Review One ring is sufficient to inhibit α-synuclein aggregation https://doi.org/10.4103/1673-5374.320973 Samuel Peña-Díaz1, 2, Salvador Ventura1, 2, 3,* Date of submission: January 17, 2021 Abstract Date of decision: February 4, 2021 Parkinson’s disease, the second most prevalent neurodegenerative disorder worldwide, Date of acceptance: March 17, 2021 is characterized by a progressive loss of dopaminergic neurons in substantia nigra pars compacta, causing motor symptoms. This disorder’s main hallmark is the formation of Date of web publication: August 4, 2021 intraneuronal protein inclusions, named Lewy bodies and neurites. The major component of these arrangements is α-synuclein, an intrinsically disordered and soluble protein that, in pathological conditions, can form toxic and cell-to-cell transmissible amyloid structures. Preventing α-synuclein aggregation has attracted significant effort in the search for a disease-modifying therapy for Parkinson’s disease. Small molecules like SynuClean-D, epigallocatechin gallate, trodusquemine, or anle138b exemplify this therapeutic potential. Here, we describe a subset of compounds containing a single aromatic ring, like dopamine, ZPDm, gallic acid, or entacapone, which act as molecular chaperones against α-synuclein aggregation. The simplicity of their structures contrasts with the complexity of the aggregation process, yet the block efficiently α-synuclein assembly into amyloid fibrils, in many cases, redirecting the reaction towards the formation of non-toxic off-pathway oligomers. Moreover, some of these compounds can disentangle mature α-synuclein amyloid fibrils. Their simple structures allow structure-activity relationship analysis to elucidate the role of different functional groups in the inhibition of α-synuclein aggregation and fibril dismantling, making them informative lead scaffolds for the rational development of efficient drugs. Key Words: amyloid; aromatic rings; dopamine; inhibition; neurodegeneration; oligomers; Parkinson’s disease; protein aggregation; α-synuclein Introduction on PubMed for periods between 2000 and 2020 by using key Parkinson’s disease (PD) is a neurodegenerative disorder terms listed in the keywords section. In PubMed, the Related that affects > 4% of people over 60 years worldwide, Citations function and citation tracking was used to retrieve being the second most prevalent neurodegenerative additional articles. Additionally, the reference lists of selected disease after Alzheimer’s disease. PD belongs to a group of articles were used to identify further significant studies. All pathologies named synucleinopathies, which also includes articles were manually checked for the relevant parts by both Dementia with Lewy Body and Multiple System Atrophy, authors. The last search was conducted in January 2021. among others. Although significant differences in terms of symptomatology, progression, and the affected cellular and α-Synuclein and Parkinson’s Disease anatomical compartments, these disorders share a common α-Syn is an intrinsically disordered protein whose sequence histopathological hallmark: proteinaceous deposits, whose can be dissected in three different regions (Fusco et al., 2014). main component are amyloid fibrils of α-synuclein (α-syn) The N-terminal domain is a highly conserved region that (Spillantini et al., 1997). In PD, these inclusions accumulate contains the KTKGEV repeats and an amphipathic character in the body (Lewy bodies) and processes (Lewy neurites) of that regulates membrane-binding; there map all the missense dopaminergic neurons in the substantia nigra pars compacta, mutations associated with early-onset and higher penetrance causing progressive cell death and dopamine decline, which of PD. The central domain, named the non-amyloid translates into motor symptoms like bradykinesia, rigidity, component, is characterized by a highly hydrophobic character or resting tremor. Moreover, cortical Lewy bodies are also that drives α-syn amyloid aggregation and constitutes mature observed, especially in dementia with Lewy bodies patients aggregates’ core. The