Significance Parkinson’s disease is characterized by the accumulation of amyloid deposits in dopaminergic neurons, mainly composed of the protein α-synuclein. The disordered nature of α-synuclein and its complex aggregation reaction complicate the identification of molecules able to prevent or revert the formation of these inclusions and the subsequent neurodegeneration. By exploiting a recently developed high-throughput screening assay, we identified SynuClean-D, a small compound that inhibits α-synuclein aggregation, disrupts mature amyloid fibrils, prevents fibril propagation, and abolishes the degeneration of dopaminergic neurons in an animal model of Parkinson’s disease.

Abstract Parkinson’s disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D–treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein–induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson’s disease.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer’s disease (AD) and is still incurable (1). PD is the most common synucleinopathy, a group of neurodegenerative disorders that includes dementia with Lewy bodies and multiple system atrophy (MSA), among others (2, 3). Although the synucleinopathies are multifactorial disorders, the molecular events triggering the pathogenic breakthrough of the disease converge to the abnormal aggregation of α-synuclein (α-Syn) in dopaminergic neurons (4, 5). α-Syn aggregation also occurs in oligodendrocytes in patients with MSA (6). α-Syn is an intrinsically disordered protein, which is expressed at high levels in the brain. α-Syn function is thought to be related to vesicle trafficking (7). This wild-type protein is the main component of cytoplasmic Lewy bodies (LB) and Lewy neurites (LN) in sporadic PD (8). In addition, dominantly inherited mutations in α-Syn, as well as multiplications of the gene encoding for α-Syn (SNCA), cause familial forms of PD (9).

Interfering with α-Syn aggregation has been envisioned as a promising disease-modifying approach for the treatment of PD (1). However, the disordered nature of α-Syn precludes the use of structure-based drug design for the discovery of novel molecules able to modulate α-Syn aggregation. Therefore, many efforts have focused on the analysis of large collections of chemically diverse molecules to identify lead compounds (10). Recently, we have developed an accurate and robust high-throughput screening methodology to identify α-Syn aggregation inhibitors (11). Here, we describe the properties of SynuClean-D (SC-D), a small molecule identified with this approach (SI Appendix, Fig. S1). We first performed a detailed in vitro biophysical characterization of the inhibitory and disaggregation activities of SC-D and tested its performance in human neural cells. Finally, we validated the effects in vivo in two well-established Caenorhabditis elegans models of PD, which express α-Syn either in muscle cells or in dopaminergic neurons. The inhibitor reduced α-Syn aggregation, improved motility, and protected against neuronal degeneration.

Discussion α-Syn aggregation plays a major pathophysiological role in the development of PD. Since its discovery and subsequent identification as the most abundant protein in Lewy bodies (3), α-Syn was shown to be important for a number of cellular processes (7). Therapeutic strategies targeting the aggregation of α-Syn thus hold the promise to result in disease modification and mitigate pathology in PD (33). The screening of large chemical libraries has rendered promising molecules that inhibit the progression of PD by targeting the aggregation of α-Syn, like BIOD303 (34) or anle138b (35). Here, by exploiting a previously developed high-throughput methodology, we identified SC-D as a metabolically stable compound able to inhibit >50% of α-Syn amyloid formation in vitro when employed in a 0.7:1 (protein:SC-D) ratio. The ability of SC-D to inhibit α-Syn aggregation was confirmed by light-scattering and TEM assays. SC-D was also active against the aggregation of α-Syn mutants that cause familial forms of the disease. The activity of SC-D was concentration-dependent and still evident at a substoichiometric 7:1 protein:compound ratio. This already suggested that, unlike other compounds, SC-D does not bind to monomeric α-Syn, supported by NMR experiments. From a therapeutic perspective, this is an important advantage, as SC-D is not expected to interfere with the physiological functions of the soluble protein. PMCA experiments indicated that SC-D might disrupt α-Syn amyloid assemblies, a property that was confirmed by the ability of SC-D to reduce the amount of formed amyloid fibrils, almost independent of the stage of the aggregation reaction at which it was added. Indeed, SC-D is very effective at disassembling clusters of aged α-Syn amyloid fibrils, conceptually similar to the α-Syn amyloid inclusions recurrently observed in the dopaminergic neurons of PD patients. This is important, because the disassembly of preformed amyloid fibrils has been traditionally challenging, and very few molecules have been reported to break down amyloid fibrils (27) because of their high stability. Computational analysis suggests that the disrupting activity of SC-D is mediated by its binding to an inner cavity in α-Syn fibrils. The disassembly of large fibrils has been seen traditionally as a risky strategy for the amelioration of aggregation-linked diseases, because it may increase the population of smaller toxic species (12). However, our in vivo experiments demonstrate that this is not the case for SC-D. The ability of SC-D to target preformed fibrils might have important implications for the prion-like pathological spreading of α-Syn aggregates in the brain (4). By disentangling transmissible fibrillary assemblies, SC-D might reduce templated seeding and thus aggregate-catalyzed conversion of soluble α-Syn molecules into their insoluble forms. It has been suggested that to attain a sustainable spreading and prevent dilution of aggregates as they propagate from cell to cell, a process of aggregate amplification is required, in addition to templated seeding (36). PMCA emulates these particular conditions in vitro. The potency of SC-D in blocking PMCA-induced amplification of α-Syn fibrils is thus promising. SC-D displayed low toxicity for neural cells and displayed cellular permeability. Indeed, SC-D treatment at concentrations as low as 1 µM significantly reduced the number of α-Syn inclusions and the number of inclusions per cell. These data prompted us to assess the effects of SC-D treatment in in vivo models of α-Syn aggregation. First, we selected a well-validated C. elegans model of PD that expresses human α-Syn in muscular cells (21). When worms were treated with SC-D at preadult stages, we observed an important decrease in the number of α-Syn inclusions and a significant recovery of motility in adult PD worms. However, independent of whether a compound targets the early stages of aggregation or disrupts mature protein inclusions, or both, the final aim of a PD-oriented therapy is not to interfere with α-Syn aggregation per se but instead to prevent the neuronal degeneration associated with this phenomenon. It is in this therapeutic context where SC-D stands out, since it is able to increase by more than threefold the number of animals with intact DA neurons in a C. elegans model in which the expression of human α-Syn is directly connected to dopaminergic degeneration (31).

