Discovery of amyloid binders as chemical chaperone to block pathological α-synuclein aggregation in synucleinopathies

Discovery of amyloid binders as chemical chaperone to block pathological α-synuclein aggregation in synucleinopathies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Discovery of amyloid binders as chemical chaperone to block pathological α-synuclein aggregation in synucleinopathies Li Tan, Cheng Yan, Qinyang He, Shenqing Zhang, Chunting Qi, Huaijiang Xiang, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6562222/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The prion-like propagation and accumulation of pathogenic α-synuclein (α-syn) aggregates in the central nervous system are central drivers of Parkinson’s disease (PD) and related synucleinopathies, and remain an important therapeutic target. However, most reported anti-fibrillogenesis agents show limited efficacy in cells or in vivo, and their mechanisms are often poorly defined. Here, we report the unexpected finding that Pittsburgh compound B (PiB), the first clinically used amyloid tracer, selectively suppresses α-syn aggregation. Using an optimized PiB analog, HQY1027, we demonstrate that these compounds function as chemical chaperones. By directly binding polymorphic α-syn fibrils, they remodel the fibril surface and enhance Hsp40 recognition of fibril cores, thereby suppressing fibril formation both in vitro and in cells. HQY1027 is highly effective in neuronal models and significantly reduced α-syn aggregate propagation and motor deficits in a PD mouse model. Our findings establish a chemical chaperone strategy for targeting pathogenic α-syn and identify HQY1027 as a promising therapeutic lead for synucleinopathies. These results further highlight the potential of phenotype-based discovery and optimization of fibril-binding ligands to target intractable protein aggregates in neurodegenerative disorders. Biological sciences/Drug discovery Physical sciences/Chemistry/Chemical biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The global rise in aging populations has intensified the societal impact of neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Despite their complex and heterogeneous etiologies, protein misfolding, aggregation, and accumulation have been identified as key drivers of ND onset and progression 1,2 . Notably, pathogenic aggregates - including tau, α-synuclein (α-syn), and TDP-43 - can spread in a prion-like manner through the central nervous system (CNS), making aggregated proteins compelling therapeutic targets. Recent clinical advances with β-amyloid (Aβ) antibodies further underscore the therapeutic tractability of targeting protein aggregates 3,4 . However, current antibody therapies are associated with a non‑negligible risk of amyloid‑related imaging abnormalities (ARIA) and microhemorrhages in AD patients 5 , while still failing to efficiently penetrate neuronal membranes and clear intracellular tau or α‑syn aggregates 6 . In addition to antibodies, small-molecule inhibitors targeting pathogenic protein aggregates have attracted significant attention in ND drug development, owing to their potential advantages in CNS penetration and low immunogenicity. The most advanced candidates are those derived from screening hits capable of directly inhibiting or even reversing protein aggregation in vitro. For instance, leucomethylene blue, a tau aggregation inhibitor, has progressed to Phase 3 clinical trials for AD 7,8 , while emrusolmin 9 and minzasolmin 10 , both reported to inhibit α-syn fibrillogenesis, have entered Phase 2 trials for PD and multiple system atrophy (MSA). However, the clinical translation of these inhibitors has yet to be realized, as exemplified by the recent Phase 2 trial failure of minzasolmin to improve PD symptoms. Fundamental questions also persist regarding the mechanism of action (MOA) and in‑vivo efficacy of many purported anti‑fibrillogenesis agents, have subsequently been shown to act as non‑specific redox modulators or autophagy inducers, or else to lack sufficient brain exposure 8,11,12 , 13 . Consequently, the development of diverse strategies to target pathogenic protein aggregates remains an urgent unmet need in the field. Recent advances to eliminate pathogenic aggregates in ND have introduced strategies for targeted protein degradation, such as molecular glue degraders 14 and proteolysis-targeting chimeras (PROTACs) 15 . Concurrently, inhibitors disrupting aggregate-membrane receptor interactions have been developed to block α-syn propagation 16,17,18 . However, these alternative approaches remain largely at the proof-of-concept stage and require further validation. Meanwhile, cellular or phenotypic screening continues to reveal biologically active compounds with unexpected MOA 19,20 . In this study, we serendipitously discovered that the amyloid PET tracer Pittsburgh compound B (PiB) inhibits α-syn aggregation. Although amyloid PET tracers are well-established in the clinical diagnosis of ND, their potential cellular functions beyond fibril binding have been largely unexplored. We demonstrate that PiB and its optimized analog, HQY1027, function via a previously unrecognized chemical chaperone mechanism. They enhance interactions between chaperone proteins and polymorphic α-syn fibrils, thereby impeding fibril propagation. HQY1027 exhibits favorable oral bioavailability and brain penetration, and shows remarkable efficacy in halting the spread of α-syn pathology in both cultured neurons and in vivo. This work establishes a new co-chaperone strategy for combating proteinopathies using drug-like small molecules. Results Serendipitous discovery and lead optimization of α -syn aggregation inhibitors To enable targeted degradation of pathological α-syn aggregates, we designed a series of PROTACs incorporating known amyloid-binding ligands, E3 ligase ligands, and diverse linkers (Extended Data Fig. 1a). In α-syn-overexpressing (α-syn-OE) 293T cells, most PROTACs showed limited effectiveness against wildtype (WT) pre-formed fibrils (PFF)-induced α-syn aggregation, as assessed by insoluble α-syn levels and Ser129 phosphorylation (p-S129) (Extended Data Fig. 1b). Notably, PROTAC 1 , comprising PiB-derived α-syn-targeting moiety conjugated to a von Hippel-Lindau (VHL) E3 ligase ligand, emerged as the most effective at reducing α-syn aggregation (Fig. 1a and Extended Data Fig. 1b). To verify the PROTAC mechanism, we synthesized control compounds including a diastereomer of 1 ( 2 ), and PEGylated derivatives of PiB ( 3 , 4 ). Surprisingly, while 2 was inactive as predicted, 3 , 4 , and PiB itself outperformed PROTAC 1 in anti-aggregation assays (Fig. 1a,b). Furthermore, VHL knockout failed to attenuate the activity of either 1 or the controls (Extended Data Fig. 1c). These unexpected results suggest that the observed activity stems from the intrinsic inhibitory properties of PiB rather than PROTAC-mediated degradation. Building on this serendipitous discovery, we synthesized over fifty PiB derivatives to explore the structure-activity relationships and to optimize both potency and drug-like properties. Several analogs exhibited substantially improved activity over PiB. Among them, HQY1027, which incorporates a benzoxazole-for-benzothiazole substitution and two additional methyl groups, proved to be the most potent. It achieved an IC₅₀ of 50 nM against WT PFF-induced α-syn aggregation, demonstrating at least a tenfold increase in potency over PiB. (Fig. 1c,d and Extended Data Fig. 2a). Notably, other reported binders of α-syn fibrils, such as BF227 and C0503 21 , showed minimal activity at 10 µM (Fig. 1c). In cellular assays, HQY1027 effectively reduced α-syn aggregation at 0.2 µM and nearly abolished the stimulated aggregation at 0.5 µM. Remarkably, even when administrated 48 h post-PFF seeding, 0.5 µM HQY1027 still effectively suppressed aggregation within 24 h (Fig. 1e). Given the structural polymorphism and varying pathological properties of α-syn fibrils 22,23 , we next evaluated HQY1027 and PiB against fibrils designed to mimic endogenous conformations observed in synucleinopathies. These fibrils incorporated hereditary mutations associated with familial synucleinopathies (A53T and G51D) 24,25 as well as an engineered mutation at Glu57, a residue known to influence fibril polymorphs 26 . Cryo-EM analysis revealed that one PFF containing the A53T/G51D double mutation formed a polymorph (PDB: 9JC3) closely resembling MSA Type-II 2 α-syn fibril (PDB: 6XYQ) 27 , while another incorporating the A53T/G51D/E57A triple mutation exhibited high structural similarity (PDB: 9KAL) to the singlet α-syn fibril found in juvenile-onset synucleinopathy (JOS) (PDB: 8BQV) (Extended Data Fig. 2b) 28 . Although both MSA-like and JOS-like PFFs induced stronger α-syn aggregation than WT PFFs in α-syn-OE 293T cells, HQY1027 consistently and effectively suppressed this aggregation with IC₅₀ values around 200 nM, 3 to 4 times lower than those of PiB (Fig. 1d and Extended Data Fig. 2a). Compared to the clinical-stage α-syn fibrillation inhibitors, emrusolmin and minzasolmin, HQY1027 exhibited markedly higher potency in cellular assays (Fig. 1f). At a concentration of 0.5 µM, HQY1027 effectively suppressed α-syn aggregation induced by either wild-type (WT) or MSA/JOS-like PFFs in α-syn-OE 293T cells. In contrast, emrusolmin and minzasolmin showed minimal activity at 1 µM and were only effective at 10 µM, a concentration associated with non-negligible cytotoxicity. Furthermore, while PiB exhibits pan-amyloid affinity, HQY1027 demonstrated no inhibitory activity against PFF-induced tau aggregation in tau-OE 293T cells (Extended Data Fig. 3a), highlighting its selectivity for α-syn aggregation. HQY1027 also demonstrated significantly improved metabolic stability, with a microsomal half-life (T 1/2 ) of 23.4 min versus only 8.4 min for PiB, addressing the rapid clearance typical of most PET tracers (Extended Data Fig. 3b). Collectively, these data establish HQY1027 as a potent and selective lead inhibitor of α-syn aggregation. Target identification of HQY1027 HQY1027’s superior cellular activity against α-syn aggregation suggests a pharmacological mechanism distinct from that of conventional fibrillation inhibitors. To elucidate its mechanism of action, we first tested its ability to directly inhibit fibril growth in vitro, a hallmark of conventional aggregation inhibitors. However, HQY1027 showed no inhibitory activity even at high concentrations (Fig. 2a). Unlike the reference inhibitor GSD-16-24 16 , HQY1027 also failed to disrupt α-syn fibril binding to membrane receptors (LAG3 or RAGE) (Extended Data Fig. 4a). Mechanistic studies revealed that HQY1027’s anti-aggregation activity remained unaffected by either proteasome inhibition (PS-341) or lysosome blockade (bafilomycin A1, BafA1) regardless the PFF type used (Fig. 2b and Extended Data Fig. 4b), indicating a mechanism independent of major protein degradation pathways. Although HQY1027 suppresses α-syn Ser129 phosphorylation, comprehensive kinome profiling at 1 µM revealed no significant kinase interactions (Supplementary Table 1), excluding direct kinase inhibition as its mechanism. To identify the direct targets, we designed and synthesized photocrosslinking probes derived from PiB, HQY1027, and related analogs - including the inactive analog 5 (Fig. 2c). These probes contain diazirine groups that generate reactive carbenes upon UV irradiation, enabling covalent bond formation with target proteins via C-H insertion. The labeled proteins can then be enriched through click chemistry and affinity pulldown. Based on their inhibitory activities, we selected probe 6 (with potency comparable to HQY1027) and inactive control probe 7 for further studies (Fig. 2c,d). Photocrosslinking was performed in live α-syn-OE 293T cells that had been pretreated with PFF for 48 h. Proteins labeled by either probe 6 or 7 were separately enriched from cell lysates and analyzed by mass spectrometry (MS)-based proteomics (Fig. 2e,f and Supplementary Tables 2,3). Proteomic analysis revealed that probe 6 labeled many cellular proteins, consistent with the expected promiscuous reactivity of carbene-based probes (Fig. 2e and Supplementary Table 2). Importantly, 6 specifically labeled α-syn, confirming its maintained affinity for α-syn aggregates as a PiB-derived analog. Notably, multiple molecular chaperones including Hsp40s, Hsp70s, and Hsp90s were among the top significantly labeled hits. Using inactive probe 7 for background subtraction, we identified a subset of proteins specifically labeled by 6 (Fig. 2f and Supplementary Table 3). Several chaperones remained highly enriched in this specific subset, with the Hsp40 family member Hdj1 emerging as one of the most prominent interactors. Western blot analysis validated these findings, demonstrating specific labeling of α-syn, Hsp40, and Hsp70 by probe 6 but not control probe 7 (Fig. 2g). This labeling was substantially reduced by HQY1027 pretreatment, but remained unaffected by the inactive analog 5 . Furthermore, in PFF-pretreated 293T cells expressing α‑syn fused to TurboID - enabling biotinylation of proximate or interacting proteins 29 - treatment with HQY1027, but not analog 5 , led to significant biotinylation of Hsp40 and Hsp70 (Fig. 2h). Collectively, these results indicated that HQY1027 likely exerts its inhibitory effects by simultaneously engaging both α-syn aggregates and cellular chaperone proteins. HQY1027 directly binds to α -syn fibrils and Hsp40 We next assessed HQY1027’s direct interactions with α-syn fibrils and recombinant chaperone proteins using surface plasmon resonance (SPR). HQY1027 displayed high binding affinity for WT α-syn PFF ( K D = 0.65 µM) and moderate affinity for Hdj1 ( K D = 5.41 µM), while showing weak binding to Hsc70 and Hsp90α ( K D >10 µM) (Fig. 3a,b and Extended Data Fig. 5a). In contrast, the inactive analog 5 displayed similar binding to chaperones but significantly reduced affinity for α-syn PFF (15-fold weaker than HQY1027) (Fig. 3b and Extended Data Fig. 5b). Furthermore, protein thermal shift assay results indicated that HQY1027 directly binds to Hsp40 but not to Hsp70/90, as HQY1027 substantially destabilized Hdj1 (ΔTm = -4.6°C) but minimally affected the thermostability of Hsc70 or Hsp90α (Fig. 3c and Extended Data Fig. 6). Cryo-EM analysis of WT α-syn PFF preincubated with HQY1027 revealed additional densities at two binding pockets previously reported for PiB 21 (Fig. 3d). The 2.9 Å reconstruction (Extended Data Fig. 7 and Extended Data Table 1) confirmed HQY1027 occupancy at both the N-terminal (N-pocket) and C-terminal (C-pocket) binding sites along the fibril axis (Fig. 3e). HQY1027 exhibits distinct binding geometries within these pockets: in the C-pocket, it tilts approximately 45° and contacts multiple stacked α-syn layers; in the N-pocket, it adopts a nearly horizontal orientation rather than a diagonal one (Fig. 3f). Electron densities corresponding to HQY1027 are relatively weak, suggesting dynamic rather than rigidly fixed interactions with the fibrils. Notably, no additional ligand density was observed in MSA- or JOS-like PFFs incubated with HQY1027, further supporting a highly dynamic binding mode for HQY1027. Detailed structural analysis revealed that HQY1027 binding within the N-pocket promotes an inward orientation of α-syn Tyr39 (Fig. 3g). This ‘closed’ N-pocket conformation has been observed with PiB and thioflavin T (ThT), but not with BF227 21 . HQY1027 binding also triggered an inward re-orientation of the negatively charged Glu83 side chain within the N-pocket, likely reducing local polarity and hydrophilicity. This Glu83 rearrangement contrasts sharply with its outward-facing orientation in both apo α-syn fibrils and other ligand-bound states 21,31 . To examine whether introducing hydrophilic or charged groups would alter the activity of HQY1027, we designed and synthesized a series of derivatives bearing positively charged ( 8 ‑ 10 ), hydrophilic ( 11 , 12 ), or negatively charged ( 13 ) moieties, and compared them with HQY1027 (Fig. 3h). In α-syn‑overexpressing 293T cells, all derivatives showed markedly reduced anti‑aggregation activity at 0.5 µM compared with HQY1027 (Fig. 3h and Extended Data Fig. 8a). According to SPR analysis, the positively charged derivatives bound α‑syn fibrils much more weakly, while the other three derivatives displayed binding affinities similar to HQY1027 (Fig. 3h and Extended Data Fig. 