C-terminal domain contains several (Mattila et al., 2000). Mutations and multiplications of the negatively charged residues that seem to counteract and SNCA gene, which encodes for α-syn, are linked to early-onset regulate the aggregation propensity, as it can be inferred from and familial PD cases, evidencing the connection between the higher aggregation capacity of the C-terminally truncated α-syn aggregation and PD (Lazaro et al., 2014). α-syn. In normal conditions, α-syn is a soluble monomer that can adopt a helical structure by interacting with membranes, Search Strategy and Selection Criteria consistent with its role in vesicle trafficking. Besides, α-syn For the present review, we searched the literature published can also be found as a soluble tetramer (Bartels et al., 2011). 1 Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Spain; 2Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain; 3Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain *Correspondence to: Salvador Ventura, PhD, salvador.ventura@uab.cat. https://orcid.org/0000-0002-9652-6351 (Salvador Ventura) How to cite this article: Peña-Díaz S, Ventura S (2022) One ring is sufficient to inhibit α-synuclein aggregation. Neural Regen Res 17(3):508-511. 508 ｜NEURAL REGENERATION RESEARCH｜Vol 17｜No. 3｜March 2022 However, under pathological conditions, α-syn self-assembles animal models, such as epigallocatechin gallate, NPT200-11, into toxic supramolecular conformations. CLR01, trodusquemine, or SynuClean-D (Pujols et al., 2018, 2020). Many of these molecules present complex structures Recombinant technology has allowed reproducing intracellular characterized by a planar hydrophobic core from which they α-syn aggregation in vitro, revealing that it follows sigmoidal project polar ramifications. The hydrophobic moiety often kinetics as in other amyloidogenic proteins. During this has an aromatic character, and it is thought to drive the process, monomeric α-syn assembles into oligomeric interaction with apolar exposed regions in α-syn assemblies, and protofibrillar species, which precedes amyloid fibrils’ which are essential for fibril elongation. Polar projections formation. Recent studies demonstrate that these metastable would interfere with hydrophobic packing and might disrupt species, rather than mature fibrils, are responsible for α-syn hydrogen bonding networks, interfering with elongation toxicity in neuronal cells (Winner et al., 2011). Moreover, and propagation and, eventually, dismantling pre-formed oligomeric and protofibrillar structures can be transmitted to aggregates. Some of the most potent inhibitors exhibit several neighbouring cells and seed the aggregation of native α-syn aromatic rings, like SynuClean-D, epigallocatechin gallate, in a prion-like manner, thus propagating the aggregation or CLR01, containing two, three, and five aromatic rings, throughout the brain. Notably, α-syn seeds could present respectively. It seems to be a certain connection between different conformations, as previously described for the the number of aromatic rings and the molecule’s inhibitory prion protein strains. Distinct α-syn strains seem to target potential, as illustrated by CLR03, a short and inactive version different brain regions and cell types and might explain of the CLR01 molecular tweezer. A larger aromatic area is neuropathological differences between synucleinopathies (Lau assumed to result in a tighter binding to α-syn hydrophobic et al., 2020). assemblies. However, potent α-syn aggregation inhibitors consisting of a single aromatic ring have been described in Targeting α-Synuclein Aggregation recent years (Figure 1). α-Syn aggregation has become an important target to develop disease-modifying therapies for PD. Strategies include Single-Ring Polyphenolic Inhibitors of accelerating aggregate clearance by promoting autophagy, gene-silencing to reduce protein concentration, or developing α-Synuclein Aggregation small molecules that interfere with the polymerization (Pujols Polyphenols constitute one of the primary sources of et al., 2020). α-Syn is an abundant brain