Threonine Cavities Are Targetable Motifs That Control Alpha-Synuclein Fibril Growth

Abstract

Recent high-resolution structures of alpha-synuclein (aSyn) fibrils offer promise for rational approaches to drug discovery for Parkinson’s disease and Lewy body dementia. Harnessing the first such structures, we previously used molecular dynamics and free energy calculations to suggest that threonines 72 and 75which line water-filled cavities within the fibril stacksmay be of central importance in stabilizing fibrils. Here, we used experimental mutagenesis of both wild-type and A53T aSyn to show that both threonine residues play important but surprisingly disparate roles in fibril nucleation and elongation. The T72A mutant, but not T75A, resulted in a large increase in the extent of fibrillization during primary nucleation, leading us to posit that T72 acts as a “brake” on run-away aggregation. An expanded set of simulations of five recent high-resolution fibril structures suggests that confinement of cavity waters around T72 correlates with this finding. In contrast, the T75A mutation led to a modest decrease in the extent of fibrillization. Furthermore, both T72A and T75A completely blocked the initial fibril elongation in seeded fibrillization. To test whether these threonine-lined cavities are druggable targets, we used computational docking to identify potential small-molecule binders. We show that the top-scoring hit, aprepitant, strongly promotes fibril growth while specifically interacting with aSyn fibrils and not monomer, and we offer speculation as to how such compounds could be used therapeutically.

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein

“…Some drugs greatly altered the morphology of the curve (most notably candesartan cilexetil, regorafenib and bazedoxifene acetate; Supplemental Figure S10e ), and some drugs had little to no effect on fibrillization ( Supplemental Figure S10f ). To interpret the effects observed more clearly, we calculated the fibrillization extent by subtracting the average ThioT signal of the first and last hours of aggregation, as reported previously ( Nathan Kochen et al, 2022 ) ( Figure 6b and c). In total, 18 of the 25 tested compounds induced a significant attenuation of fibrillization extent, demonstrating a strong correlation with compounds that modulate aSyn FLT-FRET in cells, compared with those that attenuate cell-free fibrillization.…”

“…Full-length monomeric aSyn was expressed and purified as described previously ( Kamboj et al, 2021 ; Nathan Kochen et al, 2022 ). PFFs were generated by incubating 2 mg/ml monomeric aSyn and a single 3 mm borosilicate glass bead for 90 hours (orbital shaking conditions and 37 °C) ( Nathan Kochen et al, 2022 ).…”

“…Misfolding and self-association of alpha-synuclein (aSyn) is at the epicenter of pathophysiological challenges in alpha-synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple systems atrophy ( Emadi et al, 2004 ; Gallardo et al, 2008 ; Lorenzen et al, 2014 ; Power et al, 2015 ; Rockenstein et al, 2014 ; Spillantini et al, 1998 ; Uversky, 2008 ; Uversky & Eliezer, 2009 ; Wirths et al, 2000 ). Identifying small molecules that can rescue these aSyn-associated cellular insults has been the focus of multiple studies ( Caruana et al, 2012 ; Casalino et al, 2022 ; Herva et al, 2014 ; Höllerhage et al, 2017 ; Macchi et al, 2016 ; Moussaud et al, 2015 ; Perni et al, 2017 ; Pujols et al, 2017 ; Sahihi et al, 2021 ; Tóth et al, 2019 ), including our recent live-cell high-throughput screens (HTS) that monitored aSyn oligomerization and misfolding via fluorescence lifetime (FLT) detection of Förster resonance energy transfer (FRET) within cellular biosensor constructs ( Braun et al, 2021 ) and computational docking HTS targeting aSyn fibril structures ( Nathan Kochen et al, 2022 ). With both approaches, we identified compounds that modulate aSyn fibrillization.…”

Post‐translational modification sites are present in hydrophilic cavities of alpha‐synuclein, tau, FUS, and TDP‐43 fibrils: A molecular dynamics study

“…In our recent paper, we determined the presence of hydrophilic fibril cavities in the recombinant rod (polymorph 1a, PDB: 6CU7), twister (polymorph 1b, PDB: 6CU8), and A53T (PDB: 6LRQ) fibril structures, as well as the brain-derived MSA Type IA and IB polymorphs (PDB: 6XYO). 15 Here, we expanded our simulations to three new recombinant aSyn fibril structures: H50Q (PDB: 6PES) and E46K (PDB: 6UFR) mutant fibrils, and the WT aSyn polymorph 2b (PDB: 6SST). As shown in Figure 1, we detected hydrophilic cavities in the new aSyn polymorphs (Figure 1F-H) as indicated by the volumes in gray, green, and red, which were identified using the last 25 ns of simulation with the Analysis of Null Areas (ANA2) package developed by Barletta et al 36…”

“…14 Furthermore, we demonstrated this effect in a set of experiments with aSyn fibrils, where we found that the amino acids that interact with cavity waters (most notably threonine 72) are capable of eliminating monomer seeding onto wildtype (WT) fibrils and enhancing primary fibril growth extent when mutated to more hydrophobic residues. 15 Other groups show that a subset of threonine residues that are located within the cavities of these fibril structures are actively O-GlcNACylated in the human brain, suggesting a role of modifying enzymes on the modulation of aSyn conformation and aggregation propensity. Indeed, the recent Parkinson's disease familial mutant T72M demonstrates both loss of post-translational modification (PTM) capability and enhanced aggregation relative to WT aSyn, leading to pathology.…”