Entacapone and Tolcapone, Two Catechol O-Methyltransferase Inhibitors, Block Fibril Formation of α-Synuclein and β-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 ␣-synuclein (␣-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 ␣-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 ␣-syn and ␤-amyloid (A␤) 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 ␣-syn and A␤, demonstrate that the catechol moiety is essential for the antiamyloidogenic activity. Our findings present the first characterization of the anti-amyloidogenic properties of tolcapone and entacapone against both ␣-synuclein and A␤42 and highlight the potential of this class of nitro-catechol compounds as antiamyloidogenic agents. Their inhibitory properties, mode of action, and structural properties suggest that they constitute promising lead compounds for further optimization. Parkinson disease (PD)2 is the second most common neurodegenerative disorder after Alzheimer disease (AD), affecting nearly 1-2% of the population 65 years and older. A characteristic early pathological change associated with PD is the selective loss of dopaminergic neurons of the substantia nigra pars compacta and other areas of the brain resulting in the degeneration of the nigro-striatal tract and loss of dopamine (DA) (1). Current therapeutic strategies for treating PD offer mainly transient symptomatic relief by aiming to restore the loss of dopamine by "dopamine replacement therapy." This is accomplished through the administration of levodopa (L-DOPA), a direct precursor of DA and other drugs that increase the lifetime of DA by slowing its metabolism. Catechol O-methyltransferase inhibitors (ICOMT), monoamine oxidase B inhibitors (IMAO B), and peripheral aromatic L-amino acid decarboxylase inhibitors (IAADC) are used as adjunctive medications to L-DOPA to slow DA degradation and increase the availability of brain DA (Scheme

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

“…These findings are also consistent with nucleated polymerization-linked toxicity as Tolcapone is known to inhibit fibril growth without disrupting the preformed fibrils. 22 Moreover, the substitution of monomeric α-syn by monomeric β-syn in the mixture composition did not promote any toxicity in primary neurons or in M17 cells (Figure 5b and Supplementary Figure S5B), indicating that α-syn fibrillar growth and seeding capacity play key roles in mediating α-syn toxicity.…”

“…The final concentration of α-syn oligomers was determined by amino acid analysis. inhibitor of α-syn fibrillization 22 or by replacing monomeric α-syn by β-synuclein (β-syn), a close α-syn homolog that lacks the NAC (non-Aβ component) region, does not form fibrils 36 and inhibits α-syn fibrillization and inclusion formation in vitro 37 and in vivo, 38 respectively. Remarkably, the addition of Tolcapone significantly reduced α-syn mixture-induced toxicity in both M17 cells and primary neurons, indicating that fibrillar growth contributes to the toxic event (Figure 5a and Supplementary Figure S5A).…”

“…These findings are not in agreement with previous reports demonstrating increased cellular toxicity in neuroblastoma cell lines upon treatment with extracellular α-syn fibrils, and low toxicity level with α-syn monomers. [22][23][24][25]43 It is noteworthy that the starting α-syn material used in these studies was not fully characterized, and that the ratio of monomeric to aggregated α-syn species was not assessed or reported. Therefore, we cannot rule out the possibility that the α-syn preparations used in these studies contained mixtures of monomers and aggregated forms (oligomers or fibrils) of α-syn.…”

“…Although the toxic effects of exogenous recombinant α-syn have been thoroughly investigated in different cellular models, 7,17,18,[22][23][24][25] the relative contribution of monomeric, oligomeric and fibrillar forms of α-syn to the overall toxicity remains controversial. Therefore, the identification of toxic α-syn species and the molecular mechanisms by which they contribute to neurodegeneration is required to better…”

“…Additional evidence for a causal role of extracellular α-syn in PD come from in vivo studies and cell culture models: (1) intracranial injections of pathological forms of α-syn, isolated from LBs or old mice, as well as recombinant α-syn fibrils, were shown to nucleate further α-syn aggregation, pathology spreading and trigger neurodegeneration in vivo in wildtype (WT) or transgenic mice [11][12][13][14] and rhesus monkeys; 15 (2) recombinant extracellular α-syn aggregates are internalized in cultured cells and seed the aggregation of endogenous α-syn; 12,[16][17][18] and (3) extracellular α-syn activates microglia that initiates or enhances nigral neurodegeneration. [19][20][21] Although the toxic effects of exogenous recombinant α-syn have been thoroughly investigated in different cellular models, 7,17,18,[22][23][24][25] the relative contribution of monomeric, oligomeric and fibrillar forms of α-syn to the overall toxicity remains controversial. Therefore, the identification of toxic α-syn species and the molecular mechanisms by which they contribute to neurodegeneration is required to better understand how extracellular α-syn contributes to PD pathogenesis and to develop novel strategies for the diagnosis and treatment of PD and other synucleinopathies.…”