Immobilized cholinesterases capillary reactors on-flow screening of selective inhibitors.

G Model CHROMB-18648; No. of Pages 7

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Immobilized cholinesterases capillary reactors on-flow screening of selective inhibitors夽 Adriana Ferreira Lopes Vilela a , Joyce Izidoro da Silva a , Lucas Campos Curcino Vieira b , Gilberto C.R. Bernasconi c , Arlene Gonçalves Corrêa b , Quezia Bezerra Cass b , Carmen Lúcia Cardoso a,∗ a

Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil Departamento de Química, Universidade Federal de São Carlos, Cx. Postal 676, São Carlos, 13565-905, SP, Brazil c Centro Avançado de Estudos e Pesquisas – CAEP, 13087-567, Campinas, SP, Brazil b

a r t i c l e

i n f o

Article history: Received 22 September 2013 Received in revised form 14 November 2013 Accepted 17 November 2013 Available online xxx Keywords: Selective inhibitor Screening Statistical comparation Butyrylcholinesterase Acetylcholinesterase Enzymatic inhibition assays

a b s t r a c t The discovery of selective inhibitors for acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) is extremely important for the development of drugs that can be used in the treatment of patients diagnosed with the Alzheimer’s disease (AD). For this reason, there is a growing interest in developing rapid and effective assays techniques for cholinesterases (ChE) enzymes ligand screening. Herein is presented the results of selective screening assays of a coumarin derivatives library using BChE and AChE covalently immobilized onto silica fused capillaries (ICERs, 15 cm × 0.1 mm ID). The statistical comparison of the ICERs screening assay with that of the free enzymes is reported and highlights the advantages of the onflow ICERs assay. Two out of 20 coumarin derivatives could be highlighted: compound 17 is more active toward BChE (IC50 = 109 ± 21 ␮M) and 19 showed activity against both enzymes (BChE IC50 = 128 ± 28 ␮M and hu-AChE IC50 = 144 ± 40 ␮M). The statistical evaluation of the results of the ICERs and free enzyme assays showed no difference between them, further validating the ICERs assay model. The ICERs ability to recognize selective ligands and its use for characterization of the inhibition mechanisms of the hits consolidates the approach here reported. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Butyrylcholinesterase (BChE, EC 3.1.1.8) and acetylcholinesterase (AChE, EC 3.1.1.7), are serine hydroxylases classically associated with the hydrolysis of the neurotransmitter acetylcholine (ACh), a reaction that yields choline and acetic acid. In the healthy human brain, BChE plays a secondary part in the hydrolysis of ACh, since the activity of AChE predominates [1–3]. In the brain of patients with the Alzheimer’s disease (AD), the levels of AChE decline, rapidly, whereas the levels of BChE rise and the role of BChE becomes more important [2–5]. This imbalance in AChE and BChE activity modifies the supportive role of BChE and represents the rationale behind the synthesis of new selective and brain-targeted inhibitors of BChE for mild to moderate AD forms [6]. A balance between AChE and BChE inhibition might result in optimal therapeutic efficacy of

夽 This paper is part of the special issue “Chiral Separations 2013” edited by Ruin Moaddel. ∗ Corresponding author. Tel.: +55 16 36024686; fax: +55 16 36024838. E-mail addresses: [email protected], [email protected] (C.L. Cardoso).

