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Acetylcholinesterase immobilization on microplates for high-throughput

screening of inhibitors

Poster · June 2018

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Karla Ramírez Sánchez Ricardo Starbird

Costa Rican Institute of Technology (ITCR) Costa Rican Institute of Technology (ITCR)
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 Acetylcholinesterase immobilization on microplates for
high-throughput screening of inhibitors
Inés Ardao,a,(1) Karla Ramírez-Sánchez,b,(1) María Isabel Cadavid,a Ricardo Starbird-Pérez,c María Isabel Lozaa
a BioFarma group, Center for Research in Molecular Medicine and Chronic Diseases-CiMUS,
Universidade de Santiago de Compostela, Santiago de Compostela, Spain
b Escuela de Biología, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica
c Escuela de Química, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica

ines.ardao@usc.es

Introduction
Acetylcholinesterase (AChE) catalyzes the rapid hydrolysis of the neurotransmitter acetylcholine at
cholinergic
h li i synapses. AChE inhibition
i hibiti l d to
leads t acetylcholine
t l h li accumulation,
l ti h
hyperstimulation
ti l ti off nicotinic
i ti i
and muscarinic receptors and disrupted neurotransmission. Hence, this enzyme is the primary target
of acetylcholinesterase inhibitors applied as relevant drugs (e.g. reversible inhibitors applied in
Alzheimer’s and Parkinson’s diseases or autism disorders) and toxins (e.g. irreversible inhibitors such as
organophosphorus compounds used as insecticides and nerve agents). [1]
Robust and reliable assays amenable for high-throughput screening are necessary in the discovery and
development of AChE inhibitors. Enzyme immobilization on microplates facilitates carrying out Figure 1. Scheme of the determination of AChE activity via quantification
of the formed thiocholine with DTNB
reversion assays, re-use of microplates and reduction of costs in high-throughput drug screening
campaigns.

Experimental
1) Immobilization: AChE was immobilized on 96-well microplates via
entrapment in a polymeric matrix of a mixture of poly(vinyl alcohol) (PVA) and
PVA-SbQ (stilbazole quaternized) (Fig. 2).
2) Activity assay: AChE activity was measured by conversion of the substrate
ATCh to thiocholine and subsequent reaction with DTNB (Ellman’s reagent) and
spectrophotometric determination of the formed product (Fig. 1). Enzymatic Figure 2. Experimental procedure of formation of a PVA/PVA-SbQ matrix in 96-well microplates
assay was performed in an automatic liquid handling workstation (Fig. 3).
3) AChE inhibition assay: Immobilized AChE was preincubated for 10 min with
the organophosphate pesticide Chlorpyrifos (an irreversible AChE inhibitor) and
the drug Donepezil used in the treatment of Alzheimer’s disease (a reversible
AChE inhibitor) before the activity assay.
4) Reversion assay: After the inhibition assay, the plate was washed 2 times
with 100 µL of PBS and AChE activity was measured again. This procedure was
performed 2 times.
Figure 3. Experimental procedure of AChE enzymatic assay

Results
1) AChE immobilization 2) AChE activity assay 3) Inhibition and reversion assays
- The solubility in PBS and viscosity of different - The determination of thiocholine by reaction with - Concentration-response curves of a known
mixtures of PVA and PVA-SbQ was evaluated. A DTNB was adapted for detection in irreversible inhibitor (Chlorpyrifos) and a know
mixture of 4% w/v PVA-SbQ (final SbQ 384-well plates. Compound concentration range, reversible inhibitor (Donepezil) with immobilized
concentration of 1.32% w/v) was selected since assay linearity, suitable stop solution and AChE were studied (Fig. 5).
this mixture was insoluble after 15 h of reaction time were optimized using cysteine as - Activity of the enzyme control was maintained
incubation in PBS and presented a suitable standard. after the reversion analysis (Fig. 6a) and was
viscosity
y for handling.
g p g strategy
- A suitable sampling gy ((sampling
p g volume,, recovered after reversion analysis
y ((Fig.g 6b).
)
- SbQ crosslinking was confirmed by FTIR sampling time intervals and total reaction time) (a) (b)
analysis (1730 cm-1) (Fig. 4). was implemented using soluble enzyme to
% Inhibition

% Inhibition

(a) ensure the measurement of initial reaction rates.

- Activity assay with immobilized AChE was
performed and the Z’ was evaluated (Table 1).
An enzyme loading of 3.75 U/mL was selected.

Figure 5. Concentration-response curves with immobilized

Table 1. Z’ factor after 4,5 and 10 minutes of reaction with AChE of (a) Chlorpyrifos and (b) Donepezil
(b) immobilized AChE in a 4% w/v PVA-SbQ matrix.
(a) (b)
% of control activity
% of initial activity

Figure 4. FTIR spectra of a 4% w/v PVA-SbQ matrix (a)

before and (b) after 1.5 h of UV irradiation
Figure 6. (a) Activity after washes of immobilized AChE
without inhibitor and (b) incubated with 6ꞏ10-6 M Donepezil
Conclusions
An assay for determination of AChE inhibitors using immobilized AChE in 96-well microplates has been developed (Z’=0.74). The assay has been tested with
two known inhibitors (irreversible and reversible) and showed a suitable enzyme stability and performance in reversion assays.

Acknowledgments: This work was financially supported by the School of Biology, Costa Rica Institute of Technology (ITCR) and project: “Desarrollo de electrodos para la identificación
de pesticidas y/o herbicidas en medio acuoso” from FEES funding (CONARE), Costa Rica Institute of Technology (ITCR)
References: [1] M. B. Čolović, D. Z. Krstić, T. D. Lazarević-Pašti, A. M. Bondžić, V. M. Vasić, Curr. Neuropharmacol., 2013, 11(3), 315–335
(1) These authors contributed equally to the work
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