An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening

Article abstract of DOI:10.1016/j.ab.2018.03.012

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H]⁺ m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC₅₀ values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results.

Full text of DOI:10.1016/j.ab.2018.03.012

Accepted Manuscript
An improved immobilized enzyme reactor-mass spectrometry-based label free assay
for butyrylcholinesterase ligand screening
Adriana Ferreira Lopes Vilela, Cláudia Seidl, Juliana Maria de Lima, Carmen Lúcia
Cardoso
PII:
S0003-2697(18)30268-9
10.1016/j.ab.2018.03.012
YABIO 12965
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 24 November 2017
Revised Date: 8 March 2018
Accepted Date: 12 March 2018
Please cite this article as: A.F.L. Vilela, Clá. Seidl, J.M. de Lima, Carmen.Lú. Cardoso, An improved
immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand
screening, Analytical Biochemistry (2018), doi: 10.1016/j.ab.2018.03.012.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
An improved immobilized enzyme reactor-mass spectrometry-based label free
assay for butyrylcholinesterase ligand screening
a†
a†
a†
Adriana Ferreira Lopes Vilela , Cláudia Seidl , Juliana Maria de Lima , Carmen
a
Lúcia Cardoso *
a
Departamento de Química, Grupo de Cromatografia de Bioafinidade e Produtos
Naturais, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de
São Paulo, 14040-901, Ribeirão Preto-SP, Brazil
†
Author contributions: these authors contributed equally to this work
*
Corresponding author. Tel: +55 16 36024686; fax: +55 16 36024838. E-mail:
ccardoso@ffclrp.usp.br
1

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ABSTRACT
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes
responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process
for the restoration of the cholinergic neuron. The loss of cholinergic function in the central
nervous system contributes to the cognitive decline associated with advanced age and
Alzheimer’s disease (AD). Inhibitions assays represent a significant role in the drug discovery
process. Herein, we describe an improved label free method to screen and characterize new
BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme
reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer
+
(
MS). The enzyme activity was evaluated by monitoring the choline’s precursor ion [M + H]
m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors
tacrine and galanthamine. The IC50 values were 0.03 ± 0.006 µM and 0.88 ± 0.2 for tacrine and
galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination
of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the
reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product
contributes to the reduction of false positive and negative results.
2

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1
. INTRODUCTION
Enzyme inhibitors still represent an important first line of treatment for several clinical
conditions. This can be evidenced by the considerable number of commercially
available drugs that act as enzyme inhibitors, such as antibiotics, antifungal and
antiprotozoal agents [1].
Butyrylcholinesterase (BChE, EC 3.1.1.8) and acetylcholinesterase (AChE, E.C.
3
.1.1.7) are the two types of cholinesterase enzymes present in the body under
physiological conditions. Both enzymes are responsible for the catalysis of
acetylcholine (ACh), a cholinergic neurotransmitter, yielding choline and acetic acid.
The reduction in ACh levels is one of the known hallmarks of Alzheimer's disease (AD)
(cholinergic hypothesis) [2]. AD is a neurodegenerative disease with no current cure
that is increasing dramatically as more people enter the major risk period for this age-
related disorder due to the aging of the world population [3]. AChE and BChE are
important AD therapeutic targets and their inhibition contributes for the delay in
cognitive and behavioral symptoms related to AD [2,4]. AChE plays a first line role in
ACh hydrolysis, but with the progress of the disease, AChE levels rapidly diminish
whereas BChE levels rises. Therefore, BChE stops playing a secondary role, and
consequently, a significant effort has been made towards the discovery of BChE
inhibitors [4,5]. Currently there is one agent described as BChE inhibitor undergoing
Phase I clinical trial. Scientists at the NIA/NIH have developed a BChE inhibitor called
Bisnorcymserine (BNC), and pre-clinical evidence suggests that BNC is a safe
treatment for AD [6].
A valuable tool for drug discovery is the automated high throughput screening (HTS),
however, it is well known that there are several common sources of variation within
HTS, such as the presence of non-selective binders and aggregates which can result in
3