nonselective cholinesterase inhibitors (ChEI) to be used on moderate forms of AD [2]. The rational design of selective inhibitors is based on the differences in the internalized binding pockets of the two enzymes into which ACh diffuses and undergoes hydrolysis [5–7]. Selective BChE inhibitors enhance the ACh levels, reduce the formation of ␤-amyloid plaques in the brain [8], and cause fewer peripheral side effects [9]. For this reason, there is growing interest in developing rapid and effective techniques to identify new drug candidates [10–14]. The BChE also has pharmacological and toxicological importance due to its ability to hydrolyze drugs containing ester and kidnap cholinesterase inhibitors including organophosphorus agents [15]. The BChE plays an important role in cholinergic nerve impulse transmission in insects. Its inhibition causes the ACh receptor desensitization leading to blockage of the transmission with consequent death of the insect. Thus, this enzyme is also the subject of studies in the quest of novel ligands aiming insecticides with increasingly efficiency and less harmful both to the environment and human health [16–18]. The majority of techniques used to identify cholinesterase inhibitors in natural or synthetic libraries are based on colorimetric methods applied to enzymes solutions [19]. The biochemical assay based on Ellman’ reaction [20] is by far the most often used. In this assay, the Ellman’ reagent (5,5 -dithiobis(2-nitrobenzoic

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A.F.L. Vilela et al. / J. Chromatogr. B xxx (2013) xxx–xxx

2 CH3

O N

H

eserine O

OH

H

N

5

O

H3CO

6

O

Br

10

O

11

O

O

O

O

7

H3CO

O

O

O

HO

OH

O

HO

O

O

O OCH3

9 O O

O

HO

13

O

O

O

HO N

O OH

OCH3

14

O

O

17 O 16

15

O

O O

O

O

NO2

Br

O

O

O

8

O

4 O

12

O HO

OH

O O

O

O

O

O

O

O

O

O

O O

HO

O

3 N H

HO

OH

OH

O

N

O

O

O

2 O

O

Cl

O

OH H3CO

O

O

N

O

O

1

O

galanthamine

HO

HO

O

O

OH

N

O

H3OC

O

O

H N

N

O

O

N

O

O

O N

19

18

O

O

O

20

Fig. 1. Chemical structures of tested cumarin derivatives and reference compounds.

acid)) reacts with thiocholine, formed in the enzymatic reaction, yielding 5-thio-2-nitro-benzoic acid [yellow anion (YA)] which is monitored by UV–vis spectroscopy. In the literature, the assay has been reported with a series of modifications [11,12,21] that produce varied results, especially when potency inhibition (IC50 ) is being determined [22]. Recently, we reported an online AChE capillary enzyme assay (AChE-ICER) for a small coumarin derivatives library. Inhibition constant (Ki ) and mechanism for the identified hits were also carried out [23,24]. Coumarins are secondary plant metabolites with many biological activities [25–28]. The first study on the inhibitory activity of coumarins on cholinesterase involved 4-hydroycoumarin derivatives and dates back to 1950 [29]. IC50 values ranging from 3 to 100 ␮M have been reported for a 17 coumarin small library, as non-competitive AChE inhibitors [30]. The immobilization of BChE has been reported at different supports [21,31]. For avoiding drawbacks such as secondary interactions between inhibitors and the support, in this work it is reported for the first time the preparation of immobilized capillary enzyme reactors (ICERs) based on BChE and their use in the screening of our coumarin derivatives library (Fig. 1) [23,24,32]. To differentiate BChE from other cholinesterases, we preferred butyrylthiocholine iodide (BTChI) as the substrate since BChE catalyzes also the hydrolysis of acetylthiocholine, but in a less efficient way than AChE [33]. To validate the BChE-ICER online screening assay, eserine and galanthamine were selected as standard reversible inhibitors. The data obtained in the screening assays of the coumarin derivatives (Fig. 1) either with BChE, huAChE- and ee-AChE-ICERs or free enzyme respectively were carried out and the results is herein discussed.

