Discovery of a butyrylcholinesterase-specific probe via a structure-based design strategy

Article abstract of DOI:10.1039/c7cc00577f

We report herein the structure-based design and application of a fluorogenic molecular probe (BChE-FP) specific to butyrylcholinesterase (BChE). This probe was rationally designed by mimicking the native substrate and optimized stepwise by manipulating the steric feature and the reactivity of the designed probe targeting the structural difference of the active pockets of BChE and AChE. The refined probe, BChE-FP, exhibits high specificity toward BChE compared to AChE, producing about 275-fold greater fluorescence enhancement upon the catalysis by BChE. Thus, BChE-FP is a specific BChE probe identified by the structure-based design and it can discriminate BChE from AChE. Furthermore, it has been successfully applied for imaging the endogenous BChE in living cells, as well as BChE inhibitor screening and characterization under physiological conditions.

Full text of DOI:10.1039/c7cc00577f

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Discovery of A Butyrylcholinesterase-specific Probe via Structure-
based Design Strategy
Shu-Hou Yang,ac Qi Sun,ac Hao Xiong,a Shi-Yu Liu,a Behrooz Moosavi,a Wen-Chao Yang,a* and
Guang-Fu Yangab*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report herein the structure-based design and application of a cholinesterases, or the direct product which could be further
fluorogenic molecular probe (BChE-FP) specific to converted into a colored product with a thiol-capping agent
butyrylcholinesterase (BChE). This probe was rationally designed such as Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid),
by mimicking the native substrate and optimized stepwise by DTNB); 2) fluorometric assays with fluorogenic substrates or
manipulating the steric feature and the reactivity of the designed indirect detection that yields fluorescence enhancement; 3)
3
probe targeting the structural difference of the active pocket of radiometric assays with 14C or H labeled substrates such as
BChE and AChE. The refined probe, BChE-FP, exhibits a high acetylcholine; 4) calorimetric assays with natural substrates by
specificity toward BChE compared to AChE, producing about 275- isothermal titration calorimetry.4 The radiometric and
fold higher fluorescence enhancement upon the catalysis by BChE. calorimetric methods are accurate and are based on direct
Thus, BChE-FP is a specific BChE probe identified by structure- measurement, but not suitable for high-throughput analysis;
based design and it can discriminate BChE from AChE. the common UV/Vis and fluorometric methods are fast,
Furthermore, it has been successfully applied for imaging the sensitive and compatible with high-throughput experiments,
endogenous BChE in living cells, as well as the BChE inhibitor but both mainly utilize a second reaction to generate the
screening and characterization under physiological conditions.
optical signal. Recently, extensive studies have been carried
out to develop new fluorescent probes for assessment of the
cholinesterases activity, but only in few of them fluorophore-
based probes were developed for direct cholinesterases
activity evaluation in a one-step reaction.5 Most importantly,
very few reported fluorescent probe6 could discriminate BChE
from AChE, due to the substantially similar biochemical
properties of both cholinesterases. Therefore, development of
a sensitive and specific probe for assessment of BChE activity
and inhibitors screening is highly desirable; a crucial issue that
we sought to solve in the current study.
Acetylcholinesterase (AChE; EC 3. 1.1.7)
and
butyrylcholinesterase (BChE; EC 3. 1.1.8) are two major human
esterases and the focus of rigorous studies in pharmacology
and neurobiology, due to their significant roles in human
physiology and health.1 The BChE have been implicated in lipid
metabolism and various human diseases such as liver damage,
diabetes, Alzheimer’s disease (AD) and liver metastasis.2 BChE
is also responsible for detoxifying xenobiotics such as
organophosphates and cocaine, and its engineered mutants
have drawn
a great deal of interest as detoxifying
We analyzed the catalytical mechanism7 especially the
difference on the active sites of BChE (PDB entry: 1P0M) and
AChE (PDB entry: 1B41) (Fig. 1)8. Inspired by the previous
studies on the development of fluorogenic probes targeting
the esterases in vitro and in vivo,9 we designed the probes (Fig.
