Entacapone and tolcapone, two catechol O-methyltransferase inhibitors, block fibril formation of alpha-synuclein and beta-amyloid and protect against amyloid-induced toxicity

Abstract

Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease (AD). There is considerable consensus that the increased production and/or aggregation of alpha-synuclein (alpha-syn) plays a central role in the pathogenesis of PD and related synucleinopathies. Current therapeutic strategies for treating PD offer mainly transient symptomatic relief and aim at the restitution of dopamine levels to counterbalance the loss of dopaminergic neurons. Therefore, the identification and development of drug-like molecules that block alpha-synuclein aggregation and prevent the loss of dopaminergic neurons are desperately needed to treat or slow the progression of PD. Here, we show that entacapone and tolcapone are potent inhibitors of alpha-syn and beta-amyloid (Abeta) oligomerization and fibrillogenesis, and they also protect against extracellular toxicity induced by the aggregation of both proteins. Comparison of the anti-aggregation properties of entacapone and tolcapone with the effect of five other catechol-containing compounds, dopamine, pyrogallol, gallic acid, caffeic acid, and quercetin on the oligomerization and fibrillization of alpha-syn and Abeta, demonstrate that the catechol moiety is essential for the anti-amyloidogenic activity. Our findings present the first characterization of the anti-amyloidogenic properties of tolcapone and entacapone against both alpha-synuclein and Abeta42 and highlight the potential of this class of nitro-catechol compounds as anti-amyloidogenic agents. Their inhibitory properties, mode of action, and structural properties suggest that they constitute promising lead compounds for further optimization.

SCHEME 1.

Working model illustrating that protein aggregation, i.e. α-syn in PD and Aβ in AD, has a central role in the generation of the cascade of events that result in neurodegeneration and disease.

FIGURE 1.

Chemical structure of compounds examined as inhibitors of human WT α-syn and Aβ42 fibril formation.

FIGURE 2.

Compounds abolish α-syn fibril formation and increase the soluble forms of α-syn after incubation for 72 h. A, samples of 100 μm α-syn were incubated at 37 °C, with continuous shaking, with and without compounds, at molar ratios of α-syn/inhibitor of 1:0.1, 1:0.5, and 1:1. The time course of protein fibrillization was measured by ThT fluorescence assay after incubation for 72 h. The bar graph represents the amount of fibril formation in absence and in presence of the compounds P, GA, CA, Q, T, E, and DA. The figure shows means of three independent experiments ± S.D. (n = 6). The inhibitory effect of the compounds on α-syn aggregation was evaluated by SDS-PAGE. Samples of α-syn (100 μm) were incubated with and without compounds, at molar ratios of α-syn/inhibitor of 1:1. The bands represent the amount of monomeric form of α-syn before incubation and after incubation for 72 h in the absence and in the presence of the compounds. B, electron micrographs of negatively stained quaternary structures deposited from solutions of α-syn before incubation and after 72 h incubation at 37 °C in the absence and in the presence of 100 μm compound. Scale bar, 200 nm.

FIGURE 3.

Compounds have an inhibitory effect on the α-syn seeding polymerization. A, samples of monomeric α-syn (100 μm) were incubated with the seeds (2 μm) at 37 °C, with continuous shaking, without (panel a) and with (panels b–h) 10 and 50 μm of compounds P, GA, CA, Q, T, E, and DA. The time course of protein fibrillization was measured every 30 min by ThT fluorescence assay for 3 h. B, electron micrographs of negatively stained quaternary structures deposited from solutions of seeds and α-syn + seeds after 3 h of incubation in the absence and in the presence of the compounds (50 μm). Scale bar represents 200 nm.

FIGURE 4.

Analysis of compound binding to monomeric α-syn by NMR spectroscopy. Changes in individual cross-peak positions (A–C) and intensities (D–F) of backbone ¹⁵N-¹H resonances of α-syn (60 μm) in two-dimensional ¹H-¹⁵N HSQC spectra in the presence of compounds E (A and D), T (B and E), and Q (C and F). For compounds E and F, molar ratios of 1:10 α-syn/compound were used. Compound Q is less soluble, and only the 1:2 α-syn/compound ratio could be measured. Horizontal lines indicate the average variation of chemical shifts observed for α-syn from sample to sample due to slightly different buffer conditions.

FIGURE 5.

Protective effect of the compounds against α-syn-induced toxicity in PC12 cells. PC12 cells were treated with preincubated α-syn (40 μm) alone or co-incubated with 5 μm GA, CA, Q, T, and E and 40 μm P and DA. The cellular viability was evaluated by MTT assay, and the data were expressed as percentage of the control (nontreated cells). The control treatment is set to 100%. Bars are means ± S.E. We used # to compare the data to the control and * with respect to the treatments with α-syn. ##, p < 0.001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.

FIGURE 6.

Compounds inhibit LMW Aβ42 fibril formation. A, samples of LMW Aβ42 were incubated at 37 °C with and without compounds, at molar ratios of Aβ42/inhibitor of 1:2. The time course of protein fibrillization was measured by ThT fluorescence assay. The bar graph represents the amount of fibril formation in the absence and presence of the compounds P, GA, CA, Q, T, E, and DA. The samples containing the compounds showed a decrease of the ThT fluorescence signal after 48 h of incubation. The figure shows means of three independent experiments ± S.D. (n = 6). B, electron micrographs of negatively stained quaternary structures deposited from solutions of LMW Aβ42 (10 μm) before and after 48 h of incubation at 37 °C in the absence and in the presence of 20 μm of the compounds listed. Scale bar, 200 nm.

FIGURE 7.

Compounds prevent the conversion of Aβ42 protofibrils into mature fibrils in a specific and concentration-dependent manner. A, samples of PF Aβ42 stock solutions were prepared by dissolving the peptide in 5% DMSO, 2 m Tris base, pH 7.6. Samples of Aβ42 were incubated at 37 °C with and without compounds, at molar ratios of Aβ42/inhibitor of 1:2. The time course of protein fibrillization was measured by ThT fluorescence assay. The bar graph represents the amount of fibril formation in the absence and presence of the compounds P, GA, CA, Q, T, E, and DA. The samples containing the compounds showed a decrease of the ThT fluorescence signal after 48 h of incubation. The figure shows means of three independent experiments ± S.D. (n = 6). B, electron micrographs of negatively stained quaternary structures deposited from solutions of PF Aβ42 (10 μm) before incubation and after 48 h of incubation at 37 °C in the absence and presence of 20 μm of the compounds listed. Scale bar, 200 nm.

FIGURE 8.

Aβ42 monomeric seeding polymerization assays revealed that Q, E, and T have an inhibitory effect on the kinetics. Samples of monomeric Aβ42 (10 μm) were incubated with the seeds (2 μm) at 37 °C, with continuous shaking, without (A) and with (B–H) 20 μm of compounds P, GA, CA, Q, T, E, and DA. The time course of protein fibrillization was measured every 30 min by ThT fluorescence assay for 3 h.

FIGURE 9.

Protective effect of the compounds against Aβ42-induced toxicity in PC12 cells. PC12 cells were treated with Aβ42 (40 μm) or co-treated in presence of the compounds P, GA, CA, Q, T, E, and DA. The cells were treated with two different compound concentrations, 5 and 20 μm for 24 h. The cellular viability was evaluated by MTT assay, and the data were expressed as percentage of control (nontreated cells). The control treatment is set to 100%. Error bars are means ± S.E. We used # to compare the data to the control and * with respect to the treatments with Aβ42 crude preparation. ##, p < 0.001; ***, p < 0.001; **, p < 0.01.