Back-scattering interferometry: An ultrasensitive method for the unperturbed detection of acetylcholinesterase-inhibitor interactions

Article abstract of DOI:10.1002/anie.201203640

A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22 000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer's disease, by detecting dual-binding interactions. E=enzyme, I=inhibitor, S=substrate. Copyright

Full text of DOI:10.1002/anie.201203640

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DOI: 10.1002/anie.201203640
Biosensors
Back-Scattering Interferometry: An Ultrasensitive Method for the
Unperturbed Detection of Acetylcholinesterase–Inhibitor
Interactions**
Gabrielle L. Haddad, Sherri C. Young, Ned D. Heindel, Darryl J. Bornhop,* and
Robert A. Flowers II*
In recent years, the emphasis of drug discovery and optimi-
zation has shifted from traditional methods to rapid micro-
fluidic screening to minimize cost and maximize output.
Back-scattering interferometry (BSI),[1] a technique which
quantifies refractive index (RI) changes arising from inter-
molecular binding interactions, is a novel biosensing platform
that may be useful in drug discovery. Acetylcholinesterase
(AChE), a widely studied serine hydrolase that plays pivotal
roles in Alzheimerꢀs disease (AD),[2] inflammatory process-
es,[3] and nerve-agent poisoning,[4] is an interesting system
with which to study the use of BSI for the rapid detection of
drug candidates. The search for potent AChE inhibitors
(AChEI) has been driven largely by the need for an effective
AD treatment, and is based on the long-standing cholinergic
hypothesis[2] and the more recent amyloid hypothesis.[5] It is
well-established that AChE accelerates amyloid-beta (Ab)
peptide deposition, a process which may be mediated by an
interaction between plaque precursors and the peripheral
anionic site (PAS) of the enzyme.[6] Recent drug discovery
efforts have focused on the design of AChEIs that are able to
interact with the PAS to target both cholinergic and non-
cholinergic AD pathologies.[7] Seminal work in this field has
led to the discovery of dual-binding inhibitors, such as bis-
galantamine[8] and pseudo-irreversible carbamate inhibitors.[9]
Given the large number of studies on several diverse classes
of AChEIs, this system is a widely accepted benchmark for
the development and testing of novel screening techniques.
Traditional methods used to detect interactions with
AChE focus on the indirect measurement of substrate
hydrolysis and require specialized probes and laborious
procedures.[10] To explore the utility of BSI to detect
AChEIs, several known and novel inhibitors with diverse
potencies and inhibitory mechanisms have been screened.
Herein, we show that BSI can: 1) quantify AChE–inhibitor
interactions in the absence of substrate (S); 2) detect signal
changes at enzyme concentrations lower than previously
reported techniques; 3) sense and deconvolute multiple bind-
ing events; and 4) discern between different types of inhib-
itors by comparing BSI binding data with half-maximal
inhibitory concentrations (IC50).
The straightforward setup of BSI utilizes a multipass
configuration,[1] where the refraction of monochromatic light
within a microfluidic channel generates an interference fringe
pattern. Because one of the surfaces of the channel is curved,
the light deviates from its original path and hence refracts
several times within the channel prior to exiting.[1f] The
resulting high-contrast fringes are then reflected off a mirror
and directed onto a charge-coupled device array camera,
which acts as the detector. This unique optical train increases
the effective pathlength and allows for the analysis of pM to
mm receptor and ligand concentrations for an optical probe
volume of 360 pL.[1a,f] The high-sensitivity interference fringe
pattern allows the quantification of RI changes based on
phase shifts of the back-scattered laser beam. Several studies
have shown that these shifts correlate well with ligand–
receptor binding interactions and can result from conforma-
tional changes, solvation/desolvation, changes in dipole
moments, and polarizability.[1] Because RI changes may
arise from molecular interactions unrelated to target binding,
ligand and receptor blanks must be prepared to ensure that
the measured changes reflect an actual binding event.
Furthermore, a temperature-controlled stage is employed to
avoid minor RI shifts arising from temperature fluctuations.
From the BSI-generated data, an equilibrium dissociation
constant (KD) can be obtained by fitting the average signal
shift generated at each concentration to an exponential rise to
max equation.