chemicals used during the immobilization procedure were analytical grade and were supplied by Sigma, Merck (Darmstdt, Germany), Synth (São Paulo, Brazil), or Acros (Geel, Belgica). The water used in all the preparations was obtained from a MILLI-Q® system (Millipore, São Paulo, Brazil). The fused silica capillary (0.375 mm × 0.1 mm I.D) was acquired from Polymicro Technologies (Phoenix, AZ, USA). All the buffer solutions were filtered through 0.45 ␮m membrane filters of cellulose nitrate Phenomenex® and degassed before being used in the LC experiments. The samples were prepared in methanol/water (1:1, v/v). 2.2. Apparatus BChE was immobilized onto the capillary using a syringe-pump 341B (Sage instruments, Boston, USA). The resulting BChE-ICER was placed in a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of two LC 20AD pumps. One of the pumps had an FCV-20AL valve for low-pressure gradient, a UV–vis detector (SPD-M20AV), an autosampler equipment (SIL-20A), and a six-port switching sample valve from Valco® . Data acquisition was accomplished on a Shimadzu CBM-20A system interfaced with a computer, using the Shimadzu-LC Solutions (LC Solution 2.1) software (Shimadzu, Kyoto, Japan). A microplate reader system (Elisa readers) Versa Max-Molecular Device (Silicon Valley, CA, USA) was used in the assays involving the free enzyme in solution. The program Sigma Plot 12.0 was employed to construct the kinetic curves. 2.3. Preparation of the ICERs

2. Materials and methods 2.1. Materials The enzyme BChE (EC 3.1.1.8, from the human serum, 50 units/mg) and its substrate butyrylthiocholine iodide (BTChI); 5,5 -dithiobis(2-nitrobenzoic acid) (Ellman’s reagent); galanthamine; and eserine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Glutaraldehyde, buffer components, and all the

Hu-AChE-ICER and ee-AChE-ICER were prepared, as previously described [24,34,35], by covalent enzyme immobilization onto fused silica capillary (0.1 mm I.D × 0.375 mm × 15 cm) using glutaraldehyde as spacer. For preparing the BChE-ICERs, a 0.34 mg mL−1 , 16.93 units mL−1 , enzyme solutions in 50 mM phosphate buffer, pH 8.0, were used and the formed Schiff bases reduced using sodium borohydride solution (5 mg mL−1 ) in 50 mM phosphate buffer, pH 8.0, for 1 h. They were washed, for 5 min, with 50 mM phosphate buffer and, then, for 1 h with a 0.1 M



ARTICLE IN PRESS A.F.L. Vilela et al. / J. Chromatogr. B xxx (2013) xxx–xxx

monoethanolamine solution in 50 mM phosphate buffer, pH 8.0, at room temperature. Finally, a 5 min wash with 50 mM phosphate buffer and 1.0 mL of working buffer was carried out. The immobilization process was done by using a syringe pump at a flow rate of 5 ␮L min−1 . A mobile phase consisting of 0.1 M Tris (pH 8.0 adjusted with hydrochloric acid (HCl) 10%, v/v) and 0.126 mM Ellman’s reagent; designated working buffer, was used as the mobile phase for all the chromatographic BChE-ICER analyses. The prepared ICERs (BChE-ICER, hu-AChE-ICER and ee-AChEICER) were kept with their ends immersed in the working buffer, at 4 ◦ C, when not in use. 2.4. Chromatographic conditions The influence of mobile phase pH and flow rate on the BChEICER activity were evaluated by injecting 10 ␮L aliquots of a 50 mM BTChI solution at different mobile phase pH (7–9) and flow rates (0.01–0.1 mL min−1 ). For evaluating the effect of the pH as a function of time, three BChE-ICERs were prepared and each one evaluated at the following pH values 7.5, 8.0, and 8.5. Analyses were carried out in duplicate in 2-h intervals on a daily basis. For the analyses, the samples (10 ␮L) were injected into the HPLC system operating at a flow rate of 0.05 mL min−1 , with UV detection at = 412 nm, at 25 ◦ C, and the working buffer used as mobile phase.