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false positives and/or negative results [7,8]. Over the years, mass spectrometry-based
screening has allowed the use of native substrates for screening, screen previously
intractable targets, and eliminate the need of coupled reactions. Thus, improving assays
optimal conditions, sensitivity, reproducibility and finally cost [9]. Furthermore, the
introduction of an immobilized enzyme capillary reactors (ICER) can considerably
reduce costs due to enzyme reutilization [10], increase reproducibility by using always
the same enzyme batch and increase throughput, thus allowing routine access and
automation to this technology.
A variety of cholinesterase inhibitor´s assays with either free [11–13] or immobilized
[
14–17] enzymes have been developed using the coupled-assay with the indirect
colorimetric detection as proposed by Ellman et al. [19].
Previously, we have reported [17] a zonal bioaffinity chromatography screening assay
using human serum butyrylcholinesterase (huBChE)-based ICER with detection by the
Ellman´s procedure. This method was established as an alternative to 96-well plates
assays, representing a considerable improvement particularly regarding automation,
enzyme reutilization, tolerance to pH, temperature variations as well as presence of
organic solvent [20]. The main drawback were the occurrence of false-positive
measurement due to spontaneous and non-enzymatic induced substrate hydrolysis
[
21,22]. MS-based screening inhibitors assays for AChE have already been reported and
successfully used [23,24]. For this, choline (Ch) the hydrolysis product of the native
substrate acetylcholine (ACh) is directly quantified as the product of the enzymatic
reaction.
Herein, we describe a label-free ligand screening using a huBChE-ICER in a liquid
chromatographic system coupled to an ion trap mass spectrometer. The
differences/similarities to the AChE-ICER published method [24] will be discussed.
4

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2
. Material and Methods
.1 Materials
2
-1
Butyrylcholinesterase (BChE, EC 3.1.1.8, 5 units.mg ) from human serum as
lyophilized powder; substrate, acetylcholine iodide (ACh) and butyrylcholine iodide
(BCh), choline (Ch) standard; galanthamine hydrobromide; and 9-amine-1,2,3,4-
tetrahydroacridine hydrochloride (tacrine) were obtained from Sigma-Aldrich (St.
Louis, USA). For the immobilization procedure: glutaraldehyde 25%, 3-
aminopropyltriethoxysilane (APTES), potassium phosphate monobasic, and ammonium
acetate were supplied by Sigma-Aldrich, Merck (Darmstdt, Germany), Synth (São
Paulo, Brazil), or Acros (Geel, Belgium). Ultrapure water used throughout the study
®
was obtained from a Millipore Milli-Q ultra-filtration system (Millipore, São Paulo,
Brazil). Fused silica capillary (0.362 mm E.D x 0.1 mm I.D) was acquired from
Polymicro Technologies (Phoenix, USA). The mobile phases were prepared daily.
Substrates ACh and BCh (1 mM) were prepared in 15 mM ammonium acetate pH 5.0
and standards inhibitors (1 mM) stock solutions were prepared in water. All solutions
when not in used were kept and stored at 4°C.
Uleine extraction and purification from Himatanthus lancifolius bark was carried out as
reported elsewhere [25].
2
.2 huBChE immobilization
huBChE was immobilized into the internal surface of an open tubular silica capillary
100 cm x 0.362 mm x 0.1 mm I.D) in according to previous protocols [17,23,16] with
(
-1
minor modifications. Briefly, 500 µL of huBChE enzyme solution (0.5 units.mL in 50
-1
mM phosphate solution, pH 7.4) was infused throughout the capillary at a 50 µL.min .
5