protein and molecules α-syn aggregation inhibitors, and most of the single-ring interacting with its monomeric form could impact its function; inhibitors fall into this group. Dopamine, epinephrine, therefore, inhibitors that only interact with aggregated and and norepinephrine, along with several analogues, were toxic species are preferred. the first single-ring inhibitors described (Li et al., 2004). These catecholamines interfered with the in vitro and in The lack of a well-defined three-dimensional structure for vivo aggregation of both Aβ and α-syn, driving the reaction monomeric, oligomeric, and protofibrillar α-syn precludes towards off-pathway oligomers’ formation. However, the lack the rational design and optimization of drugs that bind these of inhibition in anaerobic and acidic conditions demonstrated species. In this context, high-throughput screening protocols that these catecholamines’ oxidative products were the have become the primary strategy to identify such kinds of most active structures, consistent with catecholamines’ molecules. This strategy has rendered different compounds susceptibility to oxidation (Li et al., 2004). The mechanism that inhibit α-syn amyloid formation in vitro and/or in behind this effect is still unclear, and probably different A B H I L M C J K N D E O P Q F G Figure 1 ｜ Structure of single-ring inhibitors of α-synuclein (α-syn) aggregation. In blue, catechol and catecholamine inhibitors norepinephrine (A), epinephrine (B), dopamine (C), entacapone (D), pyrogallol (E), hydroxytyrosol (F), and hydroquinone (G). In pink, phenolic acids, protocatechuic (H), ferulic (I), gallic (J), and caffeic (K) prevent α-syn amyloid aggregation. In gray, other phenolic structures as safranal (L), 576755 (M), and thymoquinone (N) with the capacity to inhibit α-syn aggregation. Non-phenolic inhibitors scyllo-inositol (O), selegiline (P), and ZPDm (Q) in yellow. NEURAL REGENERATION RESEARCH｜Vol 17｜No. 3｜March 2022｜509 Review processes coexist. Some studies indicate that inhibition is α-syn in vitro and an MPTP-induced mice model of PD (Ardah due to a covalent interaction of the compound with tyrosine et al., 2019). Another compound that deserves mention is or lysine residues, while others suggest that the compound- safranal, a carotenoid with inhibitory and disaggregation mediated oxidation of methionine in α-syn is necessary for capacity, at high doses, whose hydrophobic interactions with inhibition and off-pathway oligomers formation (Oliveri, 2019). α-syn increase its solubility (Save et al., 2019). One of the last Non-covalent interactions with the non-amyloid component single-ring phenolic compounds found is compound 576755, domain and the C-terminal YEMPS α-syn sequence have a quinone-based structure discovered by applying a new high- also been proposed for dopamine and norepinephrine. throughput strategy based on chemical microarray surface The lower potency of tyramine, which lacks one of the two plasmon resonance imaging (Toth et al., 2019). The compound hydroxyl groups of dopamine, illustrates aromatic ring polar exhibited a significant but undeciphered inhibitory effect in projections’ critical role in inhibition (Latawiec et al., 2010). fibril and oligomers formation, also acting in seeded reactions. Single-ring phenolic acids such as ferulic acid, caffeic acid, Treatment with 576755 attenuated the A53T α-syn mediated protocatechuic acid, and gallic acid were also found to be dopaminergic loss in cellular models, without impacting cell- active molecules (Ono and Yamada, 2006; Di Giovanni et al., to-cell transmission. 