Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Abstract

Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.

Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays

“…Cell membranes, whole cells, antibodies, enzymes, nucleic acids, and other proteins can be immobilized on diverse surfaces using microarray technology without losing their functional structure. As a result, they are used in immunochemistry, autoradiography, radioligand and binding investigations, mitochondrial toxicity assays, as well as other techniques such as colorimetry and mass spectrometry [ 33 , 34 , 35 , 36 , 37 ]. Microarrays allow the reduction of the number of samples, medications, chemicals, and residues.…”

Quantification of PD-1/PD-L1 Interaction between Membranes from PBMCs and Melanoma Samples Using Cell Membrane Microarray and Time-Resolved Förster Resonance Energy Transfer

“…This microarray technology maintains the function of receptors, enzymes, and other proteins, providing a powerful tool for the screening of drugs and antibodies. To this end, several assays, including radioligand binding studies, enzymatic analyses, and immunoassays, have been performed using CMMAs [13][14][15] (Appendix A); however, to date, none have been used to study membranemembrane interactions using proteins expressed in membranes in close proximity.…”

“…CMMA can be used to support membrane protein studies [12], since the integrity of the cell membrane and the conservation of the lipid environment remain stable, allowing a physiological analysis of receptor functional states [13]. It should be noted that CMMA has already been successfully used in several studies to measure the expression and functional activity of membrane proteins [13][14][15]. In addition, this miniaturized high-throughput tool allows the use of scarce samples to generate hundreds of microarrays that can be employed to perform consecutive analyses.…”

Study of Tissue-Specific Reactive Oxygen Species Formation by Cell Membrane Microarrays for the Characterization of Bioactive Compounds

“…This study has described the application of cell membrane microarrays to determine the specific actions of antimalarial drugs on human tissues by monitoring the production of superoxide, one of the main ROS, as indicator of celular stress. For this purpose, firstly, the experimental protocol was validated using CMMA from a set of rat brain areas, whose stability and functionality has already been confirmed by different methods, together with specific inhibitors of the oxidative phosphorylation [53][54][55][56][57].…”