8b). Notably, HQY1027 was substantially more effective than any derivative in enhancing the hydrophobic surface exposure of α‑syn fibrils, as indicated by a pronounced increase in fluorescence polarization of a widely used hydrophobic‑sensitive dye, SYPRO Orange (Fig. 3i). Collectively, these data suggest that HQY1027 modifies the surface physicochemical properties of α‑syn fibrils, which may in turn affect their interactions with cellular proteins. HQY1027 promotes Hsp40’s recognition of α-syn fibrils Given HQY1027's dual binding affinity for both α-syn fibrils and Hsp40, we next investigated its effects on their interactions. SPR-based kinetic profiling of ternary complex formation 32 revealed that Hdj1 significantly enhanced HQY1027 binding to α-syn fibrils, as evidenced by increased responses and prolonged dissociation kinetics (Fig. 4a). Reciprocally, HQY1027 markedly stabilized the interaction between Hdj1 and WT PFF, reducing the dissociation rate constant ( k off ) by >90% and consequently decreasing the K D value by approximately 60-fold (Fig. 4b,c and Extended Data Fig. 9a). It also consistently stabilized the association of Hdj1 with both MSA- and JOS-like PFFs, decreasing the K D value by at least 40-fold. Furthermore, while truncation of the α-syn C-terminal nearly abolished Hdj1 binding, HQY1027 facilitated significant, dose-dependent interactions between Hdj1 and α-syn 1-100 PFF. This provides additional evidence that HQY1027 modulates the surface characteristics of α-syn fibril cores and influences their dynamic engagement with Hsp40. These findings were corroborated by quantitative enzyme-linked immunosorbent assay (ELISA), where HQY1027 significantly increased Hdj1 binding to α-syn fibrils, while the inactive analog 5 showed minimal activity (Fig. 4d). Notably, consistent with its selective binding profile among Hsp family members, HQY1027 did not enhance α-syn fibril binding to Hsc70 (Extended Data Fig. 9b). To directly visualize HQY1027-enhanced Hsp40-α-syn fibril interactions, we employed single-molecule fluorescence analysis with optical tweezers, which enables real-time measurement of binding dynamics between Alexa555-labeled Hdj1 (Hdj1-Alex555) and individual α-syn fibrils under controlled tension 33 (Fig. 5a). Control experiments with 10 nM Hdj1-Alex555 alone exhibited only sparse fluorescence signals along suspended α-synuclein fibrils. In contrast, preincubation of α-syn fibrils with HQY1027 significantly enhanced Hdj1 binding, as evidenced by rapid accumulation of persistent fluorescence signals (Fig. 5b,c). To validate this mechanism in neuronal context, we generated an SH-SY5Y human neuroblastoma cell line stably expressing GFP-tagged α-syn and quantitatively assessed α-syn aggregate-Hsp40 colocalization. Confocal microscopy showed that while PFF treatment of these cells converted diffuse α-syn monomers into punctate aggregates, whereas Hsp40 displayed only minimal baseline colocalization with these aggregates under control conditions (Fig. 5d). Notably, treatment with 1 µM HQY1027 significantly enhanced this colocalization by at least five-fold as quantified by Pearson correlation coefficients, whereas 5 showed no detectable effects (Fig. 5e). These results provide direct evidence that HQY1027 enhances Hsp40 recognition of α-syn aggregates across experimental systems, establishing a clear mechanistic basis for its anti-aggregation activity. HQY1027 impedes α-syn fibril propagation in the presence of Hsp40 Although HQY1027 alone showed minimal inhibition of α-syn fibril growth in vitro, it exhibited remarkable inhibitory activity combined with Hsp40. While low concentrations of Hdj1 protein alone had no significant effects, co-treatment with 10 µM HQY1027 achieved near-complete suppression of α-syn fibril elongation. Strikingly, 5 remained almost inactive even at 100 µM under identical conditions (Fig. 6a,b). To determine whether Hsp40 and downstream chaperones are essential for the cellular activity of HQY1027, we employed the Hsp70 inhibitor VER-155008 34 for pharmacological perturbation, in light of the current lack of inhibitors targeting Hsp40 directly. As expected, co-treatment with VER-155008 largely abolished the anti-aggregation effect of HQY1027 across all PFF types in 293T cells (Fig. 6c). These findings support the mechanism whereby HQY1027 binds to α-syn fibrils, modifying their surface architecture and thereby facilitating chaperone recruitment to block fibril growth. While HQY1027 effectively inhibited α-syn fibril growth in the 293T OE system, we further evaluated its efficacy in physiologically relevant models. Using an established neuronal propagation assay 35 , we treated primary rat cortical neurons with α-syn PFF seeds for 14 days, which induced significant pathological fibril formation as quantified by p-S129 levels. HQY1027 treatment significantly reduced p-S129 levels in PFF-seeded neurons without affecting normal neuronal morphology (assessed by MAP2 levels), whereas 5 showed minimal effects (Fig. 6d,e and Extended Data Fig. 10). Remarkably, HQY1027 at 0.5 µM reduced α-syn aggregation by 50%, demonstrating exceptional potency compared to recently reported state-of-the-art α-syn propagation inhibitors 16,17,18 . Taken together, these results establish HQY1027’s consistent anti-aggregation effectiveness across both reconstituted assays and native neuronal systems through leveraging chaperones. HQY1027 mitigates pathological α-syn aggregation in a mouse PD model To evaluate the in vivo potential of HQY1027, we performed comprehensive pharmacokinetic (PK) profiling in mice. Given the limitations of forced oral gavage for chronic ND studies, we implemented a validated peanut butter-based voluntary feeding protocol 36,37 . Oral administration of HQY1027 at 25 mg/kg (mpk) achieved sustained plasma exposure over 24 h, reaching a peak concentration (C max ) of 7.4 µM with a total exposure (AUC) of 41.4 µM·h and complete oral bioavailability (109%) (Extended Data Fig. 11a,b). Given that this exposure level substantially exceeds the required therapeutic concentration, a lower dose of 5 mpk was evaluated. At 5 mpk, HQY1027 provided a moderate plasma exposure (C max = 1.87 µM, AUC = 2.90 µM·h) and demonstrated exceptional blood-brain barrier penetration, reaching a peak brain concentration of 1.58 µM with a brain-to-plasma ratio of 2.5:1 (Extended Data Fig. 11c). This profile confirms its excellent CNS distribution and establishes 5 mpk as an efficacious dose. We next evaluated HQY1027’s efficacy in a PFF-induced mouse PD model. In this established model 38 , unilateral striatal injection of α-syn PFF induces bilateral α-syn pathology within three months, ultimately leading to motor dysfunction. HQY1027 administration (5 mpk/day via voluntary food consumption for 120 days) showed no adverse effects on body weight or signs of toxicity (Fig. 7a). Behavioral assessment revealed that PFF-injected mice developed significant hindlimb grip strength impairment compared to α-syn monomer-injected controls, consistent with synucleinopathy phenotypes. While performance in rotarod, pole climbing, and open field tests remained unaffected at this intermediate disease stage (Extended Data Fig. 12), HQY1027 treatment significantly rescued grip strength deficits (Fig. 7b). Immunohistochemical (IHC) analysis demonstrated that HQY1027 robustly reduced PFF-induced α-syn pathology in the substantia nigra and motor cortex bilaterally (Fig. 7c). Quantitative analysis confirmed >50% clearance of α-syn aggregates (Fig. 7d), demonstrating superior efficacy compared to current benchmark compounds targeting α-syn propagation. These results establish HQY1027 as an orally bioavailable, well-tolerated compound that effectively inhibits α-syn propagation and mitigates pathological progression in vivo. Discussion Numerous studies have demonstrated that upregulating or activating specific chaperones can mitigate neurodegeneration 39,40,41 . Yet, pathogenic aggregates frequently evade chaperone surveillance and propagate throughout CNS during disease progression. While chemical strategies to induce proximity between fibrils and protein degradation machinery start to emerge, analogous approaches targeting chaperone recruitment remain unexplored. Our study reveals that PiB, the first amyloid PET tracer, unexpectedly functions as a chemical chaperone that suppresses α-syn aggregation by enhancing interactions between α-syn fibrils and Hsp40. Hsp40 inherently suppresses the liquid-to-solid phase transition during fibrillation and recruits Hsp70 and other proteostasis-maintaining components 42 . This provides a plausible mechanistic basis for the substantially greater activity of HQY1027, an optimized PiB analog, in cellular systems than in cell-free assays. More broadly, this work highlights the value of phenotype-driven screening for uncovering aggregation modulators with noncanonical mechanisms. Although HQY1027 binds both α-syn fibrils and Hsp40, we could only determine a high-resolution structure for the HQY1027-α-syn fibril complex. The transient, heterogeneous nature of chaperone-client interactions remains a major barrier to structural characterization of Hsp40-substrate assemblies, and current cryo-EM methods are still limited in resolving dynamic complexes involving repetitive fibril layers at high resolution. Building upon established strategies including hydrophobic tagging for targeted protein destabilization 43 and molecular glue mechanisms 44 , we propose that HQY1027 modulates α-syn fibril surface architecture. This perturbation likely reduces both fibril surface order and charge density, thereby enhancing their susceptibility to chaperone recognition. Our structural observation that HQY1027 induces a distinctive inward conformation of Glu83 - a charged residue located in the N-pocket region of the fibril surface- directly supports this mechanism. Consistently, only HQY1027, but not its more hydrophilic or charged derivatives, significantly enhances the binding of a hydrophobic-sensitive dye to the fibril surface. Further corroboration comes from truncation studies, which demonstrate that HQY1027 enables Hsp40 to recognize the fibril core instead of the natively targeted C-terminal region. Notably, HQY1027 can bind to and remain active against other α-syn PFF types, suggesting that this mechanism is applicable to polymorphic fibrils. Nevertheless, this chemical chaperone approach may be generalizable to other protein aggregates through strategic screening or modification of fibril-binding compounds. Comparative analysis demonstrated HQY1027’s superior potency over clinical-stage aggregation inhibitors emrusolmin and minzasolmin in cellular assays. This enhanced activity was consistently observed in both neuronal cultures and animal models, where HQY1027 effectively reduced pathogenic aggregation of endogenous α-syn. Although motor deficits typically manifest only after extensive α-syn pathology in PD models, we detected significant impairment in paw grip strength at mid-stage pathology - a deficit substantially ameliorated by HQY1027 treatment. These findings underscore the therapeutic potential of our chemical chaperone strategy, which leverages endogenous protein quality control systems while causing minimal physiological disruption. It is worth noting that while the clinical relevance of widely used preformed fibril (PFF)-based synucleinopathy models is sometimes questioned due to the structural polymorphism of α-syn fibrils, a growing body of evidence indicates that synthetic fibrils can adopt conformations similar to endogenous fibrils - which are typically scarce and difficult to obtain - and recapitulate key aspects of clinical pathology in mice 45 . Our preliminary validation using MSA/JOS-like PFFs further supports the therapeutic potential of HQY1027, positioning it as a promising lead compound for ND drug development. Further investigation is warranted to evaluate the efficacy of HQY1027 in animal models associated with familial synucleinopathies and to optimize its preclinical profile to facilitate clinical translation. In summary, whereas conventional drug development based on biochemical screening targeting fibrillogenesis faces bottlenecks in clinical translation, this study - starting from a serendipitous observation in a cellular model - establishes a chemical chaperone strategy for inhibiting pathogenic α-syn aggregation and identifies HQY1027 as a promising therapeutic lead. Our findings demonstrate the potential of repurposing and optimizing existing fibril-binding compounds to target intractable protein aggregates in ND, and further reinforce the value of phenotype-driven discovery in revealing unexpected pharmacological mechanisms. Methods Preparation of recombinant α-syn monomer/PFF and protein chaperones α-Syn monomer and PFF (WT; mutants (G51DA53T, G51DA53TE57A/Q, G51DA53TE58Q/S/G/N); α-syn 1-100 ) were produced as described previously 16,31,46,47 . Briefly, human or mouse α-syn was cloned into pET22b and expressed in E. coli BL21(DE3). Acetylated α-syn (Ac-α-syn) was produced by co-transformation with pACYCDuet-1-NatB. FLAG-α-syn was generated by cloning the N-terminal FLAG tag into pET22b-α-syn. Cells were lysed, boiled, nucleic acids removed, and pH adjusted, followed by overnight dialysis. Purification was performed using SP column or ion exchange followed by size exclusion chromatography (SEC). WT, C-terminal truncated and JOS like α-syn fibrils were assembled by incubating 400 μM monomer in SEC buffer (0.02% NaN 3 ) at 37 °C with agitation (900 rpm, 5 days). The MSA-like α-syn fibrils were assembled from six distinct α-syn triple mutants mixed at an equimolar ratio, with the additional incorporation of the G51D/A53T double mutant. Fibrils were sonicated to generate seeds, and new fibrils were formed by adding seeds to fresh monomer. After removing the monomer, the fibrils were sonicated again to generate α-syn PFF. Hdj1, Hsc70, and Hsp90α was expressed and purified as previously reported 42,48,49 , using Ni-NTA and Superdex 75 or 200 columns. Purified proteins were concentrated, flash-frozen, and stored at -80 °C. Protein labelling For fluorescence labeling, purified Hdj1 was conjugated with Alexa Fluor 555 C2 maleimide (Invitrogen, A20346) following the manufacturer's protocol. After conjugation, Hdj1-Alexa 555 conjugates were further purified by SEC. The final protein concentration was determined using the Bicinchoninic Acid (BCA) assay. Biotin-labeled α-syn fibrils were prepared by conjugating purified monomer with EZ-Link Sulfo-NHS-Biotin (Thermo Scientific, 21217) according to the manufacturer's protocol. After conjugation, biotin-α-syn conjugates were further purified using SEC. The final protein concentration was determined using the BCA assay. Once unlabeled α-syn fibrils reached 2-3 µm in length, biotin-α-syn monomer was added to continue fibril growth for several additional hours to obtain the desired biotin-labeled α-syn fibrils. In vitro α-syn fibril growth assay A mixture of 50 µM α-syn monomer, 1% (molar ratio) PFF with or without Hdj1 was incubated with indicated compounds in a buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 0.02% NaN3, and 50 µM ThT). Aggregation was monitored in black 384-well plates (Thermo Scientific, 142761) at 37 °C with orbital shaking 700 rpm. Fluorescence (ex: 440 nm, em: 485 nm) was recorded using a BMG FLUOstar Omega plate reader. ELISA According to published method 16 , high-binding 96-well plates were coated with 100 nM Hdj1 overnight at 4 °C, then blocked with 5% non-fat milk for 2 h. After washing, compounds and 100 nM PFF were added and incubated for 1 h. Bound PFF were detected using HRP-conjugated anti-FLAG antibody and quantified by the color reaction between HRP and TMB substrate, measuring absorbance at 450 nm.For receptor binding ELISA, dLAG3 and vRAGE were expressed and purified, purified, and pre-coated on plates as previously described 31,50,51 , followed by incubation with compounds and 100 nM FLAG-α-syn PFF, and then detection as above. Kinome profiling assays The KINOMEscan assays were done by DiscoverX (Fremont, CA, USA) 52 . The scores were reported as a percentage of DMSO control, with the lower score usually indicating higher probability of being a hit. Scores over 35% indicate no significant inhibition. SPR analysis SPR binding assays were conducted on a Biacore 8K system (GE Healthcare) in PBS buffer supplemented with 0.05% surfactant P20 (Cytiva, 28995084). α-Syn