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The solution was collected and this step was repeated 5 times using the same solution.
The 100 cm capillary was divided generating 3 immobilized capillary enzyme reactors
(huBChE-ICER) with 30 cm length each. While not in use, the huBChE-ICER were
maintained at 4 °C, with both extremities immersed in 50 mM Tris buffer, pH 8.0.
Syringe pump model 11 Plus advanced single (Harvard Apparatus, Holliston, USA) was
used to infuse all solutions during the immobilization procedure.
2
.3 Instrumentation
The huBChE-ICER activity and inhibition studies were performed using two LC 20AD
pumps, a SIL-20A auto-sampler, a DGU-20A degasser and an interface controller
CBM-20A all parts of a Nexera XR high performance liquid chromatography (LC)
system (Shimadzu, Japan). The huBChE-ICER was placed in the LC system and
coupled to an AmaZon Speed Ion Trap (IT) Mass Spectrometer (MS) (Bruker
Daltonics, Germany) equipped with an Electro-Spray Ionization (ESI) source (FAPESP
o
Proc. n 2014-50299-5)
.
The LC-MS system was controlled, and data was acquired
using Bruker Data Analysis software (version 4.3).
2
.4 LC-huBChE-ICER-MS conditions
1
5 mM ammonium acetate solution pH 8.0 was used as running buffer at flow rate of 50
−
1
µL.min and a second pump delivered methanol after the huBChE-ICER and before the
IT-MS throughout a “T” shaped connection, at the same flow rate as the running buffer.
Total analysis time was 3 min. IT-MS parameters were: 4.000 V capillary voltage 500
-1
V, end plate voltage, 8.0 L.min drying gas, 275 °C drying temperature and 35 psi
nebulizer. Analyses were carried out with MS operated in manual MS(n) mode under
+
positive ionization (scan 50-250 m/z), with detection of the cations [M + H] m/z 145.99
6

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+
+
(
ACh), [M + H] m/z 174.01 (BCh) and [M + H] m/z 104.17 (Ch, product of the
enzymatic catalysis). Isolation width was set at ± m/z 0.5. Fragmentation amplitude was
set to 120%. Nitrogen was used as sheath gas and helium as collision gas. Data were
analysed using Bruker Data Analysis software (version 4.3). All analyses were
performed at room temperature (21 °C).
2
.5 Analytical method validation
External calibration curves of Choline (Ch) standard with final concentrations ranging
.25 – 75 µM was carried out to quantify huBChE-ICER reaction. To this end, at least 8
different stock solutions of Ch were prepared in 15 mM ammonium acetate, pH 5.0
1.25, 2.5, 12.5, 25, 62.5, 175, 250, 375 µM). An aliquot (20 µL) of each solution was
0
(
further diluted to a final volume of 100 µL in 15 mM ammonium acetate, pH 8.0. The
solutions were vortex-mixed during 10s and aliquots of 10 µL were injected in triplicate
into the system, however, an empty fused silica was used instead of the huBChE-ICER.
+
A calibration curve was obtained by plotting the Ch [M + H] m/z 104.17 area against
the injected Ch concentration. Isolation width was set at ± m/z 0.5.
Method selectivity was evaluated using blank runs in which the target analytes are not
present and measured under identical conditions (water and 15 mM ammonium acetate
solution, pH 8.0).
2
.6 Apparent Kinetic Constant (KMapp)
The apparent kinetic constant (KMapp) was obtained independently for both substrates
ACh and BCh by varying the substrate concentration while measuring the choline
formation. To this end, at least 9 different stock solutions were prepared (2.5; 3.75; 5;
2
5; 50; 100; 250; 500; 750 µM) in 15 mM ammonium acetate, pH 5.0 and 20 µL of
7

