Identification of Compounds for Butyrylcholinesterase Inhibition

Abstract

Butyrylcholinesterase (BChE) is a nonspecific cholinesterase enzyme that hydrolyzes choline-based esters. BChE plays a critical role in maintaining normal cholinergic function like acetylcholinesterase (AChE) through hydrolyzing acetylcholine (ACh). Selective BChE inhibition has been regarded as a viable therapeutic approach in Alzheimer's disease. As of now, a limited number of selective BChE inhibitors are available. To identify BChE inhibitors rapidly and efficiently, we have screened 8998 compounds from several annotated libraries against an enzyme-based BChE inhibition assay in a quantitative high-throughput screening (qHTS) format. From the primary screening, we identified a group of 125 compounds that were further confirmed to inhibit BChE activity, including previously reported BChE inhibitors (e.g., bambuterol and rivastigmine) and potential novel BChE inhibitors (e.g., pancuronium bromide and NNC 756), representing diverse structural classes. These BChE inhibitors were also tested for their selectivity by comparing their IC₅₀ values in BChE and AChE inhibition assays. The binding modes of these compounds were further studied using molecular docking analyses to identify the differences between the interactions of these BChE inhibitors within the active sites of AChE and BChE. Our qHTS approach allowed us to establish a robust and reliable process to screen large compound collections for potential BChE inhibitors.

Keywords: BChE inhibitors; butyrylcholinesterase; colorimetric BChE assay; molecular docking; qHTS.

Figure 1.

Hierarchical clustering results of the 125 follow-up compounds. The clustering was performed using Euclidean distance with the complete linkage method based on ToxPrint fingerprints generated within the publicly available ChemoTyper application.

Figure 2.

Concentration–response curves of representative compounds in BChE and AChE assays. (A) Bambuterol hydrochloride, (B) tacrine hydrochloride, (C) physostigmine, (D) diethylumbelliferyl phosphate, (E) bentamapimod, (F) fenoverine, (G) turofexorate isopropyl, and (H) NDT 9513727. Each value represents the mean ± SD of three independent experiments.

Figure 3.

Molecular docking analyses for selected BChE inhibitors. Both (A) bentamapimod and (B) turofexorate isopropyl interacted with key residues in the BChE active site, but (C) bentamapimod and (D) turofexorate isopropyl did not bind to key residues in the AChE active site. The magenta dotted lines are the H-bond interactions with the amino acid residues (cyan lines with colored atoms) of the protein (gray cartoon), and the inhibitors shown as green sticks with colored atoms. The structures of protein–ligand docked complexes were analyzed using the PyMOL visualization tool.