeling of protein substrates with
absorption wavelength of the O-NBD and N-NBD group, the 3AZ group (lane 1). In contrast, the absence of p300
respectively.15 By contrast, PN-2 at two different pH conditions resulted in very weak fluorescence signals that were close to
showed identical absorption spectrum (380 nm), which the negative control without PN-2 (lane 2 and 4). Similar result
demonstrated that the absorption spectral change was not was observed when without 3AZ-CoA treatment (lane 3).
caused by pH (Fig. S3d). These results together proved our Additionally, the fluorescence signals became modestly
hypothesized click chemistry/N-intramolecular substitution weaker in the presence of inhibitor C646 (5 ꢀM) (lane 5), and
two-step reaction mechanism. stronger when increasing the concentration of p300 (lane 6).
Next, we explored the suitability of PN-2 as a fluorescent The modest effect of C646 may attribute to the high
reporter to quantify KAT activity. We first tested the electrophilicity of pyrazolone-furan that lowers its KAT
fluorescence response of PN-2 corresponding to KAT reaction inhibitory activity in cell lysate mixtures.17 NBD-F was also
time. For the p300-catalyzed KAT reaction, the enzymatic tested as a replacement of PN-2 and as expected, no
process was quenched at different time points with the fluorescence was observed (lane 7). Combined, these data
addition of the click reaction cocktail containing PN-2. As support that the O-NBD group is essential for the “turn-on”
expected, the fluorescence signals displayed a linear increase fluorescence, and PN-2 can be used as a specific reporter to
with time over the course of 50 min, with a total of 3.2-fold visualize p300 catalyzed histone acylation in vitro. Analogously,
increase (Fig. S5a). The fluorescence signals in response to the fluorescence response of PN-1 towards HEK 293T cell
lysate acylation showed consistent “turn-on” fluorescence
a)
b)
results, though the fluorescence signals are slightly lower than
that of PN-2 Fig. S6). The lower labelling efficiency of PN-1
(
than PN-2 is consistent with the result shown in Fig. 1. Also
given that the labeling pattern of PN-1 in the in-gel
fluorescence imaging seems to be a bit different from PN-2
(
Fig. 3 vs Fig. S6), it is quite clear that the flexible and
Fig. 2 Turn-on fluorescence of p300 catalyzed acylation. a) p300 concentration
dependent fluorescence change of PN-2. PN-2: 5 μM, H4(1-21): 5 μM, 3AZ-CoA: 5
μM. Enzymatic reaction time was 30 min and “click ”reaction time was 3 h. b)
Measurement of C646 potency for p300 inhibition.
hydrophilic PEG linker in PN-2 plays a role in fine tuning the N-
substitution reaction of the NBD group with proximal amine
groups.
varying enzyme concentrations were also conducted. As p300
concentrations increased from 0 to 0.32
intensity increased linearly by 6.5-fold (Fig. 2a and Fig. S5b)
These results support that this ‘turn-on” fluorescent assay
offers quantitative approach to detect KAT catalyzed
ꢀM, the fluorescence
.
a
acylation reaction.
We also investigated the capability of PN-2 to quantify the
potency of p300 inhibitors by exposing p300 to a known
selective small-molecule KAT inhibitor, C646. Different
concentrations of C646 were pre-incubated with p300 for 30
min prior to the addition of H4(1-21) peptide and 3AZ-CoA.
The enzymatic reaction was allowed to occur for 1 h, then the
click reaction cocktail containing PN-2 was added to quench
the enzymatic reaction and initiate the click reaction at the
meantime. The dose-dependent fluorescence signals were
measured (Fig. 2b and Fig. S5c) and the data points were fitted
to the Hill equation (Equation S1). The IC50 value was
Fig. 3 In-gel fluorescence imaging of protein labelling in 293T cell lysate by PN-2.
Labelling condition: cell lysate 20 ꢀg, 3AZ-CoA 100 ꢀM, p300 1.2 ꢀM, PN-2 150 ꢀM. ++
double the concentration of p300. * NBD-F was used instead of PN-2.
Although with tremendous success in different areas of
chemistry, the cytotoxicity of copper, however, is a potential
disadvantage of the CuAAC reaction for biomedical
applications.20 Therefore, strain-promoted alkyne-azide
cycloaddition (SPAAC) reaction has been used as the
alternative for biocompatible labeling. With this idea in mind, a
strained O-NBD probe PN-3 was synthesized in one-step from
BCN-1 with moderate yield (25%) (Fig. 4a). PN-3 displayed low
calculated to be 1.8 ± 0.1 ꢀM, which was similar to the value
reported in the literature.16 These data clearly proved that the
detection platform is effective for quantitative KAT inhibitor
evaluations.
With the above encouraging results, we performed
fluorescence imaging experiments to examine the labeling
efficiency of 3AZ-CoA on the cellular proteome by PN-1 and
PN-2. Human embryonic kidney (HEK) 293T cell lysate was
incubated with p300 and 3AZ-CoA, followed by CuAAC click
background fluorescence signals that were similar to PN-1
, PN-
2
and NBD-F (Fig. S1a). In-gel fluorescence visualization was
conducted to examine the labeling efficiency of PN-3 in vitro
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Fig. 4b). Clearly, PN-3 exhibited specific selectivity towards
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Altogether, we designed smart O-NBD probes as “turn-on”
fluorescence reporters to achieve sensitive fluorescent
detection of p300 enzymatic activity. In combination with
bioorthogonal acylation, alkyne-azide click chemistry, and
proximity-promoted intramolecular substitution reaction, this
technological platform serves as an enabling technology for
the detection, quantification, and imaging of KAT activities.
While several fluorescent methods have been explored for KAT
study,7,8,22 the mix-and-read, “turn-on” fluorescence strategy
reported herein does not need enzymatic coupling or product
separation, and is free of washing procedure, thus greatly
reducing assay complexity and expanding throughput capacity.
We anticipate this activity-based chemical labeling strategy
will find great application in exploring and imaging activities of
acetyltransferases.
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Conflicts of Interest
There are no conflicts to declare.
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