[*] G. L. Haddad, S. C. Young, N. D. Heindel, Prof. R. A. Flowers II
Department of Chemistry, Lehigh University
Bethlehem, PA 18015 (USA)
E-mail: rof2@lehigh.edu
Homepage: www.lehigh.edu/~rof2/
Prof. D. J. Bornhop
Department of Chemistry, Vanderbilt University
Nashville, TN 37235-1234 (USA)
E-mail: Darryl.bornhop@vanderbilt.edu
BSI can be used to measure binding interactions in free
solution without the need for surface immobilization, speci-
alized reagents, or fluorescent probes, thus providing a clear
advantage over techniques such as isothermal titration
calorimetry (ITC),[11] surface plasmon resonance (SPR),[12]
and fluorometric assays.[13] Furthermore, cosolvents, such as
DMSO[1b] and acetonitrile,[1c] can be used without assay
interference.
[**] We thank Dr. Esther Pesciotta for her initial contributions to this
work. We are grateful for support from the National Science
Foundation (CHE-0848788) and the CounterACT Program, National
Institutes of Health Office of the Director (NIH OD), and the
National Institute of Arthritis and Musculoskeletal and Skin
Diseases (NIAMS), grant number U54AR055073.
We first wanted to probe the utility of BSI to screen
AChEIs using a set of known and novel AChEIs (Scheme 1).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201203640.
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Table 1: Reported Ki and experimental KD values for known AChEIs.
Ligand
Ki
[mm]
BSI KD
[mm]
Ref.
Edrophonium
Physostigmine
Galantamine
Propidium
1.5–3.8
1.27Æ0.4
[16,17]
[18]
[19,8a]
[16,20]
[21,22]
0.013[a]–0.11
0.20–0.61
0.63–1.5
0.020Æ0.009
1.23Æ0.5
0.64Æ0.09
0.0084Æ0.002
BW284c51
0.0032–0.008
[a] Value listed is an IC50 value.
values. KD values for other known inhibitors also are in the
range of literature values (Table 1).
Besides KD (or inhibitor dissociation constants Ki) values,
another common measurement of inhibitor potency is an IC50
value. While KD/Ki determinations require complex graphical
methods,[23] IC50 values are easily determined and are better
suited for the screening of large compound libraries. How-
ever, IC50 measurements can vary depending on experimental
conditions, such as substrate and enzyme concentrations.[24a]
Furthermore, cases exist where IC50 values differ from KD
values by up to three orders of magnitude, a trend which
usually occurs with complex inhibitory mechanisms.[24]
We next decided to explore the utility of BSI for the
investigation and mechanistic characterization of novel non-
competitive AChEIs by comparing IC50 values (determined
Scheme 1. Structures of AChEIs screened using BSI.
Edrophonium, a competitive inhibitor, propidium, a noncom-
petitive inhibitor, and the mixed inhibitors, 1,5-bis(4-allyldi-
via Ellmanꢀs method[10a]
) to BSI-generated KD values
methylammoniumphenyl)pentan-3-one
dibromide
(Table 2). Interestingly, while the IC50 value for inhibitor 3
(BW284c51) and galantamine, were the standards used to
validate BSI as a method for the detection of AChEIs.
Physostigmine,[14] a pseudo-irreversible carbamate AChEI
used for the treatment of AD, was additionally explored to
determine whether BSI could be used to detect a range of
inhibitor types. It is worth noting that carbamates are not only
effective anticholinesterases,[9] they have also been exten-
sively investigated as inhibitors of other serine hydrolases,
such as fatty acid amide hydrolase.[15] A binding curve for the
high affinity ligand, BW284c51, is shown in Figure 1. An
enzyme concentration of 72 nm with varying ligand concen-
trations (1–100 nm) resulted in signal shifts of about 0.02
radians and a KD that is consistent with previously reported
Table 2: Correlation of BSI KD values with anticholinesterase activities.
