Discovery of Azaindolin-2-One as a Dual Inhibitor of GSK3β and Tau Aggregation with Potential Neuroprotective Activity

Abstract

The inhibition of glycogen synthase kinase 3β (GSK3β) activity through pharmacological intervention represents a promising approach for treating challenging neurodegenerative disorders like Alzheimer's disease. Similarly, abnormal tau aggregate accumulation in neurons is a hallmark of various neurodegenerative diseases. We introduced new dual GSK3β/tau aggregation inhibitors due to the excellent clinical outcome of multitarget drugs. Compound (E)-2f stands out among the synthesized inhibitors as a promising GSK3β inhibitor (IC₅₀ 1.7 µM) with a pronounced tau anti-aggregation effect in a cell-based model of tauopathy. Concurrently, (E)-2f was demonstrated to be non-toxic to normal cells, making it a promising neuroprotective lead compound that needs further investigation.

Keywords: Alzheimer’s disease; GSK3β; Tau; azaindolin-2-one; neurofibrillary tangles; neuroprotective; protein aggregation.

Figure 1

GSK3β and protein aggregation inhibitors.

Figure 2

Design of our novel compounds as GSK3β inhibitors.

Figure 3

General procedure for the synthesis of compounds 2a–f.

Figure 4

General procedure for the synthesis of compounds 3a–d.

Figure 5

Structure-activity relationship (SAR) study of compounds 2a–f and 3a–d as novel GSK3β inhibitors.

Figure 6

(A) Comparison between the binding mode of the native ligand (magenta stick model) and its superposed re-docked ligand (green stick model) within the active site of GSK3β (PDB ID: 4IQ6) predicted by Molecular Operating Environment (MOE) 2019.01. (B) The top-scoring docked pose of (E)-2f. (C) Comparison between the binding mode of the native ligand (magenta stick model) and (E)-2f (green stick model); hydrogen bonds are shown as black dashed lines, and the critical amino acid residues are displayed as a cyan stick model. (D) 2D illustration (E)-2f shows two crucial interactions with the two essential amino acid residues: (E) the key for different interactions shown in 2D depiction.

Figure 6

(A) Comparison between the binding mode of the native ligand (magenta stick model) and its superposed re-docked ligand (green stick model) within the active site of GSK3β (PDB ID: 4IQ6) predicted by Molecular Operating Environment (MOE) 2019.01. (B) The top-scoring docked pose of (E)-2f. (C) Comparison between the binding mode of the native ligand (magenta stick model) and (E)-2f (green stick model); hydrogen bonds are shown as black dashed lines, and the critical amino acid residues are displayed as a cyan stick model. (D) 2D illustration (E)-2f shows two crucial interactions with the two essential amino acid residues: (E) the key for different interactions shown in 2D depiction.

Figure 7

The top-ranked pose for compounds 2a (A), 2b (B), 2c (C), 2d (D), 2e (E) and 3c (F) (green stick model) in their E configuration within the active site of GSK3β (PDB ID: 4IQ6) predicted by MOE 2019.01. Hydrogen bonds are shown as black dashed lines, and the critical amino acid residues are displayed as a cyan stick model.

Figure 7

The top-ranked pose for compounds 2a (A), 2b (B), 2c (C), 2d (D), 2e (E) and 3c (F) (green stick model) in their E configuration within the active site of GSK3β (PDB ID: 4IQ6) predicted by MOE 2019.01. Hydrogen bonds are shown as black dashed lines, and the critical amino acid residues are displayed as a cyan stick model.

Figure 8

Comparison between the binding mode of E and Z configurations for compounds 3a (A,B), 3b (C,D), and 3d (E,F) (green stick model) within the active site of GSK3β (PDB ID: 4IQ6) predicted by MOE 2019.01. Hydrogen bonds are shown as black dashed lines, while H–π interactions are red dashed lines with the critical amino acid residues (cyan stick model).

Figure 8

Comparison between the binding mode of E and Z configurations for compounds 3a (A,B), 3b (C,D), and 3d (E,F) (green stick model) within the active site of GSK3β (PDB ID: 4IQ6) predicted by MOE 2019.01. Hydrogen bonds are shown as black dashed lines, while H–π interactions are red dashed lines with the critical amino acid residues (cyan stick model).

Figure 9

Anti-tau aggregation activity of compounds (E)-2f and 3a at 1, 3, 10, and 30 µM concentrations. (A) Tau proteins were stained with green fluorescence in HeLa cells transfected with the expression vector of tau 2N4R isoform carrying P301L mutation and incubated with the compounds. Microscopic observation was performed, and photos were shown on a 100 µm scale. Seed negative (seed−) and seed positive (seed+) indicate the absence and presence of fibrils of tau protein with P301L mutation, respectively. (B) Relative green spots per area (%) after exposure of Hela cells to compounds (E)-2f and 3a are compared to seed positive (seed+ = 100%). ImageJ counted more than 100 spots in the same area in each sample. *: denotes significant difference from control (seed+) at p < 0.05, **: denotes significant difference from control (seed+) at p < 0.01.

Figure 9

Anti-tau aggregation activity of compounds (E)-2f and 3a at 1, 3, 10, and 30 µM concentrations. (A) Tau proteins were stained with green fluorescence in HeLa cells transfected with the expression vector of tau 2N4R isoform carrying P301L mutation and incubated with the compounds. Microscopic observation was performed, and photos were shown on a 100 µm scale. Seed negative (seed−) and seed positive (seed+) indicate the absence and presence of fibrils of tau protein with P301L mutation, respectively. (B) Relative green spots per area (%) after exposure of Hela cells to compounds (E)-2f and 3a are compared to seed positive (seed+ = 100%). ImageJ counted more than 100 spots in the same area in each sample. *: denotes significant difference from control (seed+) at p < 0.05, **: denotes significant difference from control (seed+) at p < 0.01.

Figure 10

Effect of compounds (E)-2f and 3a on Tau levels. HeLa cells transfected with the expression vector of tau 2N4R isoform carrying P301L mutation and incubated with compounds (E)-2f and 3a at 10 and 30 µM concentrations for 24 h. Then Hela cells were lysed, and the lysates were analyzed by Western blotting. The relative amount of total tau expressed in the total cell lysate of Hela cells of each lane was quantified by ImageJ. The total tau level of Hela cells with positive seed (seed+) alone was calculated from total tau after GAPDH normalization and is presented as Control (1.00).