Abstract
α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson’s disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species.
Introduction
α-Synuclein (αS) is a 140 amino acid protein whose aggregation into amyloid fibrils in a subset of neuronal and glial cells lies behind the onset of a group of progressive and, ultimately, fatal neurodegenerative disorders, including Parkinson’s disease (PD)1,2,3,4, that are collectively referred to as synucleinopathies. A causative link between αS and disease is supported by the discoveries that multiplications and missense mutations in SNCA, the αS gene, cause dominantly-inherited familial forms of PD5.
Interfering with αS amyloid formation and abrogating the associated toxicity is considered a promising therapeutic strategy for synucleinopathies6,7,8. However, the design of molecular entities that target specific αS toxic assemblies is challenging because of the heterogeneous, dynamic, and transient nature of these species. High-throughput screening initiatives have rendered promising αS aggregation inhibitors9,10,11. However, these selection procedures are blind to the ligand mechanism of action. In the absence of a structure-activity relationship, it is difficult to evolve the affinity and specificity of the identified hits to generate drugs that can reach the clinics. The lack of specific and sensitive molecules to detect the pathogenic forms of αS also constrains the early diagnosis of these diseases.
The in vitro aggregation of αS displays a sigmoidal growth profile, suggesting that it follows a nucleation-polymerization mechanism12, where soluble αS undergoes a nucleation process that produces oligomers able to grow through further monomer addition to form insoluble amyloid fibrils. Oligomeric forms of αS have been detected in the brains and other tissues of patients suffering from PD, and growing evidence suggests that they constitute the primary cytotoxic agents accounting for the gain-of-toxicity associated with αS aggregation, whereas both oligomers and fibrils would be responsible for pathology dissemination in the brain2,13,14,15. We have recently identified the sequential occurrence of two conformationally distinct types of oligomers during αS in vitro fibrillation. The initial non-toxic disordered oligomers, named as type A oligomers, undergo a structural reorganization to form more stable and compact β sheet-enriched, and proteinase K-resistant species that exhibit intrinsic cytotoxicity, named as type B oligomers16. Stable, trapped analogues of these two well-defined types of transient oligomers (referred to as type A* and type B* oligomers, where the star refers to the kinetically trapped nature of these isolated oligomeric forms) have been isolated and characterized in detail13,16 and, therefore, constitute important tools for the development of specific therapeutic and diagnostic strategies.
In this work, we exploit our recent advances in the understanding of the structural determinants of toxicity of αS oligomers to rationally identify peptide molecules able to target αS toxic species. By using a time-resolved single-particle fluorescence approach, we demonstrate that short, amphipathic, and cationic α-helical peptides do not interact with the functional monomeric αS, but they bind toxic oligomers and fibrils with nanomolar affinity, resulting in the substoichiometric inhibition of αS aggregation and abrogation of oligomer-induced damage in neuronal cell models. We then use a protein engineering approach to dissect the molecular determinants accounting for this interaction, which allow us to identify a human peptide, constitutively expressed in the brain and gastrointestinal tract, that binds with low nanomolar affinity to αS toxic assemblies, thus suppressing the aggregation cascade and its associated neurotoxicity. Thus, we describe here the rational identification and characterization of a family of highly potent peptidic ligands able to bind to αS toxic species and abrogate their detrimental effects in neuronal cells. This discovery may open previously unexplored avenues for the diagnosis and/or therapeutics of PD and related disorders.