Ligand
K
ies/IC50 [mm][a]
BSI KD [mm]
1
2
3
4
5
6
0.51Æ0.02
1.36Æ0.1
2.69Æ0.1
6.34Æ0.5
2.29Æ0.9
13.9Æ0.3
0.11Æ0.05
0.21Æ0.05
2.18Æ1.1
0.97Æ0.57[b]
0.12Æ0.04
Dual-binding
[a] Previously reported IC50 values against AChE from Electrophorus
electricus.[25] [b] KD value is for second binding event for dual-binding
ligand.
correlates well with the BSI KD values for ligands 1, 2, and 5
are 5- to 10-fold lower than their corresponding IC50 values.
These results were originally perplexing, because noncompe-
titive inhibitors traditionally exhibit KD values close in
magnitude to their IC50 values.
The above KD/IC50 relationship can be rationalized by
considering the Cheng–Prusoff equation.[23,26] For noncompe-
titive and mixed inhibition, the IC50 is equal to the inhibitorꢀs
affinity for the enzyme–substrate complex (Kies) in the
presence of high substrate concentrations (that is, traditional
Ellman assay conditions). Conversely, BSI measurements are
performed in the absence of substrate and are an indication of
a given ligandꢀs affinity for the enzyme alone (KD or Kie). This
relationship is depicted in Scheme 2.
Figure 1. BSI binding curve of signal versus concentration of the
BW284c51 ligand. Signal shifts of the back-scattered laser for equili-
brated samples of AChE (72 nm) with ligand (2, 5, 10, 20, 40, 80, and
100 nm) in PBS were measured. Each data point is the average of at
least five trials, and the error bars shown indicate the full value of the
standard error of measurement.
With the Cheng–Prusoff relationship in mind, we revisited
our KD/IC50 correlation for ligands 1, 2, 3, and 5. When the
IC50 is measured at high [S], the portion of the ligand with
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dual-binding ligands. It is important to note, however, that RI
shifts may not always occur in the same direction. Rather, the
transition to a zero signal shift simply is a measureable change
in the optical properties of the system. Once the first site of
AChE is saturated and the second site starts to be occupied by
ligand, enough water molecules could be displaced to change
the RI of the bulk solution (causing the signal to change and
the phase shift to approach zero). Putting this result into
context, the active-site gorge of AChE contains a considerable
amount of easily displaced water molecules, about half of
which are in a region neighboring the active site.[27] Perhaps
once the active site is occupied, water molecules are displaced
to facilitate ligand binding to the PAS. In addition to displaced
water molecules, a conformational shift of the enzyme may
occur upon saturation of the active site to allow PAS
binding.[28] This result suggest that BSI has the potential to
screen for mixed AChEIs, compounds which have shown high
efficacy against AD.[5] Furthermore, with further optimization
of this method, both the potency and the type of inhibition
could be determined using BSI and may eliminate the need
for enzyme kinetics and fluorescence displacement assays,
which are labor-intensive and prone to error.[24]
As indicated by the IC50 and dual-binding data, BSI has
the ability to distinguish between true noncompetitive and
mixed inhibition. This is important because of the well-
established role of the PAS in AD. Blocking this site by
noncompetitive inhibition has proven very effective at treat-
ing both cholinergic and non-cholinergic AD symptoms.
Seminal work by Andrisano et al. revealed that the true PAS
inhibitor, propidium, attenuates Ab aggregation by 82%,
while mixed inhibitors only provide moderate suppression
(22–30%). Competitive inhibitors, such as edrophonium,
have no effect on AChE-induced Ab aggregation, indicating
that affinity for the PAS is required for the observed
aggregation suppression.[5] By discerning between true non-
competitive and competitive (or mixed) inhibitory mecha-
nisms, BSI may be useful as part of a rapid screen for
therapeutically important PAS inhibitors.
We next determined the detection limit of BSI for AChE
sensing. At the lowest limit, concentrations of 100 pm can be
detected, which equates to an astonishing 22000 molecules of
enzyme with an optical probe volume of 360 pL. This
detection level approaches and, in many cases, surpasses the
sensitivity of recently reported AChE detection methods,
such as chemiluminescent dioxetane probes,[10b] the aggrega-
tion-induced emission (AIE) of tetraphenylethylenes
(TPE),[10c,d] cyano-substituted poly(p-phenylenevinylene)
(PPV) probes[10e] and other fluorescence assays,[10f,g] and
electrochemical detection methods involving gold nanoparti-
cles[10h] (Table 3). Unlike many of these techniques, BSI does
not require substrate analogues, specialized probes, or
laborious procedures.
