Discovery, Biological Evaluation and Binding Mode Investigation of Novel Butyrylcholinesterase Inhibitors Through Hybrid Virtual Screening

Abstract

Butyrylcholinesterase (BChE), plays a critical role in alleviating the symptoms of Alzheimer's disease (AD) by regulating acetylcholine levels, emerging as an attractive target for AD treatment. This study employed a quantitative structure-activity relationship (QSAR) model based on ECFP4 molecular fingerprints with several machine learning algorithms (XGBoost, RF, SVM, KNN), among which the XGBoost model showed the best performance (AUC = 0.9740). A hybrid strategy integrating ligand- and structure-based virtual screening identified 12 hits from the Topscience core database, three of which were identified for the first time. Among them, piboserod and Rotigotine demonstrated the best BChE inhibitory potency (IC₅₀ = 15.33 μM and 12.76 μM, respectively) and exhibited favorable safety profiles as well as neuroprotective effects in vitro. Notably, Rotigotine, a marketed drug, was newly recognized for its anti-AD potential, with further enzyme kinetic analyses revealing that it acts as a mixed-type inhibitor in a non-competitive mode. Fluorescence spectroscopy, molecular docking, and molecular dynamics simulations further clarified their binding modes and stability. This study provides an innovative screening strategy for the discovery of BChE inhibitors, which not only identifies promising drug candidates for the treatment of AD but also demonstrates the potential of machine learning in drug discovery.

Keywords: binding mode analysis; biological evaluation; butyrylcholinesterase inhibitors; machine learning; molecular docking; molecule dynamics simulations; virtual screening.

Figure 1

(A) The ROC Curve of XGBoost model. The dotted line in the figure represented the performance of the random classifier (AUC = 0.5); (B) The confusion matrix (math.) of XGBoost model, represented True Negative (TN) = 386, False Positive (FP) = 11, False Negative (FN) = 25, and True Positive (TP) = 289, which were the number of BChEIs predicted as BChEIs, non-BChEIs predicted as non-BChEIs, non-BChEIs predicted as BChEIs, BChEIs predicted as non-BChEIs, respectively.

Figure 2

The virtual screening framework.

Figure 3

(A) Toxicity of piboserod, Metergoline, and Rotigotine on PC-12 cells at 10, 25, and 50 μM; (B) Protective effect of piboserod, Metergoline, and Rotigotine against hydrogen peroxide-induced PC-12 cell damage. (The data are expressed as mean ± SD of three independent experiments. *** means p < 0.001; **** means p < 0.0001; ns means no significant difference).

Figure 4

(A) Plots of ν vs. [BChE] for piboserod; (B) Plots of ν vs. [BChE] for Rotigotine; Lineweaver-Burk plots of inhibition of piboserod (C) and Rotigotine (D), the secondary plots represent the Slope vs. [inhibitors] plot and the Y-intercept vs. [inhibitors] plot from top to bottom, respectively.

Figure 5

(A) Fluorescence-quenching spectra of BChE (5 U·mL⁻¹) in the presence of piboserod, with varying concentrations at 298 K, c(piboserod) = 0, 10, 20, 25, 30, 40, and 50 μM for curves a → g, respectively; (B) The Stern-Volmer plots for the fluorescence quenching of BChE by piboserod at 298, 304, and 310 K; (C) Time-resolved fluorescence decay curve of the piboserod-BChE system; (D) Fluorescence quenching spectra of BChE (5 U·mL⁻¹) in the presence of Rotigotine, with varying concentrations at 298 K, c(Rotigotine) = 0, 10, 12, 15, 20, 25, and 30 μM for curves a → g, respectively; (E) The Stern-Volmer plots for the fluorescence quenching of BChE by Rotigotine at 298, 304, and 310 K; (F) Time-resolved fluorescence decay curve of the Rotigotine-BChE system; The Stern-Volmer bilogarithmic plots for the fluorescence quenching of BChE by piboserod (G) and Rotigotine (H) at 298, 304, and 310 K, the secondary plots represent Van’t Hoff plots for the interaction of each with BChE.

Figure 6

3D fluorescence spectra of BchE (A), BChE–piboserod system (B) and BChE–Rotigotine system (C), c(BChE) = 0.2 U·mL⁻¹, c(piboserod) = 50 μM, c(Rotigotine) = 50 μM; Contour plots of 3D fluorescence spectra of BchE (D), BChE–piboserod system (E) and BChE–Rotigotine system (F).

Figure 7

Docking interaction plots of piboserod (A) and Rotigotine (C) with active site residues of BChE (PDB ID 5DYW), Interactions included non-covalent bonds, π interaction, the hydrogen bonds in yellow, π-π stacking in blue, π-cation in green; 2D interaction schematic of piboserod (B) and Rotigotine (D) with BChE. Hydrogen bonds were shown as purple lines, salt bridge was shown as a pink violet gradient, π-π stacking interactions were in green lines, and π-ion bonds were in red lines.

Figure 8

(A) RMSD analysis for piboserod-BChE and Rotigotine-BChE complexes; (B) RMSF plot of each residue in piboserod-BChE and Rotigotine-BChE complexes; (C,D) RMSF plot of the key residues in complexes piboserod-BChE and Rotigotine-BChE obtained from the full RMSF plot.

Figure 9

Time course (A) and relative distribution frequency (B) of SASA in piboserod-BChE and Rotigotine-BChE complexes; Number of hydrogen bonds between BChE and piboserod (C) and Rotigotine (D).