2.5. Method validation The linearity was evaluated using external calibration curve at a concentration range of 2.5–60 mM. For that, stock solution of BTChI at 250 mM was prepared, and from that, the calibrations solutions were prepared, in triplicate, by addition of 50 ␮L of the appropriate working BTChI solution to a 50 ␮L of working buffer. The solutions were vortex-mixed for 10 s, and 10 ␮L aliquots were injected into the BChE-ICER. The calibration curve for the YA was constructed by plotting the peak area against the injected BTChI concentration. Quality controls (QC) were prepared at three different concentrations 3, 30, and 54 mM. Five samples of each concentration were prepared and analyzed on three nonconsecutive days. To assess the selectivity of the method, chromatograms of the blank buffer (water and working buffer, without BTChI) were examined. 2.6. Determination of BChE-ICERs activity For measuring the BChE-ICER activity, a 10 ␮L of a 50 mM aqueous BTChI solution was injected into the LC system, under the established chromatographic conditions, and the formation of the yellow anion (YA), generated from the reaction of thiocholine and Ellman’s reagent, quantified. The initial reaction rate of the BChE-ICER was determined by a modified Ellman’s procedure [20]. To meet this aim, 10 ␮L of BTChI solutions (1–100 mM) were injected into the BChE-ICER. After 5 min of chromatographic elution, the product of the enzymatic reaction was collected in a 96-well plate containing the Ellman’ reagent. The wells were completed to 250 ␮L with the working buffer, and the absorbance registered at 412 nm using Elisa readers. 2.7. Assays with BChE-ICER 10 ␮L of solutions containing BTChI concentrations ranging from 5 to 100 mM were injected into the system, in duplicate. To validate the method, two known reversible BChE inhibitors, eserine (2–12 nM) and galanthamine (0.005–10.0 ␮M) were evaluated in

3

the presence of 50 mM BTChI solution. The eserine and galanthamine solutions were prepared using 30 ␮L of the working buffer, 20 ␮L of 250 mM BTChI, and volumes of an aqueous stock solution of eserine or galanthamine (1 mM and 1 ␮M, respectively) ranging between 0 and 50 ␮L. The sample volume was completed to 100 ␮L, and 10 ␮L of the sample was injected into the chromatographic system, in duplicate. The peak area relative to the YA was integrated and plotted (Ai ) against the concentration of the inhibitor. Additionally, 10 ␮L of 50 mM BTChI solution without inhibitor was also injected into the chromatographic system, in duplicate, and the area relative to the YA peak was determined as A0 . The enzyme activity was calculated by integrating the area values; according to Eq. (1). % Inhibition = 100 −

A i

A0

× 100

(1)

The type of inhibition and Ki value for eserine were determined under the same experimental conditions for three different concentrations (0.5, 1, and 3 nM) and four different BTChI concentrations (10, 34, 50, and 60 mM), in duplicate. A library containing twenty coumarin derivatives (Fig. 1) [24,32] was used to screen BChE-ICER ligands. A stock solution of each inhibitor with a concentration of 1 mM (MeOH/H2 O 1:1, v/v) was prepared. For analytical purposes, a sample with a final volume of 100 ␮L was obtained by using 20 ␮L of the inhibitor stock solution 20 ␮L of 200 mM BTChI, and 60 ␮L of the working buffer, which resulted in a concentration of 200 ␮M for the candidate compound and 50 mM for BTChI in the working buffer. The samples were prepared in duplicate. Eserine was used as standard inhibitor. In the absence of inhibitor, 10 ␮L of 50 mM BTChI solution was injected into the chromatographic system under the same operating conditions. Compounds that inhibited BChE by ≥70% were used to determine IC50 . An inhibition curve was constructed by plotting the percent inhibition versus the inhibitor concentration used in the assay (5–500 ␮M). The linear regression parameters were calculated, and the IC50 was extrapolated using Sigma Plot 12.0. The type of inhibition and Ki value for the coumarin derivatives 17 and 19 were determined under the same experimental conditions for three different inhibitor concentrations (range from 30 to 165 ␮M) and four different BTChI concentrations (10, 34, 50, and 60 mM). The stability of the BChE-ICER was determined by measuring its activity under established chromatographic conditions on a daily basis, and later accomplished on a weekly basis. Analyses were carried out in duplicate. 2.8. Kinetic and ligand assays with the free enzyme The kinetic and ligand assays were carried out in accordance with the literature [20,36] and will be briefly described below. The assays were carried out using a 96-well microplate with a microplate reader Type Elisa. To a final volume of 250 ␮L, each well was filled with: 125 ␮L of Ellman’s reagent (3 mM in 0.1 M phosphate buffer pH 7.4); 75 ␮L of buffer TRIS (50 mM, pH 8.0); 20 ␮L of enzyme solution at the final concentration 0.28 U/mL (in 0.1 M phosphate buffer pH 7.4); 25 ␮L of the BTChI (30, 50, 70, 90, 110, 130, 150 ␮M). The microplate was shaken for 10 s followed by reading the absorbance at 412 nm at 30 s intervals for 2 min. For the inhibition assay, it was added also a 25 ␮L of each inhibitor sample (100 ␮M) prepared from a 1 mM stock solution. The inhibition percentage was obtained by comparing the absorbance in the presence of inhibitor (Ai ) and in the absence of inhibitor (A0 ) according to Eq. (1). The assays were carried out in duplicate.