Such low-level detection of AChE, an enzyme whose price
ranges from hundreds to thousands of dollars per mg, is also
very cost-effective. The study of AChE mutants will therefore
be more accessible with BSI, as the preparation and isolation
of large quantities of enzyme are not required. Furthermore,
one of the drawbacks with the use of the Ellman assay is non-
enzymatic hydrolysis of acetylthiocholine (ATCh), an ana-
Scheme 2. Relationship between BSI KD (Kie) and Ellman assay IC50 for
noncompetitive and mixed inhibition.[23]
affinity for the active site is not able to compete with the
substrate. However, under conditions used for BSI, these
particular ligands are free to interact with both AChE sites,
thus resulting in higher binding affinities (lower KD values).
These data, then, suggest that ligands 1, 2, and 5 are able to
bind to both the active and peripheral sites in the absence of
competing substrate. On the other hand, 3 has affinity for the
PAS alone, as the affinity of this ligand for the enzyme is
unaffected by substrate. This result is profound because it
suggests that by measuring IC50 values at high [S] along with
BSI KD values (two easily determined parameters), valuable
insight is gained into the relative affinities that a mixed
inhibitor has for the active and peripheral sites of AChE.
Furthermore, these data indicate that if an IC50 value is equal
to the BSI-generated KD, the inhibitor is acting by a true
noncompetitive or an uncompetitive mechanism and thus has
affinity for the PAS alone. Conversely, if the IC50 is greater
than the KD, it implies that the compound interacts with the
active site by a competitive or mixed interaction.
Two separate and distinct saturation curves were observed
for inhibitors 4 and 6 (Figure 2 and Supporting Information,
Figure S7, respectively). For ligand 4, the dual-binding curve
is shown in the graph inset of while the fit of the second
binding event is shown in the main graph. While the first KD
of 4 could not be accurately determined because of the close
proximity to the second curve, the KD of the second curve was
calculated as 0.97 Æ 0.57 mm. To calculate this dissociation
constant, the ligand concentrations were normalized to treat
the second curve as an isolated binding event (Figure 2).
We were initially surprised that the signal shift
approached zero prior to the second binding event for both
Figure 2. Inset: BSI dual-binding curve of signal versus concentration
of 4. Signal shifts of the back-scattered laser for equilibrated samples
of AChE (6.9 mm) with ligand (0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0,
and 3.0 mm) in PBS/methanol (9:1) were measured. The main graph
depicts the fit of the second binding curve. To fit this curve, the
second curve was normalized and treated as an isolated binding
event. In both graphs, each data point is the average of at least five
trials, and the error bars shown indicate the full value of the standard
error of measurement.
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Table 3: Comparison of the limit of detection (LOD) of BSI to previously
reported AChE detection methods.
Experimental Section
Acetylcholinesterase (Type VI-S from Electrophorus electricus), 1,5-
bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, pro-
pidium diiodide, and phosphate-buffered saline (PBS) packets were
purchased from Sigma–Aldrich (Saint Louis, MO). Methanol was
obtained from EMD Chemicals (Gibbstown, NJ) and 2(2-ethoxy-
ethoxy)ethanol was purchased from Acro¯s Organics (Geel, Belgium).
Edrophonium chloride was purchased from MP Biomedicals (Solon,
Ohio). PBS was prepared with MilliQ water (Millipore, Billerica,
MA), and both PBS and the cosolvent were filtered using 0.2 mm
membrane filters prior to use. AChE was dialyzed against PBS (3 ꢁ
300 mL) using regenerated cellulose dialysis tubing with a molecular
weight cutoff of 3500 Da (Fisher, Hampton, NH). Inhibitors 1–3, 5,
and 6 were synthesized as previously described.[25] For the synthesis of
ligand 4, see the Supporting Information.
Technique
ATCh LOD
LOD
[moles]
Ref.
(Y/N) [mUmLÀ1
]
BSI
N
Y
24
–
3.6ꢀ10À20
–
Ellman assay
Chemiluminescence
AIE of TPE
1.85ꢀ10À18 [10a,b]
2.5ꢀ10À19 [10b]
Y
Y
30-40
5
–
–
–
–
[10c]
[10d]
[10e]
[10f]
AIE of TPE
N
N
N
500
12,500
50
Cyano-PPV Fluor.