2) by mimicking the native substrate and optimized stepwise
by manipulating the steric feature and the reactivity of the
designed probes targeting the structural difference of the
active pockets of BChE and AChE. After optimization and
comprehensive characterization, the refined BChE probe
(termed as BChE-FP) showed good properties as follows: 1) it
exhibits good specificity and could discriminate BChE from
AChE; 2) about 275-fold fluorescence intensity (FI)
enhancement in pure aqueous solution; and 3) about 100-fold
higher binding affinity against BChE and comparable catalytic
therapeutics.3 Thus, the quantification of BChE activity and its
inhibition are highly important in drug discovery and clinical
diagnostics.
To date, four major methods have been developed for
assessment of the activity of cholinesterases as follows: 1)
UV/Vis assays with direct chromogenic reaction catalyzed by
a. Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College
of Chemistry, Central China Normal University, Wuhan 430079, P. R. China. E-
mail: tomyang@mail.ccnu.edu.cn, gfyang@mail.ccnu.edu.cn
b. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin
30071, P.R. China.
c. They contributed equally to this work.
†Electronic Supplementary Information (ESI) available: experiment section, UV-Vis
absorption, fluorescence emission, theoretical calculation, 1H NMR, 13C NMR and
MS spectra of all the new compounds were available in Supporting Information.
See DOI: 10.1039/x0xx00000x
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Fig. 1. (A) Superposition of the crystal structures of human BChE
(green) and AChE (orange), their active sites were displayed in
surface format. (B) The sectional view of active sites for BChE (green)
and AChE (orange). Two aromatic residues (Tyr124 and Phe337)
locating in the middle of AChE active site were indicated,
respectively.
Fig. 2 Chemical structures of the designed probes, series I and II.
efficiency when comparing with the widely used substrate
acetylthiocholine Iodide (ATC).
Starting from the natural substrate, butyrylcholine, the butyryl
group was retained, and several fluorophores with different bulky
size were introduced to construct the first series of probes I1-5 in
Fig. 2. All the probes of this series were fully characterized (See
Schemes S1-S5 and Fig. S1-S9). The time-dependent FI changes of
each reaction system, defined as the relative reaction rates (ΔF/Δt),
were measured and listed in Table S1, applying the most wildly used
natural substrate analog acetylthiocholine (ATC) as a reference. The
screening results indicated that FI enhancement of probe I5 was
about 250-fold for BChE detection. This result suggests that
carboxyfluorescein is the preferred fluorophore for BChE probe
design. Therefore, we reserved the carboxyfluorescein and built the
second set of probes to further optimize the butyryl group of probe
I5. Considering the structural diversity of recognition group
having different steric effect, various substituents were utilized
to establish the second series of probes (II1-10, Scheme S5 and
Fig. 3 (A) The time-dependent fluorescence (λex = 455 nm) of BChE-
FP (10 μM) in phosphate buffer (100 mM, pH 7.0) in the presence of
BChE (0.02 U/mL) at 30 °C; Inset shows the photos of the
corresponding reaction mixture in the absence and presence of
BChE after 30 min incubation at 30 °C under UV light (365 nm)
illumination. (B) HRMS spectrum of BChE-FP (20 µM) incubated
with BChE (0.02 U/mL) for 30 min in the same buffer at 30 °C.
and 0.02 U/mL BChE) was analyzed by both HRMS (Fig. 3B) and
HPLC (Fig. S33) methods, with similar reaction system
containing no BChE as the negative control, which indicated
that the fluorescence enhancement was the result of BChE-
catalyzed hydrolysis of BChE-FP at the ester bond.
Fig. S10-S29), including short chain alkyl and cycloalkyl
substituents. Based on the screening results in Fig. S30 and
Table S1, probe II4 with the highest relative reaction rates and
about 14.5-fold selectivity, termed as BChE-FP, was finally
chosen for the following studies.