4

Fig. 2. Chromatograms corresponding to the yellow anion formed during monitoring of enzymatic activity. (------) With Schift base reduction step. (—) Protocol previously described [24]. Mobile phase: working buffer. Flow: 0.05 mL min−1 and = 412 nm.

2.9. False-positive effects on AChE inhibition in the TLC assay based on Ellman’s method Each coumarin sample (2.5 ␮L) was eluted onto a chromatographic silica gel 60 plate using CHCl3 :MeOH:H2 O 65:30:5 (v/v/v) as mobile phase. After dried, plates were sprayed with a solution, which was incubated at 37 ◦ C for 15 min, containing AChE (0.704 mg), BSA (0.025 g) and ACThI (0.00723 g) in TRIS (19 M, pH 8) (adjusted with HCl 10%, v/v), following by the Ellman’s reagent prepared in TRIS (19 M, pH 8) (adjusted with HCl 10%, v/v). This assay was carried out as described in the literature [37]. 2.10. Data analysis The kinetic parameter values and their respective standard errors were calculated by fitting the data to the appropriate equations, using the Sigma-Plot 12.0, nonlinear regression function (SPSS, Inc.). The Kruskal–Wallis test from Microsoft Office Excel 2007 was used to treat the data obtained on the inhibitory activity assays of the coumarin library (Fig. 1) with the BChE-, hu-AChE- and eeAChE-ICERs, and with the enzymes in solution. For the BChE, only the data obtained with the coumarins 2, 3, 6, 7, 12, and 17–19 were used for the statistical test. The data used from the assays with eeAChE were the one previously reported [24] while the data from the assays with hu-AChE is here published for the first time. 3. Results and discussions 3.1. Preparation and characterization of the BChE-ICER The BChE-ICERs were prepared by covalent immobilization on fused silica capillary as previously reported for other enzymes [24,34,35]. Moreover, the protocol modification including the reduction of the formed Schiff’s bases increased the BChE-ICER activity by almost 35% as illustrated in the chromatograms at Fig. 2. The calibration curves were logarithmic in the ranges studied, with mean coefficients determination (n = 3) of 0.99 or higher. The RSD for the replicates was below 20%, and the accuracy showed a deviation below 15% of the nominal value indicating that no carry over has happened between injections. The values varied from 85 to 95.7%. The inter lot precision, with RSD (n = 5) of 3.77–9.68%, was in the range of the accepted criteria. The limit of quantification was