2010; Hornedo-Ortega et al., 2016). Ferulic acid is a potent inhibitor of α-syn aggregation in vitro, and the first phenolic Nevertheless, phenolic compounds are not the only one- compound described to destabilize mature fibrils; further ring structures with α-syn anti-aggregation capacity. Scyllo- analysis demonstrated that it drives α-syn aggregation inositol, a natural-sugar derivate displaying a planar structure towards off-pathway structures (Ono and Yamada, 2006). that inhibits aggregation of α-syn and Aβ, by diverting the Similarly, caffeic acid, gallic acid, and protocatechuic acid, reaction towards the formation of off-pathway structures, described as inhibitors of α-syn and Aβ aggregation, all exerts a neuroprotective effect in cell cultures (Ibrahim and promoted the formation of stable off-pathway amorphous McLaurin, 2016). The absence of activity in chiro-inositol aggregates, consistent with their lack of effect in the lag phase suggests that the planar structure plays a fundamental role of the reaction (Hornedo-Ortega et al., 2016). Nevertheless, in masking aggregates’ self-recognition sites (Ibrahim and only in the case of α-syn seeding was prevented, indicating McLaurin, 2016). Phenethylamine-based structures, as protein-specific activity. NMR detected no direct interaction selegiline, also prevent the formation of α-syn aggregates. with the monomeric form; instead, gallic and protocatechuic This particular single-ring compound significantly delays the acids’ showed fibril-destabilization capacity. Docking analysis lag phase, impacting the nuclei’s formation and inducing demonstrated the interaction of caffeic acid with aggregated the accumulation of amorphous and annular structures of α-syn (PDB: 1XQ8). However, as this interaction was analyzed both wild-type and A30P α-syn variants (Braga et al., 2011). using micelle-bound α-syn, it may not mimic the contacts Also, a destabilizing fibril capacity has been described for this occurring in vivo. Gallic acid also inhibited the aggregation compound. One of the latest single-ring inhibitor compounds of the familial A53T variant of α-syn by transient and low- discovered is ZPDm (Pena-Diaz et al., 2020), which shares affinity interactions. Structure-Activity Relationship (SAR) these molecules’ typical architecture, with polar, non- analysis with several molecules’ derivates demonstrates that hydroxylic projections coming out from a benzene ring. the phenyl group is not sufficient by itself to inhibit fibril This minimal structure is highly related to that of the three- formation. Besides, the number (trihydroxybenzoic acid > rings containing ZPD-2 aggregation inhibitor (Pena-Diaz et dihydroxybenzoic acid > monohydroxybenzoic acid), position, al., 2019), but their action mechanisms are different. ZPDm and conjugation of the hydroxyl groups at the benzoic acid inhibits the aggregation of α-syn at substoichiometric ratios, appendage are crucial activity determinants. Notably, a higher displaying a better pharmacological profile than ZPD-2. The effect could be observed when these polar moieties were compound was very active against the oligomer-promoting displayed consecutively. A30P and H50Q α-syn variants, suggesting that it could interact with these low-molecular-weight species. Besides, the compound significantly blocked seeded transmission in Other Phenolic and Non-Phenolic Modulators of protein misfolding cyclic amplification assays and exhibited α-Synuclein Amyloid Aggregation a strong fibril disassembling capacity, with the reactive vinyl Other single-ring phenols, such as catechols and quinones, sulfone group likely involved in this last activity. Noteworthy, have also been described as anti-aggregational compounds. the in vitro inhibitory and remodeling activities of ZPDm could Hydroxytyrosol, but not tyrosol and hydroxyphenyl acetic acid, be recapitulated in a Caenorhabditis elegans model of PD. both lacking a single hydroxyl group, inhibits the aggregation of α-syn, and as the above-described phenolic acids, they Concluding Remarks and Future Perspectives mainly stabilize off-pathway oligomers (Hornedo-Ortega et Overall, single-ring inhibitors represent small (< 300 g/mol, al., 2018). Entacapone and pyrogallol, which present multiple < 200 Å2), easy-to-synthesize structures, which often display consecutive hydroxyl groups, prevented the aggregation better ADME properties than complex anti-aggregational and seeding of α-syn up to 90% at high compound: protein compounds. Nevertheless, they can fail to cross the blood- ratios, although entacapone was more effective at low ratios brain barrier