PFF and chaperones (Hdj1, Hsc70, Hsp90α) were immobilized on a CM5 sensor chip via amine coupling. To assess the interactions of compounds with target proteins, we injected serially diluted compounds (in assay buffer: PBS + 0.05% P20) over the sensor chip at 30 µL/min (120 s association), followed by dissociation (400 s). Besides, single-cycle kinetic assays of HQY1027 to α-syn PFF were performed with Hdj1 included at specified concentrations alongside HQY1027 throughout the experiment. For Hdj1-α-syn PFF binding assays, HQY1027 was included in all running buffers (association/dissociation phases). Between the cycles, the chip was regenerated with 3 M MgCl 2 (30 s). Equilibrium dissociation constants (K D ) were derived using Biacore Insight Evaluation Software (GE Healthcare). Protein thermostability shift assay The thermal stability of Hdj1, Hsc70, and Hsp90α (each at 4 µM) was assessed in complex with HQY1027 at a 1:40 protein:compound molar ratio. As previously published method 53 , after incubation, thermal denaturation was monitored in real-time using a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) with a temperature gradient from 25 °C to 95 °C. Derivative melting curves (dF/dT) were generated and analyzed using Protein Thermal Shift™ Software (ThermoFisher) to determine thermal stability shifts. Fluorescence polarization (FP) α-Syn WT fibrils were diluted to a final concentration of 2.5 µM in reaction buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl). Test compounds were mixed at concentrations as indicated. The mixture was incubated at room temperature for 1 h in the dark. Then SYPRO™ Orange Protein Gel Stain (ThermoFisher, S6650) was added at a 1:5000 (v/v) dilution and incubated at room temperature for 30 min in the dark. Fluorescence polarization was measured using an EnVision Multilabel Reader (PerkinElmer, 2104-0010) with excitation at 485 nm and emission at 535 nm. The polarizer was set to measure both parallel (I∥) and perpendicular (I⊥) emission relative to the excitation polarization. The fluorescence polarization was calculated using the standard formula: mP = 1000 * ((I∥ - G × I⊥) / (I∥ + G × I⊥)), where G=I⊥blank/I∥blank (G-factor) was derived from the blank control group (no protein) to correct for instrumental polarization bias. NS-EM 5 μL ThT assay products were deposited onto glow-discharged carbon-coated EM grids, stained with 2% uranyl acetate, and imaged using a Tecnai T12 microscope at 120 kV. Cryo-EM Sample preparation and data collection were performed as previously described 21 . Briefly, 3 µM Ac-α-syn fibrils were incubated with HQY1027 (300 µM, 2% DMSO) for 1 h at 25 °C. Ligand-bound samples were applied to glow-discharged carbon grids, blotted, plunge-frozen in liquid ethane, and imaged on a Krios G4 microscope at 300 kV with a BioContinuum K3 detector. Automated data collection was performed using EPU software with the same parameters as described previously 21 . Image preprocessing and helical reconstruction Consistent with previous descriptions 21 , in brief, image processing was performed using MotionCor2 (v1.2.1) 54 for frame alignment, dose-weighting, and binning to 0.83 Å/pixel. CTF estimation used CTFFIND4 (v4.1.8) 55 , followed by manual fibril picking in RELION (v3.1) 56 . Helical reconstruction was conducted using RELION, through particle extraction, reference-free 2D classification, 3D classification (k=3), and 3D auto-refinement, yielding 3D density maps with optimal helical twists and rises. Finally, the maps were sharpened using RELION’s post-processing, and resolution was estimated via Fourier shell correlation and local resolution analysis. Atomic model building and refinement Three-layer models of α-syn fibrils were based on the structure of α-syn fibril (PDB accession no. 6A6B) and manually adjusted in WinCoot (v.0.8.9.2) 57 , followed by refinement against the corresponding map by the real-space refinement program in PHENIX (v.1.15) 58 . Figures of atomic models were prepared using UCSF ChimeraX 59,60 or PyMOL 61 . Optical tweezers-based single-molecule analysis A confocal fluorescence microscopy-combined dual optical traps setup (LUMICKS C-trap, Netherlands) was used to detect the dynamic interaction between Hdj1 and α-syn fibrils in a climate-controlled room at 23 °C 62 . α-Syn fibrils were pre-incubated with 0, 10, 30 µM HQY1027 in the buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl) for 30 min. For the single-molecule assay, a single α-syn fibril was suspended between two streptavidin-coated polystyrene beads (4.34-µm diameter, Spherotech) 33 . Then, the tethered fibril was swiftly transported to a channel containing 10 nM. Hdj1-Alex555 and a force of ~ 5 pN along the fibril was maintained by a high-speed feedback system during the fluorescence detection. A 532 nm excitation laser was utilized to obtain fluorescence signals of Hdj1. The confocal pixel size was set to 75 nm, with a pixel dwell time of 0.2 ms. The interframe wait time was 4s for rectangular scanning. The confocal images were obtained from LakeView software provided by LUMICKS and the fluorescence intensity of Hdj1-Alex555 on α-syn fibrils (between two beads) was analyzed using ImageJ. Cell cultures Human embryonic Kidney (HEK293T) cells and SH-SY5Y cells were cultured in DMEM (ThermoFisher, C11995500BT) and DMEM/F12 (ThermoFisher, 11320033) respectively, plus 10% (v/v) FBS (ThermoFisher, 10100147) and 1% penicillin/streptomycin at 37 °C and 5% CO 2 in a humidified incubator. VHL-KO 293T cells (generated in lab previously, via CRISPR/Cas9 using guide RNA 5′-GCCGTCGAAGTTGAGCCATA-3′) were cultured in DMEM containing 1 µg/mL puromycin. Primary cortical neurons were isolated from E15-E18 Sprague-Dawley rat embryos (SIPPR-BK) and plated at 150,000 cells/well on poly-L-lysine-coated coverslips in 24-well plates, following established protocols 16 . Neurons were maintained in Neurobasal medium supplemented with B-27 (ThermoFisher, 17504044) and 2 mM GlutaMax (ThermoFisher, 35050061). 293T-based protein aggregation assays and Western blot analysis For α-syn aggregation assays, 293T (WT or VHL-KO) cells were seeded in 12-well plates (1×10⁶ cells/well) and transfected with 0.5 µg/mL pCAGGS-α-syn WT -FLAG using PEI (1:3 ratio). After 24 h, cells were re-plated (2×10⁵ cells/well) and treated with 10 nM α-syn PFFs in PBS. Following compound treatment according to specific sections, cells were lysed in NP-40 buffer (1% NP40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, cocktail protease inhibitors). Lysates were centrifuged (15,000 g, 20 min, 4 °C) to separate soluble and insoluble fractions. Protein concentrations were determined by BCA assay (ThermoFisher, 23225). Insoluble fractions were washed with PBS and solubilized overnight in 2× loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 2% β-mercaptoethanol). Soluble/insoluble loading was resolved by SDS-PAGE and transferred to 0.2 µm nitrocellulose membranes (Cytiva, 10600001). Proteins were probed with the following primary antibodies: α-syn-pS129 (Abcam, ab51253, 1:1000), FLAG-HRP (Sigma, A8592, 1:2000), Ubiquitin (Santa Cruz, sc-8017, 1:1000), LC3B (Sigma Aldrich, L7543, 1:1000) and β-actin (TransGen, HC201-01, 1:10,000) as loading control. HRP-conjugated secondary antibodies were used for detection by enhanced chemiluminescence. For tau aggregation assays, 293T cells were transfected with a lentiviral vector encoding full-length human tau (T40). At 6 h post-transfection, cells were treated with 0.1, 0.5 or 1 µM HQY1027. Cells then were transduced with either monomeric T40 or heparin-induced T40 PFF using CRISPR-Fectin transfection reagent (GeneCopoeia, EF015) at 24 h post-plasmid transfection. After another 48-h treatment with HQY1027 or PBS, cells were harvested in 1% Triton-X 100 lysis buffer (1% Triton-X 100, 30 mM Tris-HCl pH 7.5, 150 mM NaCl) supplemented with protease/phosphatase inhibitors (1:1000). Cells were sonicated using a probe-type sonicator, then kept on ice for 1 h. Insoluble material was pelleted via centrifugation (100,000 g, 30 min, 4 °C). Pellets were resuspended in 2X loading buffer, followed by immunoblotting. Primary antibodies were used: K9JA (DAKO, A0024, 1:1000); PHF-1 (gift from Dr. Peter Davies, 1:1000); GAPDH (Proteintech, 10494-1-AP, 1:1000). Photocrosslinking, biotin-click, and pulldown Following α-syn PFF induction (10 nM, 48 h), transfected 293T cells in 6-cm dishes were treated with either: (i) DMSO control, (ii) HQY1027 (20 µM), (iii) the inactive analog 5 (20 µM) for 12 h or not, followed by 0.2 µM probe 6 or 7 for 12 h. Cells were washed with PBS and incubated with fresh solutions of the same probes/DMSO in PBS. UV crosslinking was performed on ice by being exposed to UV light (365 nm) for 30 min (Analytikjena, CL-3000). Cells were then lysed in 1% SDS lysis buffer and protein concentrations determined by BCA assay. For biotin-click chemistry, lysates were incubated with 100 µM Biotin-N 3 , 100 µM TBTA, 1 mM CuSO 4 /THPTA, and 1 mM TCEP for 30 min at 25 °C with rotation. An additional 1 mM TCEP was added for 30 min to complete the reaction. Proteins were precipitated using methanol:chloroform:water (4:1:3, v/v), centrifuged (15,000 g, 15 min, 4 °C), and washed three times with ice-cold methanol. The pellet was air-dried (4 °C, 2 h) and resuspended in 1% SDS lysis buffer.For streptavidin pulldown, lysates were processed according to previously reported approaches 63 . Heat shock proteins were detected using the following antibodies: Hsp70 (Enzo, ADI-SPA-810, 1:1000) and Hsp40 (ThermoFisher, MA5-25362, 1:1000). Immunoblotting was performed as described above. Proximity labeling based on TurboID 293T WT cells were seeded in 12-well plates (1×10⁶ cells/well) and transfected with 0.5 µg/mL pCAGGS-α-syn WT -FLAG-TurboID using PEI (1:3 ratio). After 24 h, cells were re-plated (2×10⁵ cells/well) and treated with 100 nM α-syn PFFs in PBS for another 72h. Before sample collection, cells were treated with: (i) DMSO control, (ii) HQY1027 (0.5, 1, 2 µM), (iii) the inactive analog 5 (2 µM) for 2 h, followed by 50 µM D-Biotin in DMSO labeling for another 1 h. After pre-cool PBS washing, cells were lysed in 1% SDS lysis buffer and ultrasonication for 30s. Streptavidin beads pulldown and Western blot was performed as described above. Proteomic study Following biotin pulldown, beads were processed according to previously published method 64 . Generally, protein was denatured in 8 M urea/100 mM Tris pH 8.5, reduced with 5 mM TCEP and alkylated with 10 mM iodoacetamide. Trypsin (Promega, V511A; 1:100) was used for overnight digestion (37 °C, shaking in the dark). Digestion was quenched with 90% formic acid, and the supernatant was collected after centrifugation (15,000 g, 15 min). Peptides were desalted using C18 columns. Eluates were pooled, dried by vacuum centrifugation (30 °C, 2 h), and stored at -20 °C.Samples were then analyzed using an EASY-nLC 1000 HPLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific). Mass spectra were acquired in a data-dependent mode with one full scan (m/z: 350-1500; resolution: 15,000; AGC target value: 3,000,000 and maximal injection time: 20 ms), followed by MS2 scan (32% normalized collision energy; AGC target value: 100,000; maximal injection time: Dynamic). The MS/MS raw spectra were processed using MaxQuant Software (v.1.6.0.1). The average protein intensity of each treatment group was counted with GraphPad, and differential binding of probe 6 group vs DMSO or probe 7 groups was obtained. α-Syn WT -mEGFP overexpress ing SH-SY5Y cell line establishment The pLVX-EF1α-IRES-α-syn WT -mEGFP plasmid was generated by cloning human α-synuclein and the monomeric EGFP (A206K mutant) sequences into the pLVX-EF1α-IRES backbone using EcoRI and BamHI restriction sites. Then plasmids were introduced into 293T cells via packaging plasmids and PEI. Medium containing Lentivirus was collected and filtered by 0.22 µm PVDF filter (Merck, Millex-HV), before mixed with 8 µg/mL polybrene and added into SH-SY5Y cells. Infected SH-SY5Y cells were selected with 2 µg/mL puromycin and single-cell clone formation. Stable expression of α-syn WT -mEGFP was confirmed under fluorescent microscopy and Western blot. Colocalization studies To investigate α-syn–Hsp40 colocalization, SH-SY5Y cells stably overexpressing α-syn WT -mEGFP were seeded (1×10 5 cells/well) in 12-well plates pre-coated with coverslips and stimulated with 100 nM α-syn PFF for 48 h. Before fixation, cells were treated with 1 µM HQY1027 (0.5-6 h) or 1 µM the inactive analog 5 (6 h). Fixed with 4% PFA in PBS, cells were immunostained overnight with Hsp40 antibody (ThermoFisher, MA5-25362, 1:1000), followed by Alexa Fluor 647-conjugated secondary antibody (ThermoFisher, A32728). Confocal images were acquired using a Leica SP8 microscope (100×, 1.4 NA oil objective) under consistent laser settings. Pearson colocalization coefficients between α-syn aggregates and Hsp40 were quantified using ImageJ’s JACoP plugin 65 . Neuron propagation assay Primary cortical neurons cultured for 7 days in vitro (DIV7), were treated with: (i) PBS control, (ii) 200 nM α-syn monomer, (iii) 200 nM α-syn PFF, or (iv) PFF pre-incubated with test compounds (n = 3 biological replicates). Fixed neurons were immunostained using: α-syn-pS129 (Abcam, ab51253, 1:1000) and MAP2 (Abcam, ab5392, 1:2000) primary antibodies, followed by Alexa Fluor 488 anti-chicken (ThermoFisher, A11039) and 568 anti-rabbit (ThermoFisher, A11036) secondaries. Fluorescent images were acquired using a Leica SP8 confocal microscope, and the fluorescence intensity of α-syn-pS129 and MAP2 signals were quantified using ImageJ. Pharmacokinetic characterization of HQY1027 The assays for microsomal stability were performed according to the literature 66 . For mouse PK properties, HQY1027 were dissolved in DMA (10% v/v) and Solutol HS 15 (10% w/v) in normal saline or peanut butter, then injected into male ICR mice via tail vein (IV, 1 mpk) or oral administration (PO, 25 or 5 mpk). Blood samples (30-50 µL) were collected via the retro-orbital plexus at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post-dosing into tubes with EDTA2K. Plasma samples were analyzed by an Agilent 1290 Infinity II UHPLC system coupled to a SCIEX Triple Quad™ 3500 mass spectrometer. Analyte-to-internal standard peak area ratios were calculated against an 8-point calibration curve. Pharmacokinetic parameters were derived via non-compartmental analysis (Phoenix WinNonlin, v8.1.0.3530). For brain and plasma distribution, mice received HQY1027 (PO, 5 mpk) and were euthanized 15 min post-administration. Prior to brain collection, animals were transcardially perfused with 0.9% saline. Plasma and whole brains were harvested simultaneously. Brain samples were homogenized PBS and acetonitrile containing internal standard (dexamethasone, 100 ng/mL) using a TissueLyser (30 Hz, 10 min). Following centrifugation (14,000 g, 10 min, 4 °C), supernatants were analyzed by LC-MS/MS (Agilent 1290/SCIEX 3500 system). Brain-to-plasma concentration ratios were calculated from quantified drug levels in each matrix. α-Syn PFF -induced Parkinson’s Diseases mouse model All procedures were approved by the Animal Care Committee of the Interdisciplinary Research Center on Biology and Chemistry, Chinese Academy of Sciences. Male C57BL/6J mice (6 weeks old; Shanghai Lingchang Biotechnology) were individually housed and weighed every 72 h. Before stereotaxic surgery, mice were acclimated to consume peanut butter (8 g/kg body weight) at a fixed daily time point for 3 days. According to published method 67 , Mouse α-syn PFF or monomers (5 µg in sterile PBS) were unilaterally injected into the left dorsal striatum (coordinates: +0.2 mm AP, +2.0 mm ML relative to Bregma; −2.6 mm DV) using a Hamilton syringe (10 µL) at 0.4 µL/min. After 3 days of recovery, mice received daily oral HQY1027 (5 mpk), or vehicle control mixed into peanut butter at the trained time point. Treatment ceased 4 months post-injection, and mice were monitored until study endpoint. Behavior test To minimize potential stress or physical strain on the animals, the grip strength test was conducted last. All behavioral tests were performed with consistent cohort sizes (n = 10 mice per group) to ensure statistical reliability across assessments. For the grip strength test, it was measured using a metal grid meter (KEW BASIS, KW-ZL-1). Mice were placed on the grid with either (i) all four limbs or (ii) forepaws only. The tail was