Design, Synthesis and Biological Evaluation of α-Synuclein Proteolysis-Targeting Chimeras

Abstract

α-Synuclein aggregation under pathological conditions is one of the causes of related neurodegenerative diseases. PROTACs (proteolysis targeting chimeras) are bifunctional small molecules that induce a post-translational erasure of proteins via the ubiquitination of target proteins by E3 ubiquitin ligase and subsequent proteasomal degradation. However, few research studies have been conducted for targeted protein degradation of α-synuclein aggregates. In this article, we have designed and synthesized a series of small-molecule degraders 1-9 based on a known α-synuclein aggregation inhibitor sery384. In silico docking studies of sery384 with α-synuclein aggregates were accomplished to ensure that the compounds bound to α-synuclein aggregates specifically. The protein level of α-synuclein aggregates was determined to evaluate the degradation efficiency of PROTAC molecules on α-synuclein aggregates in vitro. The results show that compound 5 had the most significant degradation effect, with DC₅₀ of 5.049 μM, and could induce the degradation of α-synuclein aggregates in a time- and dose-dependent manner in vitro. Furthermore, compound 5 could inhibit the elevation of the ROS level caused by overexpression and aggregation of α-synuclein and protect H293T cells from α-synuclein toxicity. Conclusively, our results provide a new class of small-molecule degraders and an experimental basis for the treatment of α-synuclein related neurodegenerative diseases.

Keywords: PROTAC (proteolysis-targeting chimera); protein aggregates; α-synuclein.

Figure 1

(a) Design of PROTAC compounds 1–9. (b) Docking pose of sery384 with α-synuclein aggregates (PDB: 2N0A). The image was visualized using PyMOL 1.5. (c) The PROTAC molecules (3 × 3 combination).

Scheme 1

Synthesis of compounds 1–6. (a) DHP, p-TsOH, THF, rt. (b) Pd/C, H₂, THF, 1 h, rt. (c) K₂CO₃, KI, DMF, 90 °C, 24h. (d) CF₃COOH, DCM, 0.5 h, rt. (e) DIPEA, DMF, 90 °C, 24 h. (f) DIPEA, HATU, DMF, rt. (g) 2N NaOH, (Boc)₂O, acetone, 0 °C to rt.

Scheme 2

Synthesis of compounds 7–9. (a) K₂CO₃, KI, DMF, 90 °C, 24 h. (b) CF₃COOH, DCM, 0.5 h, rt. (c) DIPEA, HATU, DMF, rt.

Figure 2

PROTACs induced degradation of α-synuclein aggregates in vitro. After the α-synuclein overexpression and aggregation model was constructed on H293T cells, the cells were treated with compounds 1–9 at different concentrations (10, 20 and 40 μM) for 24 h. The level of α-synuclein aggregates was determined by Western blot. n = 3 independent experiments from three separate cell cultures. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a loading reference. Statistical significance was assessed by one-way ANOVA (n. s.: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001).

Figure 3

Compound 5 induced degradation of α-synuclein aggregates in a dose- and time-dependent manner. (a,b) Western blot results and quantitative analysis of α-synuclein aggregates in model cells after treatments with compound 5 at different concentrations (from 1.25 to 20 μM) for 48 h. GAPDH was selected as a loading reference. n = 3 independent experiments from three separate cell cultures. (c,d) Western blot results and quantitative analysis of α-synuclein aggregates in model cells after treatments with 20 μM compound 5 for various time periods (0~48 h). GAPDH was selected as a loading reference. n = 3 independent experiments from three separate cell cultures. Statistical significance was assessed by one-way ANOVA (n. s.: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.005).

Figure 4

Compound 5 rescued the elevation of ROS level caused by α-synuclein overexpression. (a) Representative confocal LSM images of DCFH-DA-stained H293T cells under different conditions. (b) Quantitative analysis of the images in (a). The ROS level was displayed by the MFI ratio of green (DCF) to blue (Hoechst) channels. n = 3 independent experiments from three separate cell cultures. Statistical significance was assessed by one-way ANOVA (*: p < 0.05). “#” represents the p < 0.05 statistical significance between Control and Model.