To uncover the specificity of this refined probe, the
hydrolysis of BChE-FP was examined under the same
conditions in the presence of various analytes, including metal
ions (K+, Na+, Mg2+, Ca2+, Mn2+), amino acids and biothiols (Arg,
GSH, Phe, His). The data presented in Fig. S34 shows that this
molecular probe is highly selective for BChE over competing
amino acids, metal ions, and thiols. We also tested the
reactivity of BChE-FP with other common enzymes or proteins
(elastase, AChE, BChE, trypsin, chymotrypsin, phospholipase,
alkaline phosphatase (ALP) and BSA). The results are
summarized in Fig 4. We also tried the specificity study in
Krebs buffer and no obvious interference was observed. It
clearly demonstrated the relative reaction rate of BChE-
The pH profile of the hydrolysis of BChE-FP catalyzed by
BChE was firstly investigated, and the relative reaction rate
(ΔF/Δt) was accelerated by increasing the pH ranging from 4.5
to 9.0 (See Fig S31-32). Considering that the physiological
condition is neutral, the primary study was carried out at pH
7.0. Under this neutral pH, the time-dependence of the FI
enhancement for BChE-FP was observed upon the addition of
BChE, and the maximum FI at 515 nm indicated around 275-
fold increase over the basal level (Fig. 3A). The data showed
that BChE-FP could be considered a sensitive fluorescence
“off-on” system for sensing BChE activity. In addition, the
reaction mixture (100 mM phosphate buffer, 20 µM BChE-FP
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of BChE in processing BChE-FP is about 34.80 µM•min-1. In this
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study, we also tested the Km (308.07 D±OI:61.04.7103µ9M/C)7CaCn0d057k7cFat
(20095 ± 143.65 min−1) of BChE against ATC with Ellman's
method under the same condition. Based on the kinetic
parameters, the binding affinity of BChE-FP with BChE is ~100-
fold higher than that of ATC, while the catalytic constant for
BChE-FP is ~190-fold lower than that of ATC. Taken together,
the overall catalytic efficiency of BChE against BChE-FP (~34.80
µM-1• min-1) is quite comparable to that of ATC (~65.24 µM-1•
min-1). These kinetic analyses imply that BChE-FP can be a
suitable substrate for BChE activity assessment.
Fig. 4 The specificity profile of BChE-FP (10 µM) toward different
hydrolases or proteins (elastase, AChE, BChE, trypsin, BSA,
phospholipase, ALP and chymotrypsin).
Next, the BChE inhibition study was carried out using the
turn-on fluorogenic system with BChE-FP. Tacrine, a well-
known inhibitor of cholinesterases, was selected as the
reference. All the reaction systems were incubated in the 96-
well plates at 30 °C and the fluorescence at 515 nm was
continually monitored for 5 min. The FI of the samples
composed of BChE-FP (10 µM), BChE (0.02 U/mL) and different
amounts of tacrine (0.1 to 20 µM) were monitored in different
reaction time points. As expected, the FI linearly increased by
prolonging the reaction time, but the speed of the
fluorescence enhancement reduced gradually when the
concentration of tacrine increased (see Fig. S36). The Dixon
plot for inhibitory kinetics of tacrine was then obtained from
the above time-dependent profile (Fig 5B). The average values
of the Ki of tacrine toward BChE were estimated to be 10.69 ±
3.27 nM by using our assay, which is close to that obtained
with Ellman’s method (8.70 ± 0.17 nM).10 The above results
demonstrated that BChE-FP has a great potential to be used as
a reliable fluorogenic substrate for discovering BChE inhibitors
in moderate conditions.
Fig. 5 (A) Michaelis-Menten curve of BChE-FP catalyzed by
BChE (0.02 U/mL) in phosphate buffer (100 mM, pH 7.0) with
fluorometric method. (B) Dixon plot of inhibitory kinetics of
Tacrine with BChE-FP (5 µM, 10 µM) as the substrate.