gently pulled backward three times, and the maximal holding force (grams) was recorded when the mouse resisted release. For the rotarod test, mice were evaluated for motor coordination and balance using an accelerating rotarod (Ugo Basile). Following one day of habituation to the stationary rod, mice underwent three trials per day with the rod accelerating from 4 to 40 rpm over 5 min. The inter-trial intervals were ≥30 min. The latency to fall (in seconds) and the corresponding speed (in rpm) at the time of falling were recorded. For the pole test, following a 3-day acclimation period, mice were placed head-up at the top of a vertical pole (50 cm height, 1 cm diameter). The time required for each mouse to reorient downward and descend to the base was recorded (in seconds). Three trials were performed per session with ≥30 min inter-trial intervals. For the open field test, the mice were placed in the open field arena under standardized illumination and allowed to explore for 10 min. Activity levels were monitored by an automated video tracking system (Noldus, EthoVision XT). Chambers were cleaned with 70% ethanol before and after testing. The total distance moved in 10 min (in cm) were used to analysis the motor activity of mice. IHC Six months post-treatment, mice were transcranially perfused with PBS. Brains were post-fixed in 4% PFA in PBS overnight at 4 °C, paraffin-embedded, and sectioned coronally (6 µm). As reported previously 67 , after deparaffinization and rehydration, antigen retrieval was performed by boiling in 100 mM citrate buffer (pH 6.0). Endogenous peroxidases were quenched with 5% H₂O₂/methanol, followed by blocking in 2% FBS/0.1 M Tris (pH 7.6). Sections were incubated overnight at 4 °C with α-syn-pS129 antibody (Abcam ab51253; 1:10,000 in blocking buffer). Next day, sections were processed according to standard DAB staining protocol (Vector), and counterstained with hematoxylin, dehydrated, and coverslipped with Cytoseal. Slides were scanned into digital format on a PANNORAMIC 250 Flash III DX (3D HISTECH). Digitized slides were then used for quantitative pathology. Pathological α-syn-pS129 area was quantified by ImageJ. Synthesis of compounds HQY1027 Reagent condition: a) TEA, DDQ, MeOH, DCM, room temperature (R.T.), 1 h. 4-(6-methoxybenzo[ d ]oxazol-2-yl)- N -methylaniline ( HQY1027 ) A solution of 4-dimethylaminobenzaldehyde (298 mg, 2.00 mmol) and 2-amino-5-methoxyphenol (139 mg, 1.00 mmol) in anhydrous MeOH (5 mL) were added TEA (0.348 mL, 2.5 mmol). After stirring for 30 min at room temperature, a solution of DDQ (227 mg, 1.00 mmol) in DCM (5 mL) was added dropwise over 5 min. The resulting mixture was stirred at room temperature for an additional 30 min until completion. Then it was diluted by water (20 mL) and extracted with EA (3 × 15 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure. Then the residue was purified by C18 flash column chromatography using a gradient eluent (H 2 O/MeCN = 100:0 to 20:80, H 2 O contains 0.05% HCl). HQY1027 was obtained by lyophilization as yellow solid (166 mg, 0.617 mmol) with a yield of 61.7%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.94 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.6 Hz, 1H), 7.34 (s, 1H), 6.93 (d, J = 8.8 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 3.82 (s, 3H), 3.03 (s, 6H). MS-ESI: m/z calculated for C 16 H 16 N 2 O 2 , Exact Mass: 268.12, found 269.1 [M + H] + . Statistical Analysis All data were analyzed using GraphPad Prism (v10.0.2). Group comparisons were performed by t -test or one/two-way ANOVA with Tukey or Dunnett's post hoc test as indicated. Data is presented as mean ± SD unless otherwise noted. Significance thresholds were defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns indicates not significant (p ≥ 0.05). Declarations Acknowledgements We thank Prof. Junying Yuan (IRCBC, CAS) for her generous help on this work, Chinese Academy of Sciences for the continued support, Dr. Yifan Ge for her help in imaging experiments, and National Facility for Protein Science in Shanghai, Ms. Ting Li (IRCBC, CAS), and Dr. Jiang Bian (SJTU) for their help in animal studies. This work was supported by National Natural Science Foundation of China (22425704 to C.L., 82188101 to C.L. and L.T.), Shanghai Basic Research Pioneer Project (L.T., C.L., Y.Z. and Z.H.), Shanghai Municipal Science and Technology Major Project (L.T., C.L., Y.Z. and Z.H.), Shanghai Leading Talent Program of Eastern Talent Plan (Y.L.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB1060000 to L.T., and C.L.), and the Shanghai Key Laboratory of Aging Studies (19DZ2260400 to C.L.). Dr. Cong Liu is SANS Exploration Scholar. Author contributions L.T. and Y.L. conceived the project. L.T., Q.H. and K.S. designed and synthesized the compounds. C.L., Y.L., Y.C., S.Z. and Y.T. designed and performed the α-syn fibril propagation, binding, and colocalization assays. Y.C., C.Q., Q.H. and H.X. conducted the animal studies. S.Z., K.L. and D.L. performed cryo-EM sample preparation, data collection and visualization. Y.C., Y-X.L. and Y.Z. conducted the proteomic studies. Y.C., S.W., X.L., and B.S. designed and performed the optical tweezers analysis. W.Z. and Z.H. designed and performed the tau-aggregation cellular assays. L.T., C.L. and Y.L. supervised the project. L.T., C.L., Y.C., Q.H. and S.Z. wrote the manuscript; All the authors contributed to discussion of the manuscript and editing. Competing interests L.T., Q.H., Y.C., C.L., and Y.L. are inventors on patent applications relating to this work, owned by SIOC. All other authors declare they have no competing interests. Data availability Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession no. EMD-64224 for HQY1027-bound α-syn fibrils, The corresponding atomic models have been deposited in the PDB under accession no. 9UJQ for HQY1027-bound α-syn fibrils. References Ross, C. A. & Poirier, M. A. Protein aggregation and neurodegenerative disease. Nat Med 10 Suppl , S10-17 (2004). https://doi.org/10.1038/nm1066 Soto, C. & Pritzkow, S. 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J Med Chem 66 , 10917-10933 (2023). https://doi.org/10.1021/acs.jmedchem.3c00736 Schreiber, S. L. Molecular glues and bifunctional compounds: Therapeutic modalities based on induced proximity. Cell Chem Biol 31 , 1050-1063 (2024). https://doi.org/10.1016/j.chembiol.2024.05.004 Burger, D. et al. Synthetic alpha-synuclein fibrils replicate in mice causing MSA-like pathology. Nature 648 , 409-417 (2025). https://doi.org/10.1038/s41586-025-09698-1 Additional Declarations Yes there is potential Competing Interest. L.T., Q.H., Y.C., C.L., and Y.L. are inventors on patent applications relating to this work, owned by SIOC. All other authors declare they have no competing interests. Supplementary Files Syntheticproceduresandphysicalcharacterizationfornewcompounds.pdf Synthetic procedures and physical characterization for new compounds Supplementarytables.xlsx Extended Data Table 1. Statistics of cryo-EM data collection, refinement and validation ExtendedDataFig.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6562222","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":613959189,"identity":"0a3a7eb6-d0a6-48b7-876c-099b0726a47a","order_by":0,"name":"Li 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08:32:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1503279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerendipitous discovery and lead optimization of α-syn aggregation inhibitors. a\u003c/strong\u003e, Chemical structure of PiB, compound \u003cstrong\u003e3\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e, and PROTACs \u003cstrong\u003e1\u003c/strong\u003e and \u003cstrong\u003e2\u003c/strong\u003e. \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e, Cellular anti-aggregation activity of PiB and derivatives/analogs for 72 h. \u003cstrong\u003ed\u003c/strong\u003e, Inhibition curves and IC\u003csub\u003e50\u003c/sub\u003e values for HQY1027 and PiB against α-syn aggregation based on titration experiments as in panel\u003cstrong\u003e b\u003c/strong\u003e. Data represent means ± SD (n = 3). \u003cstrong\u003ee\u003c/strong\u003e, HQY1027 inhibited α-syn aggregation in a time- and dose-dependent manner. \u003cstrong\u003ef\u003c/strong\u003e, Anti-aggregation activity comparison between HQY1027 (HQY) and clinical inhibitors emrusolmin (Emru) and minzasolmin (Minza). PFF types were indicated as WT, MSA- or JOS-like.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/071cb2cfbf41164fe1ff8d02.jpg"},{"id":105794688,"identity":"2e64cdf0-cb7a-4b7e-bc00-fe800cde6ed7","added_by":"auto","created_at":"2026-03-31 08:32:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1357899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTarget identification of HQY1027. a\u003c/strong\u003e, Representative negative-staining (NS)-EM micrographs of fibrils incubated with DMSO or HQY1027 (100 µM) for 72 h. Scale bar, 1 µm. \u003cstrong\u003eb\u003c/strong\u003e, Anti-aggregation activity of HQY1027 (0.5 µM) in the presence of PS-341 or BafA1 (0.1 µM) in cells. PFF types were indicated as WT, MSA- or JOS-like. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Chemical structure of the inactive analogue and the photocrosslinking probes (\u003cstrong\u003ec\u003c/strong\u003e), and their anti-aggregation activity at 0.5 µM in cells (\u003cstrong\u003ed\u003c/strong\u003e). \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e, Volcano plots of proteins labeled by \u003cstrong\u003e6\u003c/strong\u003e(0.2 µM), compared to DMSO group (\u003cstrong\u003ee\u003c/strong\u003e) or probe \u003cstrong\u003e7\u003c/strong\u003e (0.2 µM) group (\u003cstrong\u003ef\u003c/strong\u003e), respectively. Proteins of interest are highlighted with different shapes. \u003cstrong\u003eg\u003c/strong\u003e, Western blot analysis of proteins pulled down by photocrosslinking probes (0.2 µM) with or without competition by HQY1027 or \u003cstrong\u003e5 \u003c/strong\u003e(20 µM). \u003cstrong\u003eh\u003c/strong\u003e, Western blot analysis of proteins biotinylated by α-syn-TurboID in the presence with DMSO, HQY1027, or \u003cstrong\u003e5\u003c/strong\u003e(2 µM).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/8c0b5ccdb9398f4cdad1dd3f.jpg"},{"id":105904262,"identity":"7fd61b56-09d1-4b1a-8937-eabbc846a42c","added_by":"auto","created_at":"2026-04-01 10:06:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":326847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHQY1027\u003c/strong\u003e \u003cstrong\u003edirectly binds to α-syn fibrils and Hsp40. a\u003c/strong\u003e,\u003cstrong\u003eb,\u003c/strong\u003e Representative sensorgrams and summarized \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values from SPR assays. \u003cstrong\u003ec\u003c/strong\u003e, Bar graph summarizing the melting temperatures of indicated Hsp proteins. Data represent means ± SD (n = 3). Unpaired \u003cem\u003et\u003c/em\u003e-test.\u003cstrong\u003e d\u003c/strong\u003e, Representative central slice of the 3D cryo-EM density map for HQY1027-α-syn fibril complex, with extra ligand densities indicated by arrows.\u003cstrong\u003e e\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e, Top and side views of the reconstructed density map along the fibril axis. The surface of the α-syn fibril is colored grey, and the densities of HQY1027 are colored yellow. The angles between HQY1027 and fibril axis in each binding pocket are indicated.\u003cstrong\u003e g\u003c/strong\u003e, Structural comparison of the α-syn fibril N-pocket in apo (PDB ID 6A6B) or ligand-bound states. Fibril backbone is shown as grey sticks, HQY1027 density is highlighted in yellow, and ThT (PDB ID 7YNM) and BF227 (PDB ID 7YNP) are shown as magenta sticks. Dashed arrows indicate the orientation of the Glu83 side chain in each state. \u003cstrong\u003eh\u003c/strong\u003e, Chemical structures and bioactivities of HQY1027 derivatives. Anti-α-syn aggregation activity of compounds (0.5 µM) was assessed by Western blot analysis and normalized with DMSO control (100%). \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values were determined by SPR analysis. \u003cstrong\u003ei\u003c/strong\u003e, Bar graph summarizing fluorescence polarization assay results measuring the hydrophobic surface exposure of WT α-syn fibrils in the presence of HQY1027 or its derivatives. Data represent means ± SD (n = 3). Unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/1de006e2aaa490f80e3cc6fa.jpg"},{"id":105904925,"identity":"0928f723-9367-4cee-8c40-a2730ed5678f","added_by":"auto","created_at":"2026-04-01 10:11:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":978971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHQY1027 promotes recognition of α-syn fibrils by Hsp40 in vitro. a\u003c/strong\u003e, SPR sensorgrams (single-cycle kinetics) for HQY1027 binding to α-syn PFF in the absence or presence of Hsp40 (Hdj1). \u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003ec\u003c/strong\u003e, Representative SPR sensorgrams (\u003cstrong\u003eb\u003c/strong\u003e) and summarized \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values (\u003cstrong\u003ec\u003c/strong\u003e) for Hsp40 binding to different types of α-syn PFFs in the absence or presence of HQY1027 at indicated concentrations. \u003cstrong\u003ed\u003c/strong\u003e, Bar graph showing the ELISA assay results for Hsp40 binding to α-syn fibrils. Data represent means ± SD (n = 4). Two-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/f45f7f900ff3330e1a823b2b.jpg"},{"id":105794691,"identity":"611cabe6-a185-4016-82be-b43a495ea9ac","added_by":"auto","created_at":"2026-03-31 08:32:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1705933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHQY1027 enhances Hsp40’s binding to α-syn fibrils in vitro and in neuronal cells. a\u003c/strong\u003e, Cartoons depicting the principle of the optical tweezers assay.\u003cstrong\u003e b\u003c/strong\u003e, Time-lapse fluorescence imaging of Hsp40 binding to α-syn fibrils. The azure arcs indicate part of the beads connected to each side of the single fibril, the areas between white dashes indicate where the fibril is located, and the green fluorescent dots indicate Hsp40 proteins. Scale bar, 2 µm.\u003cstrong\u003e c\u003c/strong\u003e, Curve quantitating the results from panel \u003cstrong\u003eb\u003c/strong\u003e. Data represent means ± SD (n = 6). Paired \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003ed\u003c/strong\u003e, Representative images of Hsp40 (red) colocalized with α-syn (green) in SH-SY5Y cells that stably expressing α-syn-mEGFP. Scale bar, 2 µm. \u003cstrong\u003ee\u003c/strong\u003e, Bar graph quantitating the colocalization of α-syn and Hsp40 from panel A. Data represent means ± SD based on 6 images from three independent experiments. One-way ANOVA with Dunnett’s post hoc test.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/041d4b40349a64a3d9b4d967.jpg"},{"id":105904405,"identity":"4196ad60-7717-4dfa-9f73-625b7a3e5552","added_by":"auto","created_at":"2026-04-01 10:08:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1873988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHQY1027 impedes the propagation of α-syn fibrils in the presence of Hsp40. a\u003c/strong\u003e, Representative NS-EM micrographs of fibrils after growth in the presence with 100 µM HQY1027 or \u003cstrong\u003e5\u003c/strong\u003e for 84 h. Scale bar, 1 µm.\u003cstrong\u003e b\u003c/strong\u003e, Bar graph quantitating the PFF-induced α-syn fibril formation in panel A. Data represent means ± SD (n = 4).\u003cstrong\u003e c\u003c/strong\u003e, Anti-aggregation activity of HQY1027 for 48h, in the absence or presence of VER155008 (VER, 10 mM) in cells. PFF types were indicated as WT, MSA- or JOS-like. \u003cstrong\u003ed\u003c/strong\u003e, Representative immunostaining images of α-syn p-S129 (red) and MAP2 (green) in rat primary cortical neurons treated with PBS, α-syn monomer (200 nM), or α-syn PFF (200 nM) with or without HQY1027. Scale bar, 50 µm.\u003cstrong\u003e e\u003c/strong\u003e, Bar graph quantitating the α-syn p-S129 intensity normalized to MAP2 intensity from panel \u003cstrong\u003ed\u003c/strong\u003e. Data represent means ± SD based on 6 images from three independent experiments. One-way ANOVA with Dunnett’s post hoc test.