Conjugated polymer
Fluor.
Laser-induced Fluor.
Electrochemical AuNP
detection
Y
75
–
–
[10g]
[10h]
N
1000
The previously reported instrumental setup contains a helium–
neon (HeNe) laser (l = 632.8 nm) directed onto a poly(dimethylsil-
oxane) (PDMS) chip containing a semicircular microfluidic channel
(90 mm wide, 40 mm deep, cross-sectional area of 2.9 nm2).[1] The
HeNe laser and temperature controller were allowed to equilibrate
for at least 1 h before experiments were run. Chip silanization was
performed using the procedure described in the Supporting Informa-
tion. The microfluidic channels were rinsed with MilliQ water and
PBS prior to each run. Owing to the high lipophilicty of many of the
AChEIs, BSI experiments were run in PBS with either 10% methanol
or 5% 2(2-ethoxyethoxy)ethanol as a cosolvent. The AChE concen-
tration was held constant (0.025–9.5 mm) while the ligand concen-
trations were varied based either on the IC50 value determined by
Ellmanꢀs method or based on previously determined literature Ki
values. Enzyme and ligand blanks were additionally prepared in order
to correct for concentration-dependent RI changes. Significant RI
shifts arising from changes in analyte concentration were subtracted
out from the overall BSI-generated signal for that particular
concentration. AChE:ligand samples were prepared in advance and
allowed to equilibrate at 48C for at least 3 h prior to each experiment.
The signal was measured for 45 to 60 s at 258C. Once the data were
collected for each ligand concentration, the resulting binding curve
was fit to a one site binding hyperbola function using GraphPad Prism
(Version 4).
logue of acetylcholine (ACh), which can result in misleading
cholinesterase activities. This effect has been reported in the
investigation of nucleophilic oximes.[29] Spurious signals may
presumably be observed with techniques relying on the
quantification of ATCh hydrolysis (or a derivative thereof;
Table 3). As a substrate is not required to generate a BSI
signal, this is a useful method to screen diverse compound
libraries without the potential for false signals.
Any intermolecular interactions which generate a RI
change of at least 10À8 units can be detected by BSI.[1e] There
are, however, some limitations to the current platform. For
novel and insufficiently characterized receptors, appropriate
control experiments are critical to correct for such events as
enzyme aggregation or denaturation. Furthermore, care
should be taken during the interpretation of KD values, as
they may not correlate directly with physiological action or
inhibitory activity. Finally, using BSI data alone, the site of
inhibition cannot be determined (unless the ligand is a mixed
inhibitor, as shown with AChE ligands 4 and 6).
Received: May 10, 2012
Revised: August 24, 2012
Published online: October 4, 2012
In summary, BSI provides a highly sensitive method for
the detection of anticholinesterases with a range of potencies
and inhibition types. Unlike previous methods, BSI does not
require the use of substrate (or a derivative thereof) for signal
generation, thus avoiding the possibility of false signals. Using
the multipass configuration of BSI, a detection limit of 3.6 ꢁ
10À5 fmol of AChE was obtained for an optical probe volume
of 360 pL, a sensitivity which surpasses that of previously
described colorimetric, fluorescent, chemiluminescent, and
electrochemical techniques.[10] Two distinct binding interac-
tions of inhibitors 4 and 6 were also detected in one
experiment, demonstrating that BSI is well-suited for the
rapid screening for dual-binding AChEIs. Finally, by compar-
ing IC50 values generated at high [S] to BSI KD values,
therapeutically important PAS inhibitors can be screened.
While the focus of the present study is on AChE–small-
molecule interactions, the approach described herein should
be applicable to other enzyme–inhibitor interactions and thus
may be useful to screen libraries for ligands of orphan
receptors. In a broader sense, BSI has the potential to track
events other than target binding, such as metal complexation,
liposome formation, and intermolecular interactions of med-
ically and biologically relevant molecules.
Keywords: acetylcholinesterase · analytical methods ·
.
enzyme assays · high-throughput screening · interferometry
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