2.5 mM (RSD = 6.0%, n = 3, accuracy 98%); the limit of detection was 1.0 mM. We used the initial rate of BChE-ICER for the kinetic calculations. For that, the period during which the variation in the substrate concentration was equal to or less than 5% of the initial concentration was used to obtain the initial rate. We calculated the substrate conversion rate as 1%, demonstrating that the enzyme was in the steady state, and enabled us to determine the kinetic parameters. The activity assays as well as the stability evaluations indicated pH 8 as the value to be used. The studies on the flow rate showed that at 0.05 mL min−1 the substrate conversion to product was more efficient when compared with other flow rates evaluated. As a general rule, the type and the optimal amount of an organic modifier is a compromise between the best eluting agent (chromatographic requirements) and the modifier that influences the enzymatic activity (enzymatic requirements) [21]. The effect of EtOH addition at 10, 20, and 80% (v/v) to the mobile phase culminated in a 1.5, 2 and 5-fold increase in the BChE-ICER activity, respectively, as compared to the activity observed without the organic solvent. After one week under extreme condition (80% EtOH), the BChE-ICER still maintained its activity. These results indicated the possibility of using a higher amount of organic solvent in the mobile phase without damaging the enzyme activity. The immobilized enzyme retained its activity for more than twenty months, maintaining 83% of the initial activity after this period. This allowed its use in a high number of experiments. The stability of the BChE-ICER seems to be higher than data described for ICERs based on other enzymes in previously published studies [21,31,38]. We still have not reached the total number of experiments that each ICER can allow. We assessed the repeatability of the immobilization procedure by measuring the activity of three different freshly prepared BChEICERs, in duplicate. The relative standard deviation for the initial activity of the three ICERs (RSD = 6.55%, n = 3) attested to the robustness of the immobilization procedure. 3.2. ICERs and free enzyme assays KM values of 33.60 ± 6.8 mM for BChE-ICER, and of 0.12 ± 0.02 mM for the enzyme in solution, showed that the immobilization procedure decreased the affinity of the enzyme for the substrate. This was expected if one considers the structural changes taking place in the biomolecule after the immobilization process. However, the selectivity and the enzymatic activity were maintained in the bioreactor, which was validated with the reference inhibitors. Eserine and galanthamine are reference reversible competitive inhibitors, whereas eserine is specific for BChE [2]. The BChEICER gave IC50 values for eserine (2.0 ± 0.2 nM) and galanthamine (20 ± 4 nM) that are consistent with BChE’ literature values [39,40]. In the case of eserine, BChE-ICER furnished IC50 values of the same magnitude order of the free BChE (16.0 ± 2.9 nM). The results herein reported demonstrate that the prepared BChE-ICER can identify reference ligands and provide quantitative parameters such as the inhibitory potency. To evaluate the selectivity, the same small coumarin library, previously screened with ee-AChE-ICER [23,24] (Fig. 1) was screened with BChE- and hu-AChE-ICERs. The graphics at Fig. 3 encompasses the results with the ICERs and with the free enzymes. The screened compounds did not show false positive results. Coumarin 20 is a selective AChE inhibitor and it is highly active toward hu-AChE as it is with ee-AChE [23,24] and does not inhibits BChE. In another hand, coumarin 17 is more active toward BChE, IC50 of 109 ± 21 ␮M, and coumarin 19 showed activity against both enzymes IC50 = 128 ± 28 and 144 ± 40 ␮M, respectively. The




5

Fig. 3. (A) Inhibition percentage of coumarin library at 200 ␮M by BChE-ICER and solution enzyme assay. (B) Inhibition percentage of coumarin library at 200 ␮M by huAChE-ICER and solution enzyme assay.

mechanism of action and Ki were determined for these two coumarin derivatives using the BChE-ICER, and the assay studies were validated with eserine. Fig. 4 depicts Lineweaver–Burk graphs. A Ki = 0.0022 ± 0.0002 ␮M was obtained in these experiments, and the rate of enzymatic reaction did not change even in the presence of different inhibitor concentrations. This illustrates a characteristic behavior of a competitive inhibitor [41].