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/55e8a3fbd3add6a3bff6f713.jpg"},{"id":105904382,"identity":"d66f13a1-d453-4948-8059-c2ae0e8ff589","added_by":"auto","created_at":"2026-04-01 10:07:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1175219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHQY1027 mitigates the pathological aggregation of α-syn in a mouse PD model. a\u003c/strong\u003e, Curves showing weight changes of each mice group (n = 10). \u003cstrong\u003eb\u003c/strong\u003e, Grip strength measurement results for each mice group (n = 10). Unpaired \u003cem\u003et\u003c/em\u003e-test. \u003cstrong\u003ec\u003c/strong\u003e, Representative images showing pathological α-syn (p-S129, brown staining) sections in mouse motor cortex and substantia nigra from PFF groups. Cell nuclei are indicated by hematoxylin staining (blue), typical α-syn aggregates are highlighted with red arrows, and magnification times are indicated. Scale bar, 50 µm. \u003cstrong\u003ed\u003c/strong\u003e, Quantitating results of relative levels of α-syn pathology areas based on IHC straining and ImageJ. Data represent means ± SD (n = 9). Unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/d8aaec2d64d149b3d059477a.jpg"},{"id":106153124,"identity":"ff498619-c164-488d-9491-b0667ea54eb9","added_by":"auto","created_at":"2026-04-04 14:32:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10488024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/70931d27-8067-47a8-b1d1-5b314b5de4a2.pdf"},{"id":105794687,"identity":"cc897da4-2b2b-4ebd-bf5d-d1b2988333d5","added_by":"auto","created_at":"2026-03-31 08:32:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":416699,"visible":true,"origin":"","legend":"Synthetic procedures and physical characterization for new compounds","description":"","filename":"Syntheticproceduresandphysicalcharacterizationfornewcompounds.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/a27db3e8998085fb444e5c42.pdf"},{"id":105904389,"identity":"a1012014-c0b1-46ac-97a7-f50a8a3bdc7b","added_by":"auto","created_at":"2026-04-01 10:07:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":152378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Table 1. Statistics of cryo-EM data collection, refinement and validation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Supplementarytables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/4619697363b89354078df4e3.xlsx"},{"id":105794692,"identity":"1fcb5833-21ef-416b-a37e-3e4b9c1688de","added_by":"auto","created_at":"2026-03-31 08:32:35","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4560581,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-6562222/v1/42eca04622d22f5032dc6f76.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nL.T., Q.H., Y.C., C.L., and Y.L. are inventors on patent applications relating to this work, owned by SIOC. All other authors declare they have no competing interests.","formattedTitle":"Discovery of amyloid binders as chemical chaperone to block pathological α-synuclein aggregation in synucleinopathies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe global rise in aging populations has intensified the societal impact of neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Despite their complex and heterogeneous etiologies, protein misfolding, aggregation, and accumulation have been identified as key drivers of ND onset and progression\u003csup\u003e1,2\u003c/sup\u003e. Notably, pathogenic aggregates - including tau, α-synuclein (α-syn), and TDP-43 - can spread in a prion-like manner through the central nervous system (CNS), making aggregated proteins compelling therapeutic targets. Recent clinical advances with β-amyloid (Aβ) antibodies further underscore the therapeutic tractability of targeting protein aggregates\u003csup\u003e3,4\u003c/sup\u003e. However, current antibody therapies are associated with a non‑negligible risk of amyloid‑related imaging abnormalities (ARIA) and microhemorrhages in AD patients\u003csup\u003e5\u003c/sup\u003e, while still failing to efficiently penetrate neuronal membranes and clear intracellular tau or α‑syn aggregates\u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to antibodies, small-molecule inhibitors targeting pathogenic protein aggregates have attracted significant attention in ND drug development, owing to their potential advantages in CNS penetration and low immunogenicity. The most advanced candidates are those derived from screening hits capable of directly inhibiting or even reversing protein aggregation in vitro. For instance, leucomethylene blue, a tau aggregation inhibitor, has progressed to Phase 3 clinical trials for AD\u003csup\u003e7,8\u003c/sup\u003e, while emrusolmin\u003csup\u003e9\u003c/sup\u003e and minzasolmin\u003csup\u003e10\u003c/sup\u003e,\u0026nbsp;both reported to inhibit α-syn fibrillogenesis, have entered Phase 2 trials for PD and multiple system atrophy (MSA). However, the clinical translation of these inhibitors has yet to be realized, as exemplified by the recent Phase 2 trial failure of minzasolmin to improve PD symptoms. Fundamental questions also persist regarding the mechanism of action (MOA) and in‑vivo efficacy of many purported anti‑fibrillogenesis agents, have subsequently been shown to act as non‑specific redox modulators or autophagy inducers, or else to lack sufficient brain exposure\u003csup\u003e8,11,12\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e. Consequently, the development of diverse strategies to target pathogenic protein aggregates remains an urgent unmet need in the field.\u003c/p\u003e\n\u003cp\u003eRecent advances to eliminate pathogenic aggregates in ND have introduced strategies for targeted protein degradation, such as molecular glue degraders\u003csup\u003e14\u003c/sup\u003e and proteolysis-targeting chimeras (PROTACs)\u003csup\u003e15\u003c/sup\u003e. Concurrently, inhibitors disrupting aggregate-membrane receptor interactions have been developed to block α-syn propagation\u003csup\u003e16,17,18\u003c/sup\u003e. However, these alternative approaches remain largely at the proof-of-concept stage and require further validation. Meanwhile, cellular or phenotypic screening continues to reveal biologically active compounds with unexpected MOA\u003csup\u003e19,20\u003c/sup\u003e. In this study, we serendipitously discovered that the amyloid PET tracer Pittsburgh compound B (PiB) inhibits α-syn aggregation. Although amyloid PET tracers are well-established in the clinical diagnosis of ND, their potential cellular functions beyond fibril binding have been largely unexplored. We demonstrate that PiB and its optimized analog, HQY1027, function via a previously unrecognized chemical chaperone mechanism. They enhance interactions between chaperone proteins and polymorphic α-syn fibrils, thereby impeding fibril propagation. HQY1027 exhibits favorable oral bioavailability and brain penetration, and shows remarkable efficacy in halting the spread of α-syn pathology in both cultured neurons and in vivo. This work establishes a new co-chaperone strategy for combating proteinopathies using drug-like small molecules.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSerendipitous discovery and lead optimization of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eα\u003c/strong\u003e\u003cstrong\u003e-syn aggregation inhibitors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo enable targeted degradation of pathological α-syn aggregates, we designed a series of PROTACs incorporating known amyloid-binding ligands, E3 ligase ligands, and diverse linkers (Extended Data Fig. 1a). In α-syn-overexpressing (α-syn-OE) 293T cells, most PROTACs showed limited effectiveness against wildtype (WT) pre-formed fibrils (PFF)-induced α-syn aggregation, as assessed by insoluble α-syn levels and Ser129 phosphorylation (p-S129) (Extended Data Fig. 1b). Notably, PROTAC \u003cstrong\u003e1\u003c/strong\u003e, comprising PiB-derived α-syn-targeting moiety conjugated to a von Hippel-Lindau (VHL) E3 ligase ligand, emerged as the most effective at reducing α-syn aggregation (Fig. 1a and Extended Data Fig. 1b). To verify the PROTAC mechanism, we synthesized control compounds including a diastereomer of \u003cstrong\u003e1\u003c/strong\u003e (\u003cstrong\u003e2\u003c/strong\u003e), and PEGylated derivatives of PiB (\u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e). Surprisingly, while \u003cstrong\u003e2\u003c/strong\u003e was inactive as predicted, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, and PiB itself outperformed PROTAC \u003cstrong\u003e1\u003c/strong\u003e in anti-aggregation assays (Fig. 1a,b). Furthermore, VHL knockout failed to attenuate the activity of either \u003cstrong\u003e1\u003c/strong\u003e or the controls (Extended Data Fig. 1c). These unexpected results suggest that the observed activity stems from the intrinsic inhibitory properties of PiB rather than PROTAC-mediated degradation.\u003c/p\u003e\n\u003cp\u003eBuilding on this serendipitous discovery, we synthesized over fifty PiB derivatives to explore the structure-activity relationships and to optimize both potency and drug-like properties. Several analogs exhibited substantially improved activity over PiB. Among them, HQY1027, which incorporates a benzoxazole-for-benzothiazole substitution and two additional methyl groups, proved to be the most potent. It achieved an IC₅₀ of 50 nM against WT PFF-induced α-syn aggregation, demonstrating at least a tenfold increase in potency over PiB. (Fig. 1c,d and Extended Data Fig. 2a). Notably, other reported binders of α-syn fibrils, such as BF227 and C0503\u003csup\u003e21\u003c/sup\u003e, showed minimal activity at 10 µM (Fig. 1c). In cellular assays, HQY1027 effectively reduced α-syn aggregation at 0.2 µM and nearly abolished the stimulated aggregation at 0.5 µM. Remarkably, even when administrated 48 h post-PFF seeding, 0.5 µM HQY1027 still effectively suppressed aggregation within 24 h (Fig. 1e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the structural polymorphism and varying pathological properties of α-syn fibrils\u003csup\u003e22,23\u003c/sup\u003e, we next evaluated HQY1027 and PiB against fibrils designed to mimic endogenous conformations observed in synucleinopathies. These fibrils incorporated hereditary mutations associated with familial synucleinopathies (A53T and G51D)\u003csup\u003e24,25\u003c/sup\u003e as well as an engineered mutation at Glu57, a residue known to influence fibril polymorphs\u003csup\u003e26\u003c/sup\u003e. Cryo-EM analysis revealed that one PFF containing the A53T/G51D double mutation formed a polymorph (PDB: 9JC3) closely resembling MSA Type-II\u003csub\u003e2\u003c/sub\u003e α-syn fibril (PDB: 6XYQ)\u003csup\u003e27\u003c/sup\u003e, while another incorporating the A53T/G51D/E57A triple mutation exhibited high structural similarity (PDB: 9KAL) to the singlet α-syn fibril found in juvenile-onset synucleinopathy (JOS) (PDB: 8BQV) (Extended Data Fig. 2b)\u003csup\u003e28\u003c/sup\u003e. Although both MSA-like and JOS-like PFFs induced stronger α-syn aggregation than WT PFFs in α-syn-OE 293T cells, HQY1027 consistently and effectively suppressed this aggregation with IC₅₀ values around 200 nM, 3 to 4 times lower than those of PiB (Fig. 1d and Extended Data Fig. 2a).\u003c/p\u003e\n\u003cp\u003eCompared to the clinical-stage α-syn fibrillation inhibitors, emrusolmin and minzasolmin, HQY1027 exhibited markedly higher potency in cellular assays (Fig. 1f). At a concentration of 0.5 µM, HQY1027 effectively suppressed α-syn aggregation induced by either wild-type (WT) or MSA/JOS-like PFFs in α-syn-OE 293T cells. In contrast, emrusolmin and minzasolmin showed minimal activity at 1 µM and were only effective at 10 µM, a concentration associated with non-negligible cytotoxicity. Furthermore, while PiB exhibits pan-amyloid affinity, HQY1027 demonstrated no inhibitory activity against PFF-induced tau aggregation in tau-OE 293T cells (Extended Data Fig. 3a), highlighting its selectivity for α-syn aggregation. HQY1027 also demonstrated significantly improved metabolic stability, with a microsomal half-life (T\u003csub\u003e1/2\u003c/sub\u003e) of 23.4 min versus only 8.4 min for PiB, addressing the rapid clearance typical of most PET tracers (Extended Data Fig. 3b). Collectively, these data establish HQY1027 as a potent and selective lead inhibitor of α-syn aggregation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTarget identification of HQY1027\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHQY1027’s superior cellular activity against α-syn aggregation suggests a pharmacological mechanism distinct from that of conventional fibrillation inhibitors. To elucidate its mechanism of action, we first tested its ability to directly inhibit fibril growth in vitro, a hallmark of conventional aggregation inhibitors. However, HQY1027 showed no inhibitory activity even at high concentrations (Fig. 2a). Unlike the reference inhibitor GSD-16-24\u003csup\u003e16\u003c/sup\u003e, HQY1027 also failed to disrupt α-syn fibril binding to membrane receptors (LAG3 or RAGE) (Extended Data Fig. 4a). Mechanistic studies revealed that HQY1027’s anti-aggregation activity remained unaffected by either proteasome inhibition (PS-341) or lysosome blockade (bafilomycin A1, BafA1) regardless the PFF type used (Fig. 2b and Extended Data Fig. 4b), indicating a mechanism independent of major protein degradation pathways. Although HQY1027 suppresses α-syn Ser129 phosphorylation, comprehensive kinome profiling at 1 µM revealed no significant kinase interactions (Supplementary Table 1), excluding direct kinase inhibition as its mechanism.\u003c/p\u003e\n\u003cp\u003eTo identify the direct targets, we designed and synthesized photocrosslinking probes derived from PiB, HQY1027, and related analogs - including the inactive analog \u003cstrong\u003e5\u003c/strong\u003e (Fig. 2c). These probes contain diazirine groups that generate reactive carbenes upon UV irradiation, enabling covalent bond formation with target proteins via C-H insertion. The labeled proteins can then be enriched through click chemistry and affinity pulldown. Based on their inhibitory activities, we selected probe \u003cstrong\u003e6\u003c/strong\u003e (with potency comparable to HQY1027) and inactive control probe \u003cstrong\u003e7\u003c/strong\u003e for further studies (Fig. 2c,d). Photocrosslinking was performed in live α-syn-OE 293T cells that had been pretreated with PFF for 48 h. Proteins labeled by either probe \u003cstrong\u003e6\u003c/strong\u003e or \u003cstrong\u003e7\u003c/strong\u003e were separately enriched from cell lysates and analyzed by mass spectrometry (MS)-based proteomics (Fig. 2e,f and Supplementary Tables 2,3).\u003c/p\u003e\n\u003cp\u003eProteomic analysis revealed that probe \u003cstrong\u003e6\u003c/strong\u003e labeled many cellular proteins, consistent with the expected promiscuous reactivity of carbene-based probes (Fig. 2e and Supplementary Table 2). Importantly, \u003cstrong\u003e6\u003c/strong\u003e specifically labeled α-syn, confirming its maintained affinity for α-syn aggregates as a PiB-derived analog. Notably, multiple molecular chaperones including Hsp40s, Hsp70s, and Hsp90s were among the top significantly labeled hits. Using inactive probe \u003cstrong\u003e7\u003c/strong\u003e for background subtraction, we identified a subset of proteins specifically labeled by \u003cstrong\u003e6\u003c/strong\u003e (Fig. 2f and Supplementary Table 3). Several chaperones remained highly enriched in this specific subset, with the Hsp40 family member Hdj1 emerging as one of the most prominent interactors. Western blot analysis validated these findings, demonstrating specific labeling of α-syn, Hsp40, and Hsp70 by probe \u003cstrong\u003e6\u003c/strong\u003e but not control probe \u003cstrong\u003e7\u003c/strong\u003e (Fig. 2g). This labeling was substantially reduced by HQY1027 pretreatment, but remained unaffected by the inactive analog \u003cstrong\u003e5\u003c/strong\u003e. Furthermore, in PFF-pretreated 293T cells expressing α‑syn fused to TurboID - enabling biotinylation of proximate or interacting proteins\u003csup\u003e29\u003c/sup\u003e - treatment with HQY1027, but not analog \u003cstrong\u003e5\u003c/strong\u003e, led to significant biotinylation of Hsp40 and Hsp70 (Fig. 2h). Collectively, these results indicated that HQY1027 likely exerts its inhibitory effects by simultaneously engaging both α-syn aggregates and cellular chaperone proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHQY1027 directly binds to\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eα\u003c/strong\u003e\u003cstrong\u003e-syn fibrils and Hsp40\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next assessed HQY1027’s direct interactions with α-syn fibrils and recombinant chaperone proteins using surface plasmon resonance (SPR). HQY1027 displayed high binding affinity for WT α-syn PFF (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e = 0.65 µM) and moderate affinity for Hdj1 (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e = 5.41 µM), while showing weak binding to Hsc70 and Hsp90α (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e \u0026gt;10 µM) (Fig. 3a,b and Extended Data Fig. 5a). In contrast, the inactive analog \u003cstrong\u003e5\u003c/strong\u003e displayed similar binding to chaperones but significantly reduced affinity for α-syn PFF (15-fold weaker than HQY1027) (Fig. 3b and Extended Data Fig. 5b). Furthermore, protein thermal shift assay results indicated that HQY1027 directly binds to Hsp40 but not to Hsp70/90, as HQY1027 substantially destabilized Hdj1 (ΔTm = -4.6°C) but minimally affected the thermostability of Hsc70 or Hsp90α (Fig. 3c and Extended Data Fig. 6).