Objective Used glycogen synthase kinase-3 beta (GSK-3β) as a model target to design and synthesize proteolysis-targeting chimera (PROTAC) molecules with potential radioprotective effects, and preliminarily evaluated their biological activities, thereby providing a new research idea for the development of novel radioprotective drugs.

Methods Two structurally distinct PROTAC molecules (N1 and N2) were designed and synthesized. The cytotoxicity of N1 and N2 in human intestinal epithelial cells-6 was evaluated using cell counting kit-8 assay. The cells were divided into blank group, control group (drug concentration of 0 μmol/L), N1 group treated with different concentrations (0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, and 100.0 μmol/L), N2 group treated with different concentrations (0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, and 100.0 μmol/L), and SB216763 group treated with different concentrations (1.0, 5.0, 10.0, 25.0, 50.0, and 100.0 μmol/L). Western blotting concentration dependent degradation experiment was used to detect the effect of different concentrations of N1 and N2 on the degradation activity of GSK-3β protein. The cells were divided into control group (drug concentration of 0 μmol/L), N1 group with different concentrations ( 5, 10, 15, 20, 25, and 30 μmol/L), N2 group with different concentrations (5, 10, 15, 20, 25, and 30 μmol/L), and 5 μmol/L SB216763 group. Western blotting time dependent degradation experiment was used to evaluate the effect of 15 µmol/L N2 on the degradation activity of GSK-3β protein at different incubation times. The cells were divided into control group and groups treated with 15 µmol/L N2 for different incubation times (6, 12, 24, and 48 h). Western blotting was also used to detect the expression of deoxyribonucleic acid (DNA) ligase Ⅳ. The cells were divided into irradiation group, irradiation+15 µmol/L N2 group, and irradiation+5 µmol/L SB216763 group. Colony formation assay was conducted to evaluate the long-term proliferation and colony formation ability of the cells. The cells were divided into irradiation group, irradiation+5 μmol/L SB216763 group, irradiation+N2 group with different concentrations (15, 30, and 45 μmol/L). Immunofluorescence assay was used to detect the expression of phosphorylated histone H2A variant (γ-H2AX). Independent sample t-test was used for comparison between two groups of quantitative data.

Results N1 and N2 were synthesized successfully. Compared with the control group, no significant difference of cell viability was observed in cells treated with N1 and N2 of 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, and 100.0 μmol/L (t=0.254–0.472, all P>0.05), indicating that N1 and N2 have little toxicity to cells. Western blotting concentration dependent degradation experiment showed that, no significant degradation effect of GSK-3β was observed in the 5−30 μmol/L N1 group cells compared to the control group (t=0.387–0.723, all P>0.05), while the 15 µmol/L N2 treated cells exhibited the highest degradation activity toward GSK-3β (t=6.504, P<0.05). Western blotting time dependent degradation experiment revealed that, compared to the control group, GSK-3β degradation was induced after 6 h of 15 μmol/L N2 treatment, peaked at 12 h (t=3.961, 4.095; both P<0.05), and weakened after 24 h. Colony formation assay demonstrated that the cell survival fraction in the irradiation+15 µmol/L N2 group was significantly higher than that in the irradiation group. Immunofluorescence assay indicated a significant decrease in mean fluorescence intensity in the irradiation+15 µmol/L N2 group compared to the irradiation group (t=8.764, P<0.05), demonstrating that N2 significantly alleviated radiation-induced DNA damage. Furthermore, Western blotting indicated that, compared to the irradiation group, the relative expression of DNA ligase Ⅳ was significantly increased in the irradiation+15 µmol/L N2 group (t=3.045, P<0.05).

Conclusions Two PROTAC molecules, N1 and N2 were designed and synthesized. N2 effectively induced GSK-3β degradation, alleviated radiation-induced DNA damage, and exerted significant radioprotective effects.