Binding Sites of a Positron Emission Tomography Radioligand with Nanomolar Affinity to Alpha-Synuclein Fibrils

This is the authors' abstract. We don't add key points for this paper.

Diagnosing neurodegenerative diseases currently depends on a physician’s interpretation of neurological symptoms. However, because these symptoms often overlap across disorders, patients may be misdiagnosed or only correctly diagnosed after years of ineffective treatment [1]. Additionally, some clinical symptoms only emerge in the later stages of the disease. This challenge is especially pronounced for synucleinopathies, such as Parkinson’s Disease (PD), Multiple Systems Atrophy (MSA), and Lewy Body Dementia (LBD), all of which are associated with the overexpression or aggregation of α-synuclein in neurons and glia [2]. Unfortunately, no definitive diagnostic test currently exists for these conditions. The lack of diagnostic tools is particularly concerning given that PD is the second most common neurodegenerative disorder in the U.S. after Alzheimer’s disease (AD), for which a reliable diagnostic blood test is currently being developed [3]. A recent study has shown that the use of skin biopsies to detect phosphorylated α-synuclein could aide in the diagnosis of synucleinopathies, though it remains in early development [4]. As a result, the urgent need for reliable diagnostic tools has spurred significant interest in developing early detection techniques based on disease-specific biomarkers. For synucleinopathies, α-synuclein fibrils might serve as biomarkers to differentiate disease phenotypes. α-Synuclein is a 140-amino acid protein encoded by the SNCA gene. This protein features three distinct regions: an N-terminal amphipathic region involved in lipid binding, a non-amyloid-β (NAC) domain critical for aggregation, and a C-terminal acidic domain that helps maintain its soluble form that can promote aggregation when altered [5-8]. Predominantly found in the presynaptic terminal, α-synuclein plays a role in regulating neurotransmitter release [9]. Under certain conditions, it destabilizes and aggregates into fibrils, forming Lewy Bodies, a defining feature of synucleinopathies [10]. Recent cryo-EM studies on recombinant α-synuclein fibrils have revealed multiple fibril polymorphs, influenced by factors such as incubation conditions or mutations [11-14]. Structural studies of patient-derived fibrils have further highlighted unique fibril folds corresponding to specific disease states [15, 16]. Histological studies of postmortem tissue have shown that α-synuclein inclusions are location-dependent and linked to different disease phenotypes [17]. This evidence suggests that the clinical variability of synucleinopathies may be rooted in distinct molecular forms of α-synuclein, spurring interest in developing diagnostic tools to detect these molecular differences early on. One pioneering approach involves utilizing small molecules that bind preferentially to specific α-synuclein fibril forms, which could be used as PET imaging probes for accurate and early diagnosis of specific synucleinopathies. Our collaborators at the University of Pennsylvania utilized in silico screening to identify a series of small molecules, known as exemplars, that could act as radioligands for α-synuclein fibrils [18]. Exemplar-6 (Ex-6), for example, demonstrated a strong binding affinity and could serve as a promising radioligand [18]. While initial in silico studies used a model of a single protofilament, recent structural data suggests that α-synuclein fibrils exist as multi-protofilament structures. Here we present a cryo-EM analyses of in-vitro fibrils, both in their apo form and in complex with Ex-6, that revealed important binding details (Figure 1, 2). Ex-6 was found to bind near a two-protofilament interface, stabilizing in an orientation perpendicular to the fibril axis with ligand-protein interactions that could enhance radioligand specificity (Figure 2B-2D). Favorable inter-ligand interactions, due to alternating ligand confirmations, stabilize Ex-6 in this cationic pocket (Figure 2B). This work provides crucial molecular insights that will aid in refining radioligands for more accurate and effective PET-based diagnostic tools for synucleinopathies [19]. Cryo-EM reconstruction and molecular model of the apo α-synuclein fibril. A) Multiple maps were aligned along the fibril axis to display twisting fibrils, protofilaments were segmented and colored for easier visualization. B) Close up of potential binding pocket, with lack of ligand density. C) Fibril cross-section displaying tightly packed fibril core. D) Molecular model built into the EM map, only one fibril layer is shown. Cryo-EM reconstruction and molecular model of the α-synuclein fibril and Ex-6 complex. A) Maps were segmented and aligned along the fibril axis; Ex-6 density (blue) identified in binding pocket. B) Close up of map reveals alternating ligand confirmations along the fibril axis. C) Fibril cross-section shows Ex-6 binds along a pocket near the protofilament interface. D) Molecular model showing one fibril layer built into the EM map.