PROTAC-Induced Glycogen Synthase Kinase 3β Degradation as a Potential Therapeutic Strategy for Alzheimer's Disease

Abstract

Glycogen synthase kinase 3β (GSK-3β) is a serine/threonine kinase and an attractive therapeutic target for Alzheimer's disease. Based on proteolysis-targeting chimera (PROTAC) technology, a small set of novel GSK-3β degraders was designed and synthesized by linking two different GSK-3β inhibitors, SB-216763 and tideglusib, to pomalidomide, as E3 recruiting element, through linkers of different lengths. Compound 1 emerged as the most effective PROTAC being nontoxic up to 20 μM to neuronal cells and already able to degrade GSK-3β starting from 0.5 μM in a dose-dependent manner. PROTAC 1 significantly reduced the neurotoxicity induced by Aβ_25-35 peptide and CuSO₄ in SH-SY5Y cells in a dose-dependent manner. Based on its encouraging features, PROTAC 1 may serve as a starting point to develop new GSK-3β degraders as potential therapeutic agents.

Keywords: Alzheimer’s disease; chemical knockdown; glycogen synthase kinase 3β; protein degradation; proteolysis targeting chimeras.

Figure 1

(a) Design strategy leading to GSK-3β-directed PROTAC 1–4. Black circles represent tethering points. (b) MD-extracted structure of the 1–GSK-3β (left) and 3–GSK-3β (right) complexes showing H-bond contacts with kinase residues.

Scheme 1. Synthesis of the Precursors 8 and 14

Reagents and conditions. (a) tert-butyl bromoacetate, K₂CO₃, acetone, reflux, 12 h, 50% yield; (b) ethyl chlorooxoacetate, diethyl ether, rt, 12 h, N₂, 42% yield; (c) 2-(2,4-dichlorophenyl)acetamide, KOtBu, DMF, rt, 12 h, N₂, 39% yield; (d) tert-butanol, EDCI, DMAP, THF, rt, 12 h, 77% yield; (e) K₂CO₃, H₂O/MeOH, rt, 12 h, 79% yield; (f) 1,1′-thiocarbonyldi-2(1H)-pyridone, DCM, rt, 12 h, 55% yield; (g) naphthyl isocyanate, sulfuryl dichloride, THF, rt, 12 h, N₂ then air, THF, rt, 30 min, 57% yield; (h) TFA, DCM, rt, 12 h, 81% yield.

Scheme 2. Synthesis of the Target Compounds 1–4

Reagents and conditions. (a) 8, EDCI, HOBT, DIPEA, DMF, rt, 12 h, 32% yield for 1, 25% yield for 3; (b) 14, EDCI, HOBT, DIPEA, DMF, rt, 12 h, 48% yield for 2, 44% yield for 4.

Figure 2

Degradation GSK-3β protein by compounds 1–4, SB-216763, and tideglusib in SH-SY5Y cells. (a) GSK-3β protein level after 48 h of treatment with all compounds (10 μM); (b) GSK-3β protein level after 48 h of treatment with compound 1 (0.5–10 μM); (c) GSK-3β protein level after 24 h of treatment with compound 1 (10 μM) and lactacystin (1.25–5 μM). GSK-3β protein level was measured by Western blotting. Data are expressed as mean ± SEM of three independent experiments (**p < 0.01 and ***p < 0.001 vs untreated cells, ^§§§p < 0.001 vs cells treated with compound 1, at one-way ANOVA with Dunnett or Bonferroni post hoc test).

Figure 3

Compound 1 reduced the neurotoxicity induced by CuSO₄ and Aβ_25–35 in SH-SY5Y cells. (a) Cells were incubated for 24 h with compound 1 (0.5–1 μM), SB-216763, and tideglusib (1 μM) in the presence of CuSO₄ (150 μM); (b) cells were incubated for 2 h with compound 1 (0.5–1 μM) and further 3 h in the presence of Aβ_25–35 (10 μM). At the end of incubation, the cell viability was measured by MTT assay as described in the Methods section. Data are reported as mean ± SEM of three independent experiments (*p < 0.05 and ***p < 0.001 vs cells treated with CuSO₄; *p < 0.05 and **p < 0.01 vs cells treated with Aβ_25–35 at one-way ANOVA with Dunnett post hoc test).