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each solution further diluted to a final volume of 100 µL using the same buffer solution.
Final solutions were vortex-mixed during 10s and 10 µL aliquots injected into the LC-
+
+
MS system in triplicate. Ch [M+H] 104.17 m/z; ACh [M+H] 145.99 m/z and BCh
+
[
M+H] 174.01 m/z ions were monitored. GraphPad Prism 5 software was used to
obtain Michaelis–Menten plots by nonlinear regression analysis.
2
.7 Inhibition Studies
Tacrine and galanthamine, were used as known cholinesterase inhibitors [26]. In
addition, the alkaloid uleine, previously described as a cholinesterase inhibitor [27], was
also evaluated to validate the applicability of the assay .
The half maximum inhibitory concentration (IC ) for each sample was obtained by
5
0
quantifying Ch production after the hydrolysis of ACh using different concentrations of
inhibitors. Stock solutions of tacrine (0.5 to 15 µM) and galanthamine (5 to100 µM)
were prepared in water. Reaction mixtures were prepared by mixing tacrine (0.05 to 1.5
µM) and galanthamine (0.5 to 10 µM) with ACh 70 µM. Final volume was completed
with 15 mM ammonium acetate pH 8.0. Each reaction mixture (10 µL) was injected
into the LC-MS system and the percentage of inhibition was calculated in accordance
with equation 1:
ꢈ
ꢉ
%
ꢀꢁꢂꢀꢃꢄℎꢃꢅꢃꢆꢃꢁꢄꢀ =ꢀꢇ1 −ꢀ ꢋ ꢀꢌꢀ100
Eq. 1 ꢀ
ꢈ
ꢊ
Where, P is the achieved peak area of choline produced: (i) in the presence of ligand);
0) in the absence of ligand.
For each inhibitor concentration, an inhibition curve, IC , was obtained applying
(
5
0
nonlinear regression analysis (GraphPad Prism 5) by plotting individual % inhibition
8

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versus each correspondent ligand concentration.
Mechanism of action of galanthamine as well as its steady-state inhibition constant (Ki)
were also obtained. For that, different solutions of ACh (12; 24; 40; 48; 56; 80 µM)
were containing fixed galanthamine concentration (0.25; 0.5; 1 µM) prepared in 15 mM
ammonium acetate pH 8.0 in triplicate and injected (10 µL) into the huBChE-ICER-MS
system. Reciprocal plots of 1/[Ch] versus 1/[ACh] were then obtained and Lineweaver-
Burk plots were used to determine the action mechanism for galanthamine.
The K values for galanthamine were determined from the slope of reciprocal plot
i
versus the ligand concentration (calculated to each ligand concentration). A linear replot
was traced and the quotient of division between linear/angular coefficients provided the
K value [28].
i
3
. RESULTS AND DISCUSSION
The use of huBChE-ICER in the LC-MS system allowed the direct quantification of the
+
enzymatic reaction product ion Ch [M+H] m/z 104.17. LC-MS conditions and
parameters were based on previous reports to AChE [24].
The assay proved to be robust, fast (3 min) and suited for measuring huBChE activity
using both substrates ACh and BCh (Fig 1A and 1B respectively).
Substrate solutions were freshly prepared in 15 mM ammonium acetate buffer pH 5.0 to
avoid spontaneous substrate hydrolysis. Induced substrate hydrolysis can be easily
verified injecting the reaction mixture into the LC-MS system with an empty fused
+
silica capillary. If any Ch [M+H] m/z 104 is detected, this must be subtracted from the
experimental enzymatic hydrolysis values before calculating the inhibition percentage.
No spontaneous or induced ACh or BCh hydrolysis was observed with tacrine,
galanthamine or uleine.
9

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Figure 1. A) EIC (extracted ion chromatogram) and IT-MS spectrum obtained after the
enzymatic hydrolysis using the LC-MS system with huBChE-ICER after injection of
+
ACh (70 µM), products of enzymatic hydrolysis Ch [M + H] m/z 104.17 and acetate
+
+
[
M + H] m/z 59.39; substrate ACh [M + H] m/z 145.99; B) IT-MS spectrum after
+
injection of BCh (50 µM); products of enzymatic hydrolysis Ch[M + H] with m/z
04.17 and acetate [M + H] m/z 59.41; substrate BCh [M + H] m/z 174.01. Analysis
+
+
1
conditions: described item 2.3
The assay was validated according guidelines for bioanalytic methods [29]. The
calibration curve was logarithmic in the range studied (0.25 to 75 µM), with average
1
0