catalized hydrolysis for BChE-FP is muchhigher than that of
other hydrolases and proteins, indicating that BChE-FP is
particularly selective toward BChE. Most importantly, BChE-FP
displayed over 14-fold better specificity to BChE compared to
AChE, and the AChE mediated-hydrolysis of BChE-FP was
To disclose the sensing capability of BChE-FP in cellular
comparable to the spontaneous hydrolysis that could be environment, we further applied BChE-FP to image the
neglected. In addition, the molecular docking simulation endogenous BChE in PANC-1 cells. We checked the cytotoxicity
revealed that the proximity of Ser-198 to the carbonyl group of of BChE-FP against this PANC-1 cells and found no significant
BChE-FP was about 4.3 Å in BChE active site, whereas the cytotoxicity up to 50 µM for 24 hours (See Fig S37).
proximity of Ser-203 to the carbonyl group of BChE-FP was Furthermore no fluorescence was observed without any
about 7.4 Å in AChE active site (Fig. S35). Hence, the binding of treatment (data not shown). After incubating the cells with
BChE-FP in BChE active site is much more favourable to the BChE-FP (20 µM) for 20 min, the bright green fluorescence
Ser-triggered nucleophilic attack than that in AChE active site, appeared, and the co-staining with Hoechst 33342 revealed
which provides plausible evidence for the superior specificity that the endogenous BChE was located in the cytoplasm of
to BChE. To the best of our knowledge, BChE-FP is the first PANC-1 cells (Fig. 6A-6D). Intriguingly, once the cells were pre-
selective probe that can discriminate BChE from AChE.
treated with the well- known inhibitor tacrine (50 µM) before
the addition of the probe BChE-FP (20 µM), the green
fluorescence signal was significantly diminished (Fig. 6E-6H). In
addition, we conducted the flow cytometry analysis of PANC-1
cells exposed to different concentrations of BChE-FP (0 µM to
20 µM) to obtain the quantification result of imaging without
and with the preincubation of cholinesterase inhibitor. It
turned out that tacrine had similar inhibition potency against
BChE and AChE, while the donepezil was a specific AChE
inhibitor. Based on the original data (See Fig S38), it was easily
observed that the fluorescence signal enhanced with the
increased concentration of BChE-FP without addition of any
inhibitor. More interestingly, the preincubation with tacrine
significantly decreases the FI, whereas the FI of the same cells
To further elucidate the applicability of BChE-FP as a
substrate for evaluation of BChE activity, the kinetic properties
of BChE-catalyzed hydrolysis of BChE-FP were investigated.
According to the measurement of the FI change at the
emission of 515 nm, the initial reaction rates for the enzymatic
hydrolysis of BChE-FP were obtained at various concentrations
of BChE-FP (0-50 µM). As anticipated, the plot of V versus S
obeyed the Michaelis–Menten equation (shown in Fig. 5A). On
the basis of the Michaelis–Menten curves, the Michaelis
constant (Km) and the catalytic rate constant (kcat) of BChE for
BChE-FP were determined to be 3.01 ± 0.32 µM and 104.75 ±
12.16 min-1, respectively. Thus, the catalytic efficiency (kcat/Km)
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In conclusion, a small-molecular BChE-FP, acted as a specific
fluorogenic substrate for BChE, was identified by structure
difference-oriented design and stepwise optimization. BChE-FP
showed very low background and exhibited remarkable bright
fluorescence upon the catalysis of BChE. In addition, BChE
showed about 100-fold higher binding affinity against BChE-FP
than the most widely used substrate ATC, and the overall
catalytical efficiency for BChE-FP was comparable to that of
ATC. It also exhibited >14-fold specificity for BChE over AChE,
producing 275-fold higher fluorescence enhancement upon
the catalysis by BChE. In all, BChE-FP is a typical BChE-specific
probe that can discriminate BChE from AChE. Additionally, the
BChE-FP based turn-on fluorescent assay was established for
evaluating BChE activity in aqueous solution and in living cells,
and subsequently applied in BChE inhibitors screening and
characterization. It was also found that BChE-FP could detect
the endogenous BChE in living cells. The current study
demonstrated that the BChE-FP had tremendous potential in
future high-throughput studies in drug discovery and clinical
diagnosis of BChE-associated diseases. Furthermore, this study
implies that the structure difference-oriented design strategy
offers a new approach that can be generally applied to
designing protein isoform-specific probes.
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