\u003c/p\u003e\n\u003cp\u003eCryo-EM analysis of WT α-syn PFF preincubated with HQY1027 revealed additional densities at two binding pockets previously reported for PiB\u003csup\u003e21\u003c/sup\u003e (Fig. 3d). The 2.9 Å reconstruction (Extended Data Fig. 7 and Extended Data Table 1) confirmed HQY1027 occupancy at both the N-terminal (N-pocket) and C-terminal (C-pocket) binding sites along the fibril axis (Fig. 3e). HQY1027 exhibits distinct binding geometries within these pockets: in the C-pocket, it tilts approximately 45°\u0026nbsp;and contacts multiple stacked α-syn layers; in the N-pocket, it adopts a nearly horizontal orientation rather than a diagonal one (Fig. 3f). Electron densities corresponding to HQY1027 are relatively weak, suggesting dynamic rather than rigidly fixed interactions with the fibrils. Notably, no additional ligand density was observed in MSA- or JOS-like PFFs incubated with HQY1027,\u0026nbsp;further supporting a highly dynamic binding mode for HQY1027.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetailed structural analysis revealed that HQY1027 binding within the N-pocket promotes an inward orientation of α-syn Tyr39 (Fig. 3g). This ‘closed’ N-pocket conformation has been observed with PiB and thioflavin T (ThT), but not with BF227\u003csup\u003e21\u003c/sup\u003e. HQY1027 binding also triggered an inward re-orientation of the negatively charged Glu83 side chain within the N-pocket, likely reducing local polarity and hydrophilicity. This Glu83 rearrangement contrasts sharply with its outward-facing orientation in both apo α-syn fibrils and other ligand-bound states\u003csup\u003e21,31\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo examine whether introducing hydrophilic or charged groups would alter the activity of HQY1027, we designed and synthesized a series of derivatives bearing positively charged (\u003cstrong\u003e8\u003c/strong\u003e‑\u003cstrong\u003e10\u003c/strong\u003e), hydrophilic (\u003cstrong\u003e11\u003c/strong\u003e, \u003cstrong\u003e12\u003c/strong\u003e), or negatively charged (\u003cstrong\u003e13\u003c/strong\u003e) moieties, and compared them with HQY1027 (Fig. 3h). In α-syn‑overexpressing 293T cells, all derivatives showed markedly reduced anti‑aggregation activity at 0.5 µM compared with HQY1027 (Fig. 3h and Extended Data Fig. 8a). According to SPR analysis, the positively charged derivatives bound α‑syn fibrils much more weakly, while the other three derivatives displayed binding affinities similar to HQY1027 (Fig. 3h and Extended Data Fig. 8b). Notably, HQY1027 was substantially more effective than any derivative in enhancing the hydrophobic surface exposure of α‑syn fibrils, as indicated by a pronounced increase in fluorescence polarization of a widely used hydrophobic‑sensitive dye, SYPRO Orange (Fig. 3i). Collectively, these data suggest that HQY1027 modifies the surface physicochemical properties of α‑syn fibrils, which may in turn affect their interactions with cellular proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHQY1027 promotes Hsp40’s recognition of α-syn fibrils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven HQY1027's dual binding affinity for both α-syn fibrils and Hsp40, we next investigated its effects on their interactions. SPR-based kinetic profiling of ternary complex formation\u003csup\u003e32\u003c/sup\u003e revealed that Hdj1 significantly enhanced HQY1027 binding to α-syn fibrils, as evidenced by increased responses and prolonged dissociation kinetics (Fig. 4a). Reciprocally, HQY1027 markedly stabilized the interaction between Hdj1 and WT PFF, reducing the dissociation rate constant (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e) by \u0026gt;90% and consequently decreasing the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e value by approximately 60-fold (Fig. 4b,c and Extended Data Fig. 9a). It also consistently stabilized the association of Hdj1 with both MSA- and JOS-like PFFs, decreasing the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e value by at least 40-fold. Furthermore, while truncation of the α-syn C-terminal nearly abolished Hdj1 binding, HQY1027 facilitated significant, dose-dependent interactions between Hdj1 and α-syn\u003csup\u003e1-100\u003c/sup\u003e PFF. This provides additional evidence that HQY1027 modulates the surface characteristics of α-syn fibril cores and influences their dynamic engagement with Hsp40. These findings were corroborated by quantitative enzyme-linked immunosorbent assay (ELISA), where HQY1027 significantly increased Hdj1 binding to α-syn fibrils, while the inactive analog \u003cstrong\u003e5\u003c/strong\u003e showed minimal activity (Fig. 4d). Notably, consistent with its selective binding profile among Hsp family members, HQY1027 did not enhance α-syn fibril binding to Hsc70 (Extended Data Fig. 9b).\u003c/p\u003e\n\u003cp\u003eTo directly visualize HQY1027-enhanced Hsp40-α-syn fibril interactions, we employed single-molecule fluorescence analysis with optical tweezers, which enables real-time measurement of binding dynamics between Alexa555-labeled Hdj1 (Hdj1-Alex555) and individual α-syn fibrils under controlled tension\u003csup\u003e33\u003c/sup\u003e (Fig. 5a). Control experiments with 10 nM Hdj1-Alex555 alone exhibited only sparse fluorescence signals along suspended α-synuclein fibrils. In contrast, preincubation of α-syn fibrils with HQY1027 significantly enhanced Hdj1 binding, as evidenced by rapid accumulation of persistent fluorescence signals (Fig. 5b,c).\u003c/p\u003e\n\u003cp\u003eTo validate this mechanism in neuronal context, we generated an SH-SY5Y human neuroblastoma cell line stably expressing GFP-tagged α-syn and quantitatively assessed α-syn aggregate-Hsp40 colocalization. Confocal microscopy showed that while PFF treatment of these cells converted diffuse α-syn monomers into punctate aggregates, whereas Hsp40 displayed only minimal baseline colocalization with these aggregates under control conditions (Fig. 5d). Notably, treatment with 1 µM HQY1027 significantly enhanced this colocalization by at least five-fold as quantified by Pearson correlation coefficients, whereas \u003cstrong\u003e5\u003c/strong\u003e showed no detectable effects (Fig. 5e). These results provide direct evidence that HQY1027 enhances Hsp40 recognition of α-syn aggregates across experimental systems, establishing a clear mechanistic basis for its anti-aggregation activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHQY1027 impedes α-syn fibril propagation in the presence of Hsp40\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough HQY1027 alone showed minimal inhibition of α-syn fibril growth in vitro, it exhibited remarkable inhibitory activity combined with Hsp40. While low concentrations of Hdj1 protein alone had no significant effects, co-treatment with 10 µM HQY1027 achieved near-complete suppression of α-syn fibril elongation. Strikingly, \u003cstrong\u003e5\u003c/strong\u003e remained almost inactive even at 100 µM under identical conditions (Fig. 6a,b).\u0026nbsp;To determine whether Hsp40 and downstream chaperones are essential for the cellular activity of HQY1027, we employed the Hsp70 inhibitor VER-155008\u003csup\u003e34\u003c/sup\u003e for pharmacological perturbation, in light of the current lack of inhibitors targeting Hsp40 directly. As expected, co-treatment with VER-155008 largely abolished the anti-aggregation effect of HQY1027 across all PFF types in 293T cells (Fig. 6c). These findings support the mechanism whereby HQY1027 binds to α-syn fibrils, modifying their surface architecture and thereby facilitating chaperone recruitment to block fibril growth.\u003c/p\u003e\n\u003cp\u003eWhile HQY1027 effectively inhibited α-syn fibril growth in the 293T OE system, we further evaluated its efficacy in physiologically relevant models. Using an established neuronal propagation assay\u003csup\u003e35\u003c/sup\u003e, we treated primary rat cortical neurons with α-syn PFF seeds for 14 days, which induced significant pathological fibril formation as quantified by p-S129 levels. HQY1027 treatment significantly reduced p-S129 levels in PFF-seeded neurons without affecting normal neuronal morphology (assessed by MAP2 levels), whereas \u003cstrong\u003e5\u003c/strong\u003e showed minimal effects (Fig. 6d,e and Extended Data Fig. 10). Remarkably, HQY1027 at 0.5 µM reduced α-syn aggregation by 50%, demonstrating exceptional potency compared to recently reported state-of-the-art α-syn propagation inhibitors\u003csup\u003e16,17,18\u003c/sup\u003e. Taken together, these results establish HQY1027’s consistent anti-aggregation effectiveness across both reconstituted assays and native neuronal systems through leveraging chaperones.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHQY1027 mitigates pathological α-syn aggregation in a mouse PD model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the in vivo potential of HQY1027, we performed comprehensive pharmacokinetic (PK) profiling in mice. Given the limitations of forced oral gavage for chronic ND studies, we implemented a validated peanut butter-based voluntary feeding protocol\u003csup\u003e36,37\u003c/sup\u003e. Oral administration of HQY1027 at 25 mg/kg (mpk) achieved sustained plasma exposure over 24 h, reaching a peak concentration (C\u003csub\u003emax\u003c/sub\u003e) of 7.4 µM with a total exposure (AUC) of 41.4 µM·h and complete oral bioavailability (109%) (Extended Data Fig. 11a,b). Given that this exposure level substantially exceeds the required therapeutic concentration, a lower dose of 5 mpk was evaluated. At 5 mpk, HQY1027 provided a moderate plasma exposure (C\u003csub\u003emax\u003c/sub\u003e = 1.87 µM, AUC = 2.90 µM·h) and demonstrated exceptional blood-brain barrier penetration, reaching a peak brain concentration of 1.58 µM with a brain-to-plasma ratio of 2.5:1 (Extended Data Fig. 11c). This profile confirms its excellent CNS distribution and establishes 5 mpk as an efficacious dose.\u003c/p\u003e\n\u003cp\u003eWe next evaluated HQY1027’s efficacy in a PFF-induced mouse PD model. In this established model\u003csup\u003e38\u003c/sup\u003e, unilateral striatal injection of α-syn PFF induces bilateral α-syn pathology within three months, ultimately leading to motor dysfunction. HQY1027 administration (5 mpk/day via voluntary food consumption for 120 days) showed no adverse effects on body weight or signs of toxicity (Fig. 7a). Behavioral assessment revealed that PFF-injected mice developed significant hindlimb grip strength impairment compared to α-syn monomer-injected controls, consistent with synucleinopathy phenotypes. While performance in rotarod, pole climbing, and open field tests remained unaffected at this intermediate disease stage (Extended Data Fig. 12), HQY1027 treatment significantly rescued grip strength deficits (Fig. 7b). Immunohistochemical (IHC) analysis demonstrated that HQY1027 robustly reduced PFF-induced α-syn pathology in the substantia nigra and motor cortex bilaterally (Fig. 7c). Quantitative analysis confirmed \u0026gt;50% clearance of α-syn aggregates (Fig. 7d), demonstrating superior efficacy compared to current benchmark compounds targeting α-syn propagation. These results establish HQY1027 as an orally bioavailable, well-tolerated compound that effectively inhibits α-syn propagation and mitigates pathological progression in vivo.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNumerous studies have demonstrated that upregulating or activating specific chaperones can mitigate neurodegeneration\u003csup\u003e39,40,41\u003c/sup\u003e. Yet, pathogenic aggregates frequently evade chaperone surveillance and propagate throughout CNS during disease progression. While chemical strategies to induce proximity between fibrils and protein degradation machinery start to emerge, analogous approaches targeting chaperone recruitment remain unexplored. Our study reveals that PiB, the first amyloid PET tracer, unexpectedly functions as a chemical chaperone that suppresses α-syn aggregation by enhancing interactions between α-syn fibrils and Hsp40. Hsp40 inherently suppresses the liquid-to-solid phase transition during fibrillation and recruits Hsp70 and other proteostasis-maintaining components\u003csup\u003e42\u003c/sup\u003e. This provides a plausible mechanistic basis for the substantially greater activity of HQY1027, an optimized PiB analog, in cellular systems than in cell-free assays. More broadly, this work highlights the value of phenotype-driven screening for uncovering aggregation modulators with noncanonical mechanisms.\u003c/p\u003e\n\u003cp\u003eAlthough HQY1027 binds both α-syn fibrils and Hsp40, we could only determine a high-resolution structure for the HQY1027-α-syn fibril complex. The transient, heterogeneous nature of chaperone-client interactions remains a major barrier to structural characterization of Hsp40-substrate assemblies, and current cryo-EM methods are still limited in resolving dynamic complexes involving repetitive fibril layers at high resolution. Building upon established strategies including hydrophobic tagging for targeted protein destabilization\u003csup\u003e43\u003c/sup\u003e and molecular glue mechanisms\u003csup\u003e44\u003c/sup\u003e, we propose that HQY1027 modulates α-syn fibril surface architecture. This perturbation likely reduces both fibril surface order and charge density, thereby enhancing their susceptibility to chaperone recognition. Our structural observation that HQY1027 induces a distinctive inward conformation of Glu83 - a charged residue located in the N-pocket region of the fibril surface- directly supports this mechanism. Consistently, only HQY1027, but not its more hydrophilic or charged derivatives, significantly enhances the binding of a hydrophobic-sensitive dye to the fibril surface. Further corroboration comes from truncation studies, which demonstrate that HQY1027 enables Hsp40 to recognize the fibril core instead of the natively targeted C-terminal region. Notably, HQY1027 can bind to and remain active against other α-syn PFF types, suggesting that this mechanism is applicable to polymorphic fibrils. Nevertheless, this chemical chaperone approach may be generalizable to other protein aggregates through strategic screening or modification of fibril-binding compounds.\u003c/p\u003e\n\u003cp\u003eComparative analysis demonstrated HQY1027’s superior potency over clinical-stage aggregation inhibitors emrusolmin and minzasolmin in cellular assays. This enhanced activity was consistently observed in both neuronal cultures and animal models, where HQY1027 effectively reduced pathogenic aggregation of endogenous α-syn. Although motor deficits typically manifest only after extensive α-syn pathology in PD models, we detected significant impairment in paw grip strength at mid-stage pathology - a deficit substantially ameliorated by HQY1027 treatment. These findings underscore the therapeutic potential of our chemical chaperone strategy, which leverages endogenous protein quality control systems while causing minimal physiological disruption. It is worth noting that while the clinical relevance of widely used preformed fibril (PFF)-based synucleinopathy models is sometimes questioned due to the structural polymorphism of α-syn fibrils, a growing body of evidence indicates that synthetic fibrils can adopt conformations similar to endogenous fibrils - which are typically scarce and difficult to obtain - and recapitulate key aspects of clinical pathology in mice\u003csup\u003e45\u003c/sup\u003e. Our preliminary validation using MSA/JOS-like PFFs further supports the therapeutic potential of HQY1027, positioning it as a promising lead compound for ND drug development. Further investigation is warranted to evaluate the efficacy of HQY1027 in animal models associated with familial synucleinopathies and to optimize its preclinical profile to facilitate clinical translation.