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

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 ß-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.

A novel on-flow mass spectrometry-based dual enzyme assay.

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.

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

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 ±â€¯0.006 µM and 0.88 ±â€¯0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ±â€¯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.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candidarugosa lipase, metagenomic lipase, ß-galactosidase and ß-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

Copper (II) and zinc (II) complexes with flavanone derivatives: Identification of potential cholinesterase inhibitors by on-flow assays.

Metal chelates strongly influence the nature and magnitude of pharmacological activities in flavonoids. In recent years, studies have shown that a promising class of flavanone-metal ion complexes can act as selective cholinesterase inhibitors (ChEIs), which has led our group to synthesize a new series of flavanone derivatives (hesperidin, hesperetin, naringin, and naringenin) complexed to either copper (II) or zinc (II) and to evaluate their potential use as selective ChEIs. Most of the synthesized complexes exhibited greater inhibitory activity against acetylcholinesterase (AChE) than against butyrylcholinesterase (BChE). Nine of these complexes constituted potent, reversible, and selective ChEIs with inhibitory potency (IC50) and inhibitory constant (Ki) ranging from 0.02 to 4.5µM. Copper complexes with flavanone-bipyridine derivatives afforded the best inhibitory activity against AChE and BChE. The complex Cu(naringin)(2,2'-bipyridine) (11) gave IC50 and Ki values of 0.012±0.002 and 0.07±0.01µM for huAChE, respectively, which were lower than the inhibitory values obtained for standard galanthamine (IC50=206±30.0 and Ki=126±18.0µM). Evaluation of the inhibitory activity of this complex against butyrylcholinesterase from human serum (huBChE) gave IC50 and Ki values of 8.0±1.4 and 2.0±0.1µM, respectively. A Liquid Chromatography-Immobilized Capillary Enzyme Reactor by UV detection (LC-ICER-UV) assay allowed us to determine the IC50 and Ki values and the type of mechanism for the best inhibitors.

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

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 on-flow ICERs assay. Two out of 20 coumarin derivatives could be highlighted: compound 17 is more active toward BChE (IC50=109 ± 21 µM) and 19 showed activity against both enzymes (BChE IC50=128 ± 28 µM and hu-AChE IC50=144 ± 40 µM). 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.