Conclusions In the present study, we describe how drug-screening efforts have crystallized in the discovery of a molecule able to inhibit α-Syn aggregation, both in vitro and in vivo, without interacting significantly with functional, monomeric, and soluble α-Syn. SC-D is a nontoxic molecule that exhibits a unique capability to interact with and disassemble amyloid fibrils, a property that is likely connected to its ability to prevent the α-Syn–promoted degeneration of dopaminergic neurons. Taken together, SC-D constitutes a very promising lead compound for the development of a novel therapeutic molecule for disease modification in PD and other synucleinopathies.

Materials and Methods Protein purification, metabolic stability assays, in vitro aggregation studies, protein-misfolding cyclic amplification, molecular dynamics simulations, NMR studies, cytotoxicity assays, in-cell aggregation studies, and the C. elegans models of PD are described in detail in SI Appendix.

Acknowledgments We thank the Infraestructura Científica y Técnica Singular NMR facility at Centres Científics i Tecnològics de la Universitat de Barcelona for help with NMR, Amable Bernabé at Institut de Ciència de Materials de Barcelona–Consejo Superior de Investigaciones Científicas for help with nanoparticle tracking analysis, and Anna Villar-Pique for help with plasmid construction. The worm strain UA196 used for neurodegeneration assays was a generous gift of Dr. Guy A. Caldwell. S.V. and T.F.O. are supported by Fundación La Marato de TV3 (Ref. 20144330). T.F.O. is supported by the Deutschen Forschungsgemeinschaft Center for Nanoscale Microscopy and Molecular Physiology of the Brain and by SFB1286. S.V. is supported by Ministerio de Economía y Competitividad (MINECO) (BIO2016-78310-R). J.S. is supported by MINECO (BFU2016-78232-P) and Gobierno de Aragón (E45_17R). E.D. is supported by Instituto de Salud Carlos III (PH613883/ERDF/ESF). J.G. and X.S. are supported by MINECO (BIO2015-70092-R) and the European Research Council (Contract 648201).

Footnotes Author contributions: S.V. designed research; J.P., S.P.-D., D.F.L., F. Peccati, F. Pinheiro, D.G., A.C., S.N., M.C.-G., J.G., S.G., and E.D. performed research; J.P., S.P.-D., D.F.L., E.G., X.S., J.S., M.S., T.F.O., E.D., and S.V. analyzed data; and M.S., E.D., and S.V. wrote the paper.

Conflict of interest statement: J.P., S.P.-D., M.C.-G., J.S., E.D., and S.V. are inventors on a patent application (PCT/EP2018/054540) related to the compound in this study.

This article is a PNAS Direct Submission.

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