A hyperbranched dopamine-containing PEG-based polymer for the inhibition of α-synuclein fibrillation

Abstract

Aggregation of α-synuclein is believed to play an important role in Parkinson's disease and in other neurodegenerative maladies. Small molecule inhibitors of this process are among the most promising drug candidates for neurodegenerative diseases. Dendrimers have also been studied for anti-fibrillation applications but they can be difficult and expensive to synthetize. Here we show that RAFT polymerization can be used to produce a hyperbranched polyethylene glycol structure via a one-pot reaction. This polymer included a dopamine moiety, a known inhibitor of α-synuclein fibril formation. Dopamine within the polymer structure was capable of aggregation inhibition, although not to the same degree as free dopamine. This result opens up new avenues for the use of controlled radical polymerizations as a means of preparing hyperbranched polymers for anti-fibrillation activity, but shows that the incorporation of functional groups from known small molecules within polymers may alter their biological activity.

Keywords: Dopamine; Hyperbranched polymers; Macromolecular crowding; Parkinson's disease; Protein aggregation; RAFT polymerization; α-synuclein.

Graphical abstract

Fig. 1

Schematic depiction of RAFT polymerization with 1H NMR peak assignment. RAFT co-polymerization of DMA, PEGDA and PEGMEA was carried out in DMF at 70 °C for 17 h to produce a DOPA containing polymer of Mw 18.8 kDa for anti-fibrillation applications.

Fig. 2

Effect of additives on the kinetics of α-synuclein aggregation. (A – C): Kinetic curves for fibril formation from α-synuclein (0.25 mg/mL, pH 7.5, 50 μg/mL heparin, 40 °C) in the presence of dopamine, dopamine-containing polymer (DP) and PEG 12. A – DP; B – dopamine; C PEG 12. Black – no additive, red–0.025% additive, green–0.2% additive, yellow–2% additive. (D–E): Initiation and elongation rates for fibril formation from α-synuclein in the presence of dopamine, DP and PEG 12. D – initiation rate (1/lag phase); E−fibril yield (ThT fluorescence). Black – dopamine, red – DP, green – PEG 12. Scale bars correspond to standard error between independent measurements. (F – I): EM images of aggregates obtained after 5 days of incubation. F – no additive; G – 0.5% DP; H – 0.5% dopamine; I – 0.5% PEG 12. Scale bars: 500 nm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Morphology of α-synuclein aggregates formed in the presence of dopamine at different time points. A – kinetic curve of α-synuclein aggregation in the presence of 0.1% dopamine; B – aggregates at 10 h; C – ggregates at 48 h. Time points are marked with arrows. Scale bars: 200 nm.

Fig. 4

Disaggregation of α-synuclein fibrils by dopamine and DP. A, B – kinetic curves for disaggregation of preformed α-synuclein fibrils (0.05 mg/mL, pH 7.5, 50 μg/mL heparin, 40 °C) in the presence of dopamine or DP. A – dopamine; B – DP. Black – no additive, red–0.025% additive, green–0.5% additive, yellow–2% additive. C – exponential decay rate of ThT fluorescence of α-synuclein fibrils incubated in the presence of DOPA and DP. D – E: morphology of fibrils after incubation with the additive for 15 h. D – no additives, E 0.5% dopamine, F – 0.1% DP.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)