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2
coefficient of determination (n = 3) of r = 0.99. Blank runs (15 mM ammonium acetate
pH 8.0), were the target analytes were not present, under identical experimental
conditions revealed no interference demonstrating that no carry over occurred between
analyses. The Limit of Quantification (LoQ) was 0.25 µM (RSD = 1.0 %, n=3, accuracy
=
108%) and the Limit of Detection (LoD) was 0.005 µM, calculated by the signal-to-
noise ratio.
The KMapp values were of 39.9 ± 3 and 44.8 ± 10 µM for BCh, and ACh respectively.
Although BCh is the natural substrate of huBChE, this enzyme can also hydrolyze ACh
at a lower turnover rate [26]. These KMapp values were used to calculate the optimal
substrate concentration to carry out the inhibitions and ligand screening studies.
The main goal of the herein reported assay is its application for the screening of
huBChE inhibitors. To that matter, two well-known cholinesterase inhibitors, namely
tacrine and galanthamine were used. Tacrine low IC value 0.03 ± 0.006 µM obtained
5
0
(Figure 2A) corroborated the literature reports of it being a strong huBChE inhibitor.
For galanthamine (figure 2B), a nonspecific BChE inhibitor, IC value obtained with
5
0
the assay was 0.88 ± 0.2 µM and demonstrates the capability to distinguish between
inhibitors potency. Studies on mechanism of action for galanthamine using ACh as
substrate showed to be a competitive type with steady-state inhibition constant K = 0.11
i
±
0.02 µM (Figure 3). This result showed that even after immobilization the enzyme
behavior towards known ligands has not changed.
1
1

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A
B
1
00
1
00
80
60
40
20
0
8
0
6
0
4
0
IC50 = 0.03 ± 0.006 µM
IC50 = 0.88 ± 0.2 µM
2
0
0
0.0
0.4
0.8
1.2
1.6
0
2
4
6
8
10
12
[
Tacrine] (µM)
[Galanthamine] (µM)
Figure 2. Dose response curve plots of inhibition percentage using the LC-MS system
with huBChE-ICER for tacrine (A) and galanthamine (B).
Figure 3. Reciprocal plots of 1/[Ch] versus 1/[ACh] in the presence of different
concentrations of galanthamine using the LC-MS system with huBChE-ICER.
The results demonstrate that the use of a LC-MS system using the huBChE-ICER can
not only identify but also mechanistically characterize huBChE inhibitors and provide
quantitative parameters such as IC and K . Moreover, this method is an alternative to
5
0
i
overcome the shortcomings of indirect assays widely used to screen huBChE inhibitors
21,22].To further consolidate the screening assay , the alkaloid Uleine (Figure 1S) was
also evaluated. This natural compound, extracted from Himatanthus lancifolius barks,
[
1
2

ACCEPTED MANUSCRIPT
was previously isolated and identified by França et al.[25]. The uleine IC value 35.1 ±
5
0
5
µM (Figure 1S) herein obtained is accordance with the published results that describes
this alkaloid as an huBChE inhibitor [27].
4
. CONCLUSION
The label free huBChE-ICER-based LC-MS assay herein reported offers a significant
improvement to cholinesterase colorimetric assays. For the first time, with a short
analysis time (3 minutes) not only the activity but also the inhibition of huBChE can be
easily assessed. The developed assay can be used, as demonstrated, not only for
screening inhibitors but also for characterizing them, with a short analysis time (3
minutes) high productivity can be achieved.
5
. ACKNOWLEDGEMENTS
This work was supported by the Sao Paulo State Research Foundation (FAPESP) [grant
numbers 2013/01710-5, 2014/50299-5], National Council for Technological and
Scientific Development (CNPq) and Coordinating Development of Higher Level
(CAPES). J.M.L., A.F.L.V. and C.S. also acknowledge FAPESP for scholarships (grant
numbers 2014/06907-0, 2016/02873-0, 2014/11640-3).
1
3

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6
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