Coumarin derivatives 17 and 19 followed an uncompetitive action mechanism for BChE-ICER with Ki = 108 ± 10 ␮M and 36.0 ± 5.0 ␮M, respectively (Fig. 5). It is important to call attention that coumarin 19 showed a competitive mechanism for ee-AChEICER [24]. Considering that the same coumarin derivative library were screened as inhibitors for BChE, hu-AChE and ee-AChE in two assays

Fig. 4. Dose response curve plots of inhibition percentage (A) Lineweaver–Burk reciprocal plots (B) for eserine (n = 2 for each concentration).




6

Fig. 5. Dose–response curves plots of inhibition percentage for coumarins 17 (A) and 19 (B). Inhibition mechanism studies, Lineweaver–Burk graph: (C) compound 17 and (D) compound 19 (n = 2 for each concentration).

Table 1 Kruskal–Wallis analysis for cholinesterase assays.

BChE-ICER versus free BChE ee-AChE-ICER versus free AChEee hu-AChE-ICER versus free hu-AChE solution BChE-ICER versus hu-AChE-ICER

R1

R2

H

p-Value

73.00 428.00 477.00 137.00

98.00 475.00 426.00 269.00

1.22 0.35 0.41 9.20

0.27 0.55 0.52 0.0024

format (ICERs versus free enzyme), statistical analyses were carried out by using Kruskal–Wallis test to investigate difference between the assays [42]. Table 1 depicts the statistical results obtained. The calculated test statistic H has approximately a chi-square distribution with (k − 1) degrees of freedom, as the number of samples k = 2 has (2 − 1) degrees of freedom, the value 3.84 is critical to 5% level of significance. Thus, for the calculated H values in the range of 1.22 to 0.41, the hypothesis H0 (p value = 0.27–0.52) can be accepted, and there is no difference in the results of the samples as evaluated by two assays format. The data obtained with BChE- and huAChE-ICERs assays were also statistically evaluated and furnished a calculated value H = 9.20, so the hypothesis H0 (p value = 0.0024) should be reject and demonstrates the assays differences. The statistical results validate the ICERs screening model. The result obtained with the comparison of BChE-ICER with hu-AChEICER corroborates the differences in inhibition potency observed within the coumarin library toward the two enzymes and demonstrates as the selectivity of a series of compound can be easily evaluated with ICERs’ screening approach. As advantage this approach can be used as high throughput screening technique. 4. Conclusions Considering the complexity of AD and the interest in the discovery of new therapeutic compounds from natural or synthetic

sources, novel screening procedures that help identify and select hit ligands are important. The novel BChE-ICER herein developed offers many advantages: lower costs, absence of non-specific matrix interactions, and immediate recovery of the enzymatic activity, very short analysis time, and absence of back-pressure. BChE immobilization proved to be efficient and promoted retention of the catalytic activity of the target enzyme with high stability and activity for a period exceeding seventeen months. The ability of BChE-ICER to recognize standard ligands and its application in the screening assay with determination of the inhibition mechanisms of the hits demonstrate its usefulness. The results of the statistical analysis endorsed the cholinesterase’ ICERs assays approach herein reported. Conflict of interest The author(s) declare(s) that they have no conflicts of interest to disclose. Acknowledgements This work was funded by grants of the Sao Paulo State Research Foundation (FAPESP) and the National Council for Technological and Scientific Development (CNPq). Q.B.C., C.L.C., and A.F.L.V. acknowledge CNPq and FAPESP for research and MSc fellowships. References [1] E. Perry, R. Perry, G. Blessed, B. Tomlinson, Neuropathol. Appl. Neurobiol. 4 (1978) 273–277. [2] E. Giacobini, Butyrylcholinesterase Its Function and Inhibitors, Martin Dunitz, London, 2003. [3] E. Giacobini, G. Pepeu, in: E. Giacobini, G. Pepeu (Eds.), The Brain Cholinergic System: in Health and Disease, Informa Healthcare, Oxon, 2006, p. 274. [4] E. Giacobini, Pharmacol. Res. 50 (2004) 433.




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