Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

Abstract

A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22,000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer's disease, by detecting dual-binding interactions. Keywords detection method; interferometry; acetylcholinesterase; high-throughput screening; enzyme assayIn recent years, the emphasis of drug discovery and optimization has shifted from traditional methods to rapid microfluidic screening to minimize cost and maximize output. Backscattering interferometry (BSI),[1] a technique which quantifies refractive index (RI) changes arising from intermolecular binding interactions, is a novel biosensing platform that may be useful in drug discovery. Acetylcholinesterase (AChE), a widely studied serine hydrolase that plays pivotal roles in Alzheimer's disease (AD), [2] inflammatory processes, [3] and nerve-agent poisoning, [4] is an interesting system with which to study the use of BSI for the rapid detection of drug candidates. The search for potent AChE inhibitors (AChEI) has been driven largely by the need for an effective AD treatment, and is based on the longstanding cholinergic hypothesis [2] and the more recent amyloid hypothesis. [5] It is wellestablished that AChE accelerates amyloid-beta (Ab) peptide deposition, a process which may be mediated by an interaction between plaque precursors and the peripheral anionic site (PAS) of the enzyme.[6] Recent drug discovery efforts have focused on the design of AChEIs that are able to interact with the PAS to target both cholinergic and noncholinergic AD pathologies. [7] Seminal work in this field has led to the discovery of dual-binding inhibitors, such as bisgalantamine [8] and pseudo-irreversible carbamate inhibitors. [9] Given the large number of studies on several diverse classes of AChEIs, this system is a widely accepted benchmark for the development and testing of novel screening techniques.Traditional methods used to detect interactions with AChE focus on the indirect measurement of substrate procedures. [10] To explore the utility of BSI to detect AChEIs, several known and novel inhibitors with diverse potencies and inhibitory mechanisms have been screened. Herein, we show that BSI can: 1) quantify AChE-inhibitor interactions in the We first wanted to probe the utility of BSI to screen AChEIs using a set of known and novel AChEIs (Scheme 1). Edrophonium, a competitive inhibitor, propidium, a noncompetitive inhibitor, and the mixed inhibitors, 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) and galantamine, were the standards used to validate BSI as a method for the detection of AChEIs. Two separate and distinct saturation curves were observed for inhibitors 4 and 6 (Figure 2 and Supporting Information, Figure S7, respectively). For ligand 4, the dual-binding curve is shown in the graph inset of while the fit of the second bindin...

A Label‐Free Fluorescent Sensor Based on Structure‐Switching Oligonucleotides for the Detection of Ag+, Biothiols and Acetylcholinesterase Activity

“…Based on these founds a highly sensitive and rapid method for AChE activity assay and inhibitor screening are urgently needed. Over the past decades, a number of methods have been developed for AChE quantification, such as electrochemical, colorimetric, interferometric, and chemiluminescent . However, the disadvantages of these assays still exist, for example, time‐consuming, multi‐step organic reaction, low sensitivity, multi‐step operation, and disturbances by the sample matrix.…”