or even induce significant toxicity, as shown (Di Giovanni et al., 2010). These molecules act by diverting for some polyphenols (Kobayashi et al., 2020). As a general toxic intermediates towards off-pathway nontoxic species, trend, these compounds’ effect seems to be biased towards which is confirmed by the lack of NMR binding signature the generation of off-pathway aggregates, likely because to the monomeric protein. Another catechol structure that the single-ring structure interacts with a common protein presents anti-aggregational capacity is hydroquinone, one of hydrophobic patch, despite some of them being also the tobacco components (Hong et al., 2009). It acts on both potent fibril disruptors and propagation inhibitors. Despite wild-type and A30P familial variants, inducing the formation some debate, these off-pathways structures are generally of stable off-pathway oligomers. Interaction studies revealed considered to hold low intrinsic toxicity and immunogenicity. that hydroquinone establishes covalent interactions via Overall, they are, in our opinion, under-investigated molecules Michael additions with the side chains of neighboring lysine that constitute excellent tools to decipher which groups and residues placed at the interface of two α-syn molecules in an in which position are required for the long-pursued goal of oligomer (Hong et al., 2009). Thymoquinone, a structurally developing disease-modifying compounds to treat PD and/or related quinone, also inhibited the aggregation and seeding of other synucleinopathies. 510 ｜NEURAL REGENERATION RESEARCH｜Vol 17｜No. 3｜March 2022 Author contributions: Review conception and design, data collection, Lazaro DF, Rodrigues EF, Langohr R, Shahpasandzadeh H, Ribeiro T, Guerreiro analysis and interpretation, manuscript writing, manuscript review and P, Gerhardt E, Krohnert K, Klucken J, Pereira MD, Popova B, Kruse N, review guiding: SPD and SV. Both authors approved the final version of Mollenhauer B, Rizzoli SO, Braus GH, Danzer KM, Outeiro TF (2014) this manuscript. Conflicts of interest: The authors declare no conflicts of interest. Systematic comparison of the effects of alpha-synuclein mutations on its Financial support: None. oligomerization and aggregation. PLoS Genet 10:e1004741. Copyright license agreement: The Copyright License Agreement has Li J, Zhu M, Manning-Bog AB, Di Monte DA, Fink AL (2004) Dopamine and been signed by all authors before publication. L-dopa disaggregate amyloid fibrils: implications for Parkinson’s and Plagiarism check: Checked twice by iThenticate. Peer review: Externally peer reviewed. Alzheimer’s disease. FASEB J 18:962-964. Open access statement: This is an open access journal, and articles Mattila PM, Rinne JO, Helenius H, Dickson DW, Roytta M (2000) Alpha- are distributed under the terms of the Creative Commons Attribution- synuclein-immunoreactive cortical Lewy bodies are associated with NonCommercial-ShareAlike 4.0 License, which allows others to remix, cognitive impairment in Parkinson’s disease. Acta Neuropathol 100:285- tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical 290. terms. Oliveri V (2019) Toward the discovery and development of effective modulators of alpha-synuclein amyloid aggregation. Eur J Med Chem References 167:10-36. Ardah MT, Merghani MM, Haque ME (2019) Thymoquinone prevents Ono K, Yamada M (2006) Antioxidant compounds have potent anti- neurodegeneration against MPTP in vivo and modulates alpha-synuclein fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro. aggregation in vitro. Neurochem Int 128:115-126. J Neurochem 97:105-115. Bartels T, Choi JG, Selkoe DJ (2011) alpha-Synuclein occurs physiologically as a Pena-Diaz S, Pujols J, Conde-Gimenez M, Carija A, Dalfo E, Garcia J, Navarro helically folded tetramer that resists aggregation. Nature 477:107-110. S, Pinheiro F, Santos J, Salvatella X, Sancho J, Ventura S (2019) ZPD-2, a Braga CA, Follmer C, Palhano FL, Khattar E, Freitas MS, Romao L, Di Giovanni S, small compound that inhibits alpha-Synuclein amyloid aggregation and its Lashuel HA, Silva JL, Foguel D (2011) The anti-Parkinsonian