Small molecules that bind with high affinity and specificity to fibrils of the α-synuclein (αS) protein have the potential to serve as positron emission tomography (PET) imaging probes to aid in the diagnosis of Parkinson's disease and related synucleinopathies. To identify such molecules, we employed an ultra-high throughput in silico screening strategy using idealized pseudo-ligands termed exemplars to identify compounds for experimental binding studies. For the top hit from this screen, we used photo-crosslinking to confirm its binding site and studied the structure–activity relationship of its analogs to develop multiple molecules with nanomolar affinity for αS fibrils and moderate specificity for αS over Aβ fibrils. Lastly, we demonstrated the potential of the lead analog as an imaging probe by measuring binding to αS-enriched homogenates from mouse brain tissue using a radiolabeled analog of the identified molecule. This study demonstrates the validity of our powerful new approach to the discovery of PET probes for challenging molecular targets.

Characterization of the amyloidogenic Parkinson's disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson’s disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.