\u003c/p\u003e\n\u003cp\u003eIn summary, whereas conventional drug development based on biochemical screening targeting fibrillogenesis faces bottlenecks in clinical translation, this study - starting from a serendipitous observation in a cellular model - establishes a chemical chaperone strategy for inhibiting pathogenic α-syn aggregation and identifies HQY1027 as a promising therapeutic lead. Our findings demonstrate the potential of repurposing and optimizing existing fibril-binding compounds to target intractable protein aggregates in ND, and further reinforce the value of phenotype-driven discovery in revealing unexpected pharmacological mechanisms.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePreparation of recombinant \u0026alpha;-syn monomer/PFF and protein chaperones\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-Syn monomer and PFF (WT; mutants (G51DA53T, G51DA53TE57A/Q, G51DA53TE58Q/S/G/N); \u0026alpha;-syn\u003csup\u003e1-100\u003c/sup\u003e) were produced as described previously\u003csup\u003e16,31,46,47\u003c/sup\u003e. Briefly, human or mouse \u0026alpha;-syn was cloned into pET22b and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). Acetylated \u0026alpha;-syn (Ac-\u0026alpha;-syn) was produced by co-transformation with pACYCDuet-1-NatB. FLAG-\u0026alpha;-syn was generated by cloning the N-terminal FLAG tag into pET22b-\u0026alpha;-syn.\u003c/p\u003e\n\u003cp\u003eCells were lysed, boiled, nucleic acids removed, and pH adjusted, followed by overnight dialysis. Purification was performed using SP column or ion exchange followed by size exclusion chromatography (SEC).\u003c/p\u003e\n\u003cp\u003eWT, C-terminal truncated and JOS like \u0026alpha;-syn fibrils were assembled by incubating 400 \u0026mu;M monomer in SEC buffer (0.02% NaN\u003csub\u003e3\u003c/sub\u003e) at 37 \u0026deg;C with agitation (900 rpm, 5 days). The MSA-like \u0026alpha;-syn fibrils were assembled from six distinct \u0026alpha;-syn triple mutants mixed at an equimolar ratio, with the additional incorporation of the G51D/A53T double mutant. Fibrils were sonicated to generate seeds, and new fibrils were formed by adding seeds to fresh monomer. After removing the monomer, the fibrils were sonicated again to generate \u0026alpha;-syn PFF.\u003c/p\u003e\n\u003cp\u003eHdj1, Hsc70, and Hsp90\u0026alpha; was expressed and purified as previously reported\u003csup\u003e42,48,49\u003c/sup\u003e, using Ni-NTA and Superdex 75 or 200 columns. Purified proteins were concentrated, flash-frozen, and stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein labelling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor fluorescence labeling, purified Hdj1 was conjugated with Alexa Fluor 555 C2 maleimide (Invitrogen, A20346) following the manufacturer\u0026apos;s protocol. After conjugation, Hdj1-Alexa 555 conjugates were further purified by SEC. The final protein concentration was determined using the Bicinchoninic Acid (BCA) assay.\u003c/p\u003e\n\u003cp\u003eBiotin-labeled \u0026alpha;-syn fibrils were prepared by conjugating purified monomer with EZ-Link Sulfo-NHS-Biotin (Thermo Scientific, 21217) according to the manufacturer\u0026apos;s protocol. After conjugation, biotin-\u0026alpha;-syn conjugates were further purified using SEC. The final protein concentration was determined using the BCA assay. Once unlabeled \u0026alpha;-syn fibrils reached 2-3 \u0026micro;m in length, biotin-\u0026alpha;-syn monomer was added to continue fibril growth for several additional hours to obtain the desired biotin-labeled \u0026alpha;-syn fibrils.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro \u0026alpha;-syn fibril growth assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 50 \u0026micro;M \u0026alpha;-syn monomer, 1% (molar ratio) PFF with or without Hdj1 was incubated with indicated compounds in a buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 0.02% NaN3, and 50 \u0026micro;M ThT). Aggregation was monitored in black 384-well plates (Thermo Scientific, 142761) at 37 \u0026deg;C with orbital shaking 700 rpm. Fluorescence (ex: 440 nm, em: 485 nm) was recorded using a BMG FLUOstar Omega plate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to published method\u003csup\u003e16\u003c/sup\u003e, high-binding 96-well plates were coated with 100 nM Hdj1 overnight at 4 \u0026deg;C, then blocked with 5% non-fat milk for 2 h. After washing, compounds and 100 nM PFF were added and incubated for 1 h. Bound PFF were detected using HRP-conjugated anti-FLAG antibody and quantified by the color reaction between HRP and TMB substrate, measuring absorbance at 450 nm.For receptor binding ELISA, dLAG3 and vRAGE were expressed and purified,\u0026nbsp;purified, and pre-coated on plates\u0026nbsp;as\u0026nbsp;previously\u0026nbsp;described\u003csup\u003e31,50,51\u003c/sup\u003e,\u0026nbsp;followed by incubation with compounds and 100 nM FLAG-\u0026alpha;-syn PFF, and then detection as above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKinome profiling assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe KINOMEscan assays were done by DiscoverX (Fremont, CA, USA)\u003csup\u003e52\u003c/sup\u003e. The scores were reported as a percentage of DMSO control, with the lower score usually indicating higher probability of being a hit. Scores over 35% indicate no significant inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSPR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPR binding assays were conducted on a Biacore 8K system (GE Healthcare) in PBS buffer supplemented with 0.05% surfactant P20 (Cytiva, 28995084). \u0026alpha;-Syn PFF and chaperones (Hdj1, Hsc70, Hsp90\u0026alpha;) were immobilized on a CM5 sensor chip via amine coupling.\u003c/p\u003e\n\u003cp\u003eTo assess the interactions of compounds with target proteins, we injected serially diluted compounds (in assay buffer: PBS + 0.05% P20) over the sensor chip at 30 \u0026micro;L/min (120 s association), followed by dissociation (400 s). Besides, single-cycle kinetic assays of HQY1027 to \u0026alpha;-syn PFF were performed with Hdj1 included at specified concentrations alongside HQY1027 throughout the experiment. For Hdj1-\u0026alpha;-syn PFF binding assays, HQY1027 was included in all running buffers (association/dissociation phases). Between the cycles, the chip was regenerated with 3 M MgCl\u003csub\u003e2\u003c/sub\u003e (30 s). Equilibrium dissociation constants (K\u003csub\u003eD\u003c/sub\u003e) were derived using Biacore Insight Evaluation Software (GE Healthcare).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein thermostability shift assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thermal stability of Hdj1, Hsc70, and Hsp90\u0026alpha; (each at 4 \u0026micro;M) was assessed in complex with HQY1027 at a 1:40 protein:compound molar ratio. As previously published method\u003csup\u003e53\u003c/sup\u003e, after incubation, thermal denaturation was monitored in real-time using a QuantStudio\u0026trade; 7 Flex Real-Time PCR System (Applied Biosystems) with a temperature gradient from 25 \u0026deg;C to 95 \u0026deg;C. Derivative melting curves (dF/dT) were generated and analyzed using Protein Thermal Shift\u0026trade; Software (ThermoFisher) to determine thermal stability shifts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence polarization (FP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-Syn WT fibrils were diluted to a final concentration of 2.5 \u0026micro;M in reaction buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl). Test compounds were mixed at concentrations as indicated. The mixture was incubated at room temperature for 1 h in the dark. Then SYPRO\u0026trade; Orange Protein Gel Stain (ThermoFisher, S6650) was added at a 1:5000 (v/v) dilution and incubated at room temperature for 30 min in the dark. Fluorescence polarization was measured using an EnVision Multilabel Reader (PerkinElmer, 2104-0010) with excitation at 485 nm and emission at 535 nm. The polarizer was set to measure both parallel (I∥) and perpendicular (I\u0026perp;) emission relative to the excitation polarization. The fluorescence polarization was calculated using the standard formula: mP = 1000 * ((I∥\u0026nbsp;- G \u0026times; I\u0026perp;) / (I∥\u0026nbsp;+ G \u0026times; I\u0026perp;)), where G=I\u0026perp;blank/I∥blank (G-factor) was derived from the blank control group (no protein) to correct for instrumental polarization bias.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNS-EM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5 \u0026mu;L ThT assay products were deposited onto glow-discharged carbon-coated EM grids, stained with 2% uranyl acetate, and imaged using a Tecnai T12 microscope at 120 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample preparation and data collection were performed as previously described\u003csup\u003e21\u003c/sup\u003e. Briefly, 3 \u0026micro;M Ac-\u0026alpha;-syn fibrils were incubated with HQY1027 (300 \u0026micro;M, 2% DMSO) for 1 h at 25 \u0026deg;C. Ligand-bound samples were applied to glow-discharged carbon grids, blotted, plunge-frozen in liquid ethane, and imaged on a Krios G4 microscope at 300 kV with a BioContinuum K3 detector. Automated data collection was performed using EPU software with the same parameters as described previously\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage preprocessing and helical reconstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with previous descriptions\u003csup\u003e21\u003c/sup\u003e, in brief, image processing was performed using MotionCor2 (v1.2.1)\u003csup\u003e54\u003c/sup\u003e for frame alignment, dose-weighting, and binning to 0.83 \u0026Aring;/pixel. CTF estimation used CTFFIND4 (v4.1.8)\u003csup\u003e55\u003c/sup\u003e, followed by manual fibril picking in RELION (v3.1)\u003csup\u003e56\u003c/sup\u003e. Helical reconstruction was conducted using RELION,\u0026nbsp;through particle extraction, reference-free 2D classification, 3D classification (k=3), and 3D auto-refinement, yielding 3D density maps with optimal helical twists and rises. Finally, the maps were sharpened using RELION\u0026rsquo;s post-processing, and resolution was estimated via Fourier shell correlation and local resolution analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAtomic model building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-layer models of \u0026alpha;-syn fibrils were based on the structure of \u0026alpha;-syn fibril (PDB accession no. 6A6B) and manually adjusted in WinCoot (v.0.8.9.2)\u003csup\u003e57\u003c/sup\u003e, followed by refinement against the corresponding map by the real-space refinement program in PHENIX (v.1.15)\u003csup\u003e58\u003c/sup\u003e. Figures of atomic models were prepared using UCSF ChimeraX\u003csup\u003e59,60\u003c/sup\u003e or PyMOL\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptical tweezers-based single-molecule analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA confocal fluorescence microscopy-combined dual optical traps setup (LUMICKS C-trap, Netherlands) was used to detect the dynamic interaction between Hdj1 and \u0026alpha;-syn fibrils in a climate-controlled room at 23 \u0026deg;C\u003csup\u003e62\u003c/sup\u003e. \u0026alpha;-Syn fibrils were pre-incubated with 0, 10, 30 \u0026micro;M HQY1027 in the buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl) for 30 min. For the single-molecule assay, a single \u0026alpha;-syn fibril was suspended between two streptavidin-coated polystyrene beads (4.34-\u0026micro;m diameter, Spherotech)\u003csup\u003e33\u003c/sup\u003e. Then, the tethered fibril was swiftly transported to a channel containing 10 nM. Hdj1-Alex555 and a force of ~ 5 pN along the fibril was maintained by a high-speed feedback system during the fluorescence detection. A 532 nm excitation laser was utilized to obtain fluorescence signals of Hdj1. The confocal pixel size was set to 75 nm, with a pixel dwell time of 0.2 ms. The interframe wait time was 4s for rectangular scanning. The confocal images were obtained from LakeView software provided by LUMICKS and the fluorescence intensity of Hdj1-Alex555 on \u0026alpha;-syn fibrils (between two beads) was analyzed using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman embryonic Kidney (HEK293T) cells and SH-SY5Y cells were cultured in DMEM (ThermoFisher, C11995500BT) and DMEM/F12 (ThermoFisher, 11320033) respectively, plus 10% (v/v) FBS (ThermoFisher, 10100147) and 1% penicillin/streptomycin at 37 \u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator. VHL-KO 293T cells (generated in lab previously, via CRISPR/Cas9 using guide RNA 5\u0026prime;-GCCGTCGAAGTTGAGCCATA-3\u0026prime;) were cultured in DMEM containing 1 \u0026micro;g/mL puromycin.\u003c/p\u003e\n\u003cp\u003ePrimary cortical neurons were isolated from E15-E18 Sprague-Dawley rat embryos (SIPPR-BK) and plated at 150,000 cells/well on poly-L-lysine-coated coverslips in 24-well plates, following established protocols\u003csup\u003e16\u003c/sup\u003e. Neurons were maintained in Neurobasal medium supplemented with B-27 (ThermoFisher, 17504044) and 2 mM GlutaMax (ThermoFisher, 35050061).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e293T-based protein aggregation assays\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u0026alpha;-syn aggregation assays, 293T (WT or VHL-KO) cells were seeded in 12-well plates (1\u0026times;10⁶ cells/well) and transfected with 0.5 \u0026micro;g/mL pCAGGS-\u0026alpha;-syn\u003csup\u003eWT\u003c/sup\u003e-FLAG using PEI (1:3 ratio). After 24 h, cells were re-plated (2\u0026times;10⁵ cells/well) and treated with 10 nM \u0026alpha;-syn PFFs in PBS. Following compound treatment\u0026nbsp;according to specific sections, cells were lysed in NP-40 buffer (1% NP40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, cocktail protease inhibitors). Lysates were centrifuged (15,000 g, 20 min, 4 \u0026deg;C) to separate soluble and insoluble fractions. Protein concentrations were determined by BCA assay (ThermoFisher, 23225). Insoluble fractions were washed with PBS and solubilized overnight in 2\u0026times; loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 2% \u0026beta;-mercaptoethanol). Soluble/insoluble loading was resolved by SDS-PAGE and transferred to 0.2 \u0026micro;m nitrocellulose membranes (Cytiva, 10600001). Proteins were probed with the following primary antibodies: \u0026alpha;-syn-pS129 (Abcam, ab51253, 1:1000), FLAG-HRP (Sigma, A8592, 1:2000), Ubiquitin (Santa Cruz, sc-8017, 1:1000), LC3B (Sigma Aldrich, L7543, 1:1000) and \u0026beta;-actin (TransGen, HC201-01, 1:10,000) as loading control. HRP-conjugated secondary antibodies were used for detection by enhanced chemiluminescence.\u003c/p\u003e\n\u003cp\u003eFor tau aggregation assays, 293T cells were transfected with a lentiviral vector encoding full-length human tau (T40). At 6 h post-transfection, cells were treated with 0.1, 0.5 or 1 \u0026micro;M HQY1027. Cells then were transduced with either monomeric T40 or heparin-induced T40 PFF using CRISPR-Fectin transfection reagent (GeneCopoeia, EF015) at 24 h post-plasmid transfection. After another 48-h treatment with HQY1027 or PBS, cells were harvested in 1% Triton-X 100 lysis buffer (1% Triton-X 100, 30 mM Tris-HCl pH 7.5, 150 mM NaCl) supplemented with protease/phosphatase inhibitors (1:1000). Cells were sonicated using a probe-type sonicator, then kept on ice for 1 h. Insoluble material was pelleted via centrifugation (100,000 g, 30 min, 4 \u0026deg;C). Pellets were resuspended in 2X loading buffer, followed by immunoblotting. Primary antibodies were used: K9JA (DAKO, A0024, 1:1000); PHF-1 (gift from Dr. Peter Davies, 1:1000); GAPDH (Proteintech, 10494-1-AP, 1:1000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotocrosslinking, biotin-click, and pulldown\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing \u0026alpha;-syn PFF induction (10 nM, 48 h), transfected 293T cells in 6-cm dishes were treated with either: (i) DMSO control, (ii) HQY1027 (20 \u0026micro;M), (iii) the inactive analog \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003e(20 \u0026micro;M) for 12 h or not, followed by 0.2 \u0026micro;M probe \u003cstrong\u003e6\u003c/strong\u003e or \u003cstrong\u003e7\u003c/strong\u003e for 12 h. Cells were washed with PBS and incubated with fresh solutions of the same probes/DMSO in PBS. UV crosslinking was performed on ice by being exposed to UV light (365 nm) for 30 min (Analytikjena, CL-3000). Cells were then lysed in 1% SDS lysis buffer and protein concentrations determined by BCA assay.