drug selegiline seeded polymerization. Front Mol Neurosci 12:306. delays the nucleation phase of alpha-synuclein aggregation leading to the Pena-Diaz S, Pujols J, Pinheiro F, Santos J, Pallares I, Navarro S, Conde-Gimenez formation of nontoxic species. J Mol Biol 405:254-273. M, Garcia J, Salvatella X, Dalfo E, Sancho J, Ventura S (2020) Inhibition Di Giovanni S, Eleuteri S, Paleologou KE, Yin G, Zweckstetter M, Carrupt of alpha-Synuclein aggregation and mature fibril disassembling with a PA, Lashuel HA (2010) Entacapone and tolcapone, two catechol minimalistic compound, ZPDm. Front Bioeng Biotechnol 8:588947. O-methyltransferase inhibitors, block fibril formation of alpha-synuclein Pujols J, Pena-Diaz S, Pallares I, Ventura S (2020) Chemical chaperones as and beta-amyloid and protect against amyloid-induced toxicity. J Biol Chem novel drugs for Parkinson’s disease. Trends Mol Med 26:408-421. 285:14941-14954. Pujols J, Pena-Diaz S, Lazaro DF, Peccati F, Pinheiro F, Gonzalez D, Carija A, Fusco G, De Simone A, Gopinath T, Vostrikov V, Vendruscolo M, Dobson CM, Navarro S, Conde-Gimenez M, Garcia J, Guardiola S, Giralt E, Salvatella Veglia G (2014) Direct observation of the three regions in alpha-synuclein X, Sancho J, Sodupe M, Outeiro TF, Dalfo E, Ventura S (2018) Small that determine its membrane-bound behaviour. Nat Commun 5:3827. molecule inhibits alpha-synuclein aggregation, disrupts amyloid fibrils, and Hong DP, Fink AL, Uversky VN (2009) Smoking and Parkinson’s disease: does prevents degeneration of dopaminergic neurons. Proc Natl Acad Sci U S A nicotine affect alpha-synuclein fibrillation? Biochim Biophys Acta 1794:282- 115:10481-10486. 290. Save SS, Rachineni K, Hosur RV, Choudhary S (2019) Natural compound Hornedo-Ortega R, Cerezo AB, Troncoso AM, Garcia-Parrilla MC (2018) safranal driven inhibition and dis-aggregation of alpha-synuclein fibrils. Int J Protective effects of hydroxytyrosol against alpha-synuclein toxicity on Biol Macromol 141:585-595. PC12cells and fibril formation. Food Chem Toxicol 120:41-49. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M Hornedo-Ortega R, Alvarez-Fernandez MA, Cerezo AB, Richard T, Troncoso (1997) Alpha-synuclein in Lewy bodies. Nature 388:839-840. AMA, Garcia-Parrilla MAC (2016) Protocatechuic acid: inhibition of fibril Toth G, Neumann T, Berthet A, Masliah E, Spencer B, Tao J, Jobling MF, formation, destabilization of preformed fibrils of amyloid-beta and alpha- Gardai SJ, Bertoncini CW, Cremades N, Bova M, Ballaron S, Chen XH, Mao synuclein, and neuroprotection. J Agric Food Chem 64:7722-7732. W, Nguyen P, Tabios MC, Tambe MA, Rochet JC, Junker HD, Schwizer D, Ibrahim T, McLaurin J (2016) alpha-Synuclein aggregation, seeding and et al. (2019) Novel small molecules targeting the intrinsically disordered inhibition by scyllo-inositol. Biochem Biophys Res Commun 469:529-534. structural ensemble of alpha-Synuclein protect against diverse alpha- Kobayashi H, Murata M, Kawanishi S, Oikawa S (2020) Polyphenols with anti- Synuclein mediated dysfunctions. Sci Rep 9:16947. amyloid beta aggregation show potential risk of toxicity via pro-oxidant Winner B, Jappelli R, Maji SK, Desplats PA, Boyer L, Aigner S, Hetzer C, Loher T, properties. Int J Mol Sci 21:3561. Vilar M, Campioni S, Tzitzilonis C, Soragni A, Jessberger S, Mira H, Consiglio Latawiec D, Herrera F, Bek A, Losasso V, Candotti M, Benetti F, Carlino E, Kranjc A, Pham E, Masliah E, Gage FH, Riek R (2011) In vivo demonstration that A, Lazzarino M, Gustincich S, Carloni P, Legname G (2010) Modulation of alpha-synuclein oligomers are toxic. Proc Natl Acad Sci U S A 108:4194- alpha-synuclein aggregation by dopamine analogs. PLoS One 5:e9234. 4199. Lau A, So RWL, Lau HHC, Sang JC, Ruiz-Riquelme A, Fleck SC, Stuart E, Menon S, Visanji NP, Meisl G, Faidi R, Marano MM, Schmitt-Ulms C, Wang Z, Fraser C-Editors: Zhao M, Qiu Y; T-Editor: Jia Y PE, Tandon A, Hyman BT, Wille H, Ingelsson M, Klenerman D, et al. (2020) alpha-Synuclein strains target distinct brain regions and cell types. Nat Neurosci 23:21-31. NEURAL REGENERATION RESEARCH｜Vol 17｜No. 3｜March 2022｜511