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. 2021 Jul 8:9:708374.
doi: 10.3389/fchem.2021.708374. eCollection 2021.

Co-Immobilized Capillary Enzyme Reactor Based on Beta-Secretase1 and Acetylcholinesterase: A Model for Dual-Ligand Screening

Adriana Ferreira Lopes Vilela  1 , Vitor Eduardo Narciso Dos Reis  1 , Carmen Lúcia Cardoso  1
Affiliations

Co-Immobilized Capillary Enzyme Reactor Based on Beta-Secretase1 and Acetylcholinesterase: A Model for Dual-Ligand Screening

Adriana Ferreira Lopes Vilela et al. Front Chem. .

Abstract

We have developed a dual enzymatic system assay involving liquid chromatography-mass spectrometry (LC-MS) to screen AChE and BACE1 ligands. A fused silica capillary (30 cm × 0.1 mm i.d. × 0.362 mm e.d.) was used as solid support. The co-immobilization procedure encompassed two steps and random immobilization. The resulting huAChE+BACE1-ICER/MS was characterized by using acetylcholine (ACh) and JMV2236 as substrates. The best conditions for the dual enzymatic system assay were evaluated and compared to the conditions of the individual enzymatic system assays. Analysis was performed in series for each enzyme. The kinetic parameters (KMapp) and inhibition assays were evaluated. To validate the system, galantamine and a β-secretase inhibitor were employed as standard inhibitors, which confirmed that the developed screening assay was able to identify reference ligands and to provide quantitative parameters. The combination of these two enzymes in a single on-line system allowed possible multi-target inhibitors to be screened and identified. The innovative huAChE+BACE1-ICER/MS dual enzymatic system reported herein proved to be a reliable tool to identify and to characterize hit ligands for AChE and BACE1 in an enzymatic competitive environment. This innovative system assay involved lower costs; measured the product from enzymatic hydrolysis directly by MS; enabled immediate recovery of the enzymatic activity; showed specificity, selectivity, and sensitivity; and mimicked the cellular process.

Keywords: acetylcholinesterase; beta-secretase1; co-immobilization procedure; dual enzymatic system assay; screening dual- target ligands.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Activities of huAChE-ICER and BACE1-ICER in individual enzymatic system assays as a function of the pH of the mobile phase.
FIGURE 2
FIGURE 2
Overlap of extracted ion chromatogram obtained after injection of 50 µM ACh into the huAChE-ICER-LC-MS system and 50 µM JMV2236 into the BACE1-ICER-LC-MS system. The products of enzymatic hydrolysis were monitored at pH 4.5.
FIGURE 3
FIGURE 3
Michaelis-plots of the product area obtained for huAChE-ICER by using ACh as the substrate. (A) mobile phase pH 4.5 and (B) mobile phase pH 8.0. See Table 1 for conditions.
FIGURE 4
FIGURE 4
Inhibitory potency curve obtained for huAChE-ICER by using ACh as a substrate. (A) mobile phase pH 4.5 and (B) mobile phase pH 8.0. See Table 1 for conditions.
FIGURE 5
FIGURE 5
Mass spectrum obtained after injection of JMV2236 (100 µM) into the huAChE+BACE1-ICER-LC-MS system. (A) The product of enzymatic hydrolysis ([M+H]+ m/z 464.2 and 542.1) was monitored. (B) Mass spectrum obtained after injection of ACh (70 µM) into the huAChE+BACE1-ICER-LC-MS system by monitoring the product of enzymatic hydrolysis ([M+H]+ m/z 104) (Conditions listed in Table 1).
FIGURE 6
FIGURE 6
Michaelis-Menten plots of the product area obtained for huAChE (A) and BACE1 (B) in the huAChE+BACE1-ICER-LC-MS system by using ACh and JMV2236 as substrates, respectively.
FIGURE 7
FIGURE 7
IC50 curve obtained for huAChE (A) and BACE1 (B) in the huAChE+BACE1-ICER-LC-MS system by using ACh and JMV2236 as substrates, respectively.
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