\u003c/p\u003e\n\u003cp\u003eFor biotin-click chemistry, lysates were incubated with 100 \u0026micro;M Biotin-N\u003csub\u003e3\u003c/sub\u003e, 100 \u0026micro;M TBTA, 1 mM CuSO\u003csub\u003e4\u003c/sub\u003e/THPTA, and 1 mM TCEP for 30 min at 25 \u0026deg;C with rotation. An additional 1 mM TCEP was added for 30 min to complete the reaction. Proteins were precipitated using methanol:chloroform:water (4:1:3, v/v), centrifuged (15,000 g, 15 min, 4 \u0026deg;C), and washed three times with ice-cold methanol. The pellet was air-dried (4 \u0026deg;C, 2 h) and resuspended in 1% SDS lysis buffer.For streptavidin pulldown, lysates were processed according to previously reported approaches\u003csup\u003e63\u003c/sup\u003e. Heat shock proteins were detected using the following antibodies: Hsp70 (Enzo, ADI-SPA-810, 1:1000) and Hsp40 (ThermoFisher, MA5-25362, 1:1000). Immunoblotting was performed as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximity labeling based on TurboID\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e293T WT cells were seeded in 12-well plates (1\u0026times;10⁶ cells/well) and transfected with 0.5 \u0026micro;g/mL pCAGGS-\u0026alpha;-syn\u003csup\u003eWT\u003c/sup\u003e-FLAG-TurboID using PEI (1:3 ratio). After 24 h, cells were re-plated (2\u0026times;10⁵ cells/well) and treated with 100 nM \u0026alpha;-syn PFFs in PBS for another 72h. Before sample collection, cells were treated with: (i) DMSO control, (ii) HQY1027 (0.5, 1, 2 \u0026micro;M), (iii) the inactive analog \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003e(2 \u0026micro;M) for 2 h, followed by 50 \u0026micro;M D-Biotin in DMSO labeling for another 1 h. After pre-cool PBS washing, cells were lysed in 1% SDS lysis buffer and ultrasonication for 30s. Streptavidin beads pulldown and Western blot was performed as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomic study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing biotin pulldown, beads were processed according to previously published method\u003csup\u003e64\u003c/sup\u003e. Generally, protein was denatured in 8 M urea/100 mM Tris pH 8.5, reduced with 5 mM TCEP and alkylated with 10 mM iodoacetamide. Trypsin (Promega, V511A; 1:100) was used for overnight digestion (37 \u0026deg;C, shaking in the dark). Digestion was quenched with 90% formic acid, and the supernatant was collected after centrifugation (15,000 g, 15 min). Peptides were desalted using C18 columns. Eluates were pooled, dried by vacuum centrifugation (30 \u0026deg;C, 2 h), and stored at -20 \u0026deg;C.Samples were then analyzed using an EASY-nLC 1000 HPLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific). Mass spectra were acquired in a data-dependent mode with one full scan (m/z: 350-1500; resolution: 15,000; AGC target value: 3,000,000 and maximal injection time: 20 ms), followed by MS2 scan (32% normalized collision energy; AGC target value: 100,000; maximal injection time: Dynamic). The MS/MS raw spectra were processed using MaxQuant Software (v.1.6.0.1). The average protein intensity of each treatment group was counted with GraphPad, and differential binding of probe \u003cstrong\u003e6\u003c/strong\u003e group vs DMSO or probe \u003cstrong\u003e7\u003c/strong\u003e groups was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026alpha;-Syn\u003csup\u003eWT\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e-mEGFP\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;overexpress\u003c/strong\u003e\u003cstrong\u003eing\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;SH-SY5Y cell line establishment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pLVX-EF1\u0026alpha;-IRES-\u0026alpha;-syn\u003csup\u003eWT\u003c/sup\u003e-mEGFP plasmid was generated by cloning human \u0026alpha;-synuclein and the monomeric EGFP (A206K mutant) sequences into the pLVX-EF1\u0026alpha;-IRES backbone using EcoRI and BamHI restriction sites. Then plasmids were introduced into 293T cells via packaging plasmids and PEI. Medium containing Lentivirus was collected and filtered by 0.22 \u0026micro;m PVDF filter (Merck, Millex-HV), before mixed with 8 \u0026micro;g/mL polybrene and added into SH-SY5Y cells. Infected SH-SY5Y cells were selected with 2 \u0026micro;g/mL puromycin and single-cell clone formation. Stable expression of \u0026alpha;-syn\u003csup\u003eWT\u003c/sup\u003e-mEGFP was confirmed under fluorescent microscopy and Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColocalization studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate \u0026alpha;-syn\u0026ndash;Hsp40 colocalization, SH-SY5Y cells stably overexpressing \u0026alpha;-syn\u003csup\u003eWT\u003c/sup\u003e-mEGFP were seeded (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) in 12-well plates pre-coated with coverslips and stimulated with 100 nM \u0026alpha;-syn PFF for 48 h. Before fixation, cells were treated with 1 \u0026micro;M HQY1027 (0.5-6 h) or 1 \u0026micro;M the inactive analog \u003cstrong\u003e5\u003c/strong\u003e (6 h). Fixed with 4% PFA in PBS, cells were immunostained overnight with Hsp40 antibody (ThermoFisher, MA5-25362, 1:1000), followed by Alexa Fluor 647-conjugated secondary antibody (ThermoFisher, A32728). Confocal images were acquired using a Leica SP8 microscope (100\u0026times;, 1.4 NA oil objective) under consistent laser settings. Pearson colocalization coefficients between \u0026alpha;-syn aggregates and Hsp40 were quantified using ImageJ\u0026rsquo;s JACoP plugin\u003csup\u003e65\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuron propagation\u0026nbsp;assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cortical neurons cultured for 7 days in vitro (DIV7), were treated with:\u0026nbsp;(i) PBS control, (ii) 200 nM \u0026alpha;-syn monomer, (iii) 200 nM \u0026alpha;-syn PFF, or (iv) PFF pre-incubated with test compounds (n = 3 biological replicates). Fixed neurons were immunostained using: \u0026alpha;-syn-pS129 (Abcam, ab51253, 1:1000) and MAP2 (Abcam, ab5392, 1:2000) primary antibodies, followed by Alexa Fluor 488 anti-chicken (ThermoFisher, A11039) and 568 anti-rabbit (ThermoFisher, A11036) secondaries. Fluorescent images were acquired using a Leica SP8 confocal microscope, and the fluorescence intensity of \u0026alpha;-syn-pS129 and MAP2 signals were quantified using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetic characterization\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof HQY1027\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assays for microsomal stability were performed according to the literature\u003csup\u003e66\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor mouse PK properties, HQY1027 were dissolved in DMA (10% v/v) and Solutol HS 15 (10% w/v) in normal saline or peanut butter, then injected into male ICR mice via tail vein (IV, 1\u0026nbsp;mpk) or\u0026nbsp;oral administration\u0026nbsp;(PO,\u0026nbsp;25 or 5\u0026nbsp;mpk). Blood samples (30-50 \u0026micro;L) were collected via the retro-orbital plexus at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post-dosing into tubes\u0026nbsp;with EDTA2K. Plasma samples were analyzed by\u0026nbsp;an Agilent 1290 Infinity II UHPLC system coupled to a SCIEX Triple Quad\u0026trade; 3500 mass spectrometer. Analyte-to-internal standard peak area ratios were calculated against an 8-point calibration curve. Pharmacokinetic parameters were derived via non-compartmental analysis (Phoenix WinNonlin, v8.1.0.3530).\u003c/p\u003e\n\u003cp\u003eFor brain and plasma distribution,\u0026nbsp;mice received HQY1027 (PO, 5\u0026nbsp;mpk) and were euthanized 15 min post-administration. Prior to brain collection, animals were transcardially perfused with 0.9% saline. Plasma and whole brains were harvested simultaneously. Brain samples were homogenized PBS and acetonitrile containing internal standard (dexamethasone, 100 ng/mL) using a TissueLyser (30 Hz, 10 min). Following centrifugation (14,000 g, 10 min, 4 \u0026deg;C), supernatants were analyzed by LC-MS/MS (Agilent 1290/SCIEX 3500 system). Brain-to-plasma concentration ratios were calculated from quantified drug levels in each matrix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026alpha;-Syn PFF\u003c/strong\u003e\u003cstrong\u003e-induced Parkinson\u0026rsquo;s Diseases mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Animal Care Committee of the Interdisciplinary Research Center on Biology and Chemistry, Chinese Academy of Sciences. Male C57BL/6J mice (6 weeks old; Shanghai Lingchang Biotechnology) were individually housed and weighed every 72 h.\u003c/p\u003e\n\u003cp\u003eBefore stereotaxic surgery, mice were acclimated to consume peanut butter (8 g/kg body weight) at a fixed daily time point for 3 days. According to published method\u003csup\u003e67\u003c/sup\u003e, Mouse \u0026alpha;-syn PFF or monomers (5 \u0026micro;g in sterile PBS) were unilaterally injected into the left dorsal striatum (coordinates: +0.2 mm AP, +2.0 mm ML relative to Bregma; \u0026minus;2.6 mm DV) using a Hamilton syringe (10 \u0026micro;L) at 0.4 \u0026micro;L/min. After 3 days of recovery, mice received daily oral HQY1027 (5 mpk), or vehicle control mixed into peanut butter at the trained time point. Treatment ceased 4 months post-injection, and mice were monitored until study endpoint.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavior\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo minimize potential stress or physical strain on the animals, the grip strength test was conducted last. All behavioral tests were performed with consistent cohort sizes (n = 10 mice per group) to ensure statistical reliability across assessments.\u003c/p\u003e\n\u003cp\u003eFor the grip strength test, it was measured using a metal grid meter (KEW BASIS, KW-ZL-1). Mice were placed on the grid with either (i) all four limbs or (ii) forepaws only. The tail was gently pulled backward three times, and the maximal holding force (grams) was recorded when the mouse resisted release.\u003c/p\u003e\n\u003cp\u003eFor the rotarod test, mice were evaluated for motor coordination and balance using an accelerating rotarod (Ugo Basile). Following one day of habituation to the stationary rod, mice underwent three trials per day with the rod accelerating from 4 to 40 rpm over 5 min. The inter-trial intervals were \u0026ge;30 min. The latency to fall (in seconds) and the corresponding speed (in rpm) at the time of falling were recorded.\u003c/p\u003e\n\u003cp\u003eFor the pole test, following a 3-day acclimation period, mice were placed head-up at the top of a vertical pole (50 cm height, 1 cm diameter). The time required for each mouse to reorient downward and descend to the base was recorded (in seconds). Three trials were performed per session with \u0026ge;30 min inter-trial intervals.\u003c/p\u003e\n\u003cp\u003eFor the open field test, the mice were placed in the open field arena under standardized illumination and allowed to explore for 10 min. Activity levels were monitored by an automated video tracking system (Noldus, EthoVision XT). Chambers were cleaned with 70% ethanol before and after testing. The total distance moved in 10 min (in cm) were used to analysis the motor activity of mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix months post-treatment, mice were transcranially perfused with PBS. Brains were post-fixed in 4% PFA in PBS overnight at 4 \u0026deg;C, paraffin-embedded, and sectioned coronally (6 \u0026micro;m). As reported previously\u003csup\u003e67\u003c/sup\u003e, after deparaffinization and rehydration, antigen retrieval was performed by boiling in 100 mM citrate buffer (pH 6.0). Endogenous peroxidases were quenched with 5% H₂O₂/methanol, followed by blocking in 2% FBS/0.1 M Tris (pH 7.6). Sections were incubated overnight at 4 \u0026deg;C with \u0026alpha;-syn-pS129 antibody (Abcam ab51253; 1:10,000 in blocking buffer).\u0026nbsp;Next day, sections were processed according to standard DAB staining protocol (Vector), and counterstained with hematoxylin, dehydrated, and coverslipped with Cytoseal. Slides were scanned into digital format on a PANNORAMIC 250 Flash III DX (3D HISTECH). Digitized slides were then used for quantitative pathology. Pathological \u0026alpha;-syn-pS129 area was quantified by ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of compounds HQY1027\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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width=\"679\" height=\"123\"\u003e\u003cstrong\u003e\n \u003cv:shapetype id=\"_x0000_t75\" coordsize=\"21600,21600\" o:spt=\"75\" o:preferrelative=\"t\" path=\"m@4@5l@4@11@9@11@9@5xe\" filled=\"f\" stroked=\"f\"\u003e\n \u003cv:stroke joinstyle=\"miter\"\u003e\n \u003cv:formulas\u003e\n \u003cv:f eqn=\"if lineDrawn pixelLineWidth 0\"\u003e\n \u003cv:f eqn=\"sum @0 1 0\"\u003e\n \u003cv:f eqn=\"sum 0 0 @1\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @2 1 2\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @3 21600 pixelWidth\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @3 21600 pixelHeight\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @0 0 1\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @6 1 2\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @7 21600 pixelWidth\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @8 21600 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @7 21600 pixelHeight\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @10 21600 0\"\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\u003c/v:f\u003e\u0026nbsp;\n \u003c/v:f\u003e\u0026nbsp;\n \u003c/v:f\u003e\u0026nbsp;\n \u003c/v:formulas\u003e\n \u003cv:path o:extrusionok=\"f\" gradientshapeok=\"t\" o:connecttype=\"rect\"\u003e\u0026nbsp;\u003c/v:path\u003e\u0026nbsp;\n \u003c/v:stroke\u003e\u0026nbsp;\n \u003c/v:shapetype\u003e\n \u003cv:shape id=\"_x0000_i1025\" type=\"#_x0000_t75\" o:ole=\"\"\u003e\u0026nbsp;\u003cv:imagedata src=\"file:///C%3A/Users/pgs9865/AppData/Local/Temp/msohtmlclip1/01/clip_image001.emz\" o:title=\"\"\u003e\u0026nbsp;\u003c/v:imagedata\u003e\u0026nbsp;\u003c/v:shape\u003e\u0026nbsp;\n \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReagent condition: a) TEA, DDQ, MeOH, DCM, room temperature (R.T.), 1 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(6-methoxybenzo[\u003cem\u003ed\u003c/em\u003e]oxazol-2-yl)-\u003cem\u003eN\u003c/em\u003e-methylaniline\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eHQY1027\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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width=\"268\" height=\"103\"\u003e\u003c/p\u003e\n\u003cp\u003eA solution of 4-dimethylaminobenzaldehyde (298\u0026nbsp;mg, 2.00 mmol)\u0026nbsp;and\u0026nbsp;2-amino-5-methoxyphenol (139 mg, 1.00 mmol)\u0026nbsp;in anhydrous MeOH (5 mL) were added TEA\u0026nbsp;(0.348 mL, 2.5 mmol). After stirring for 30 min\u0026nbsp;at\u0026nbsp;room\u0026nbsp;temperature, a solution of\u0026nbsp;DDQ (227 mg, 1.00 mmol) in DCM (5 mL)\u0026nbsp;was\u0026nbsp;added dropwise over 5 min. The resulting mixture was stirred\u0026nbsp;at\u0026nbsp;room\u0026nbsp;temperature for\u0026nbsp;an additional 30 min until completion.\u0026nbsp;Then it was diluted by water (20 mL) and extracted with EA (3 \u0026times; 15 mL). The combined organic layers were dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated under reduced pressure.\u0026nbsp;Then the\u0026nbsp;residue was purified by C18 flash column chromatography\u0026nbsp;using a gradient eluent\u0026nbsp;(H\u003csub\u003e2\u003c/sub\u003eO/MeCN\u0026nbsp;= 100:0 to\u0026nbsp;20:80, H\u003csub\u003e2\u003c/sub\u003eO contains 0.05% HCl). \u003cstrong\u003eHQY1027\u003c/strong\u003e was obtained by lyophilization as yellow solid (166 mg, 0.617 mmol) with a yield of 61.7%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta; 7.94 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 2H), 7.56 (d, \u003cem\u003eJ\u003c/em\u003e = 8.6 Hz, 1H), 7.34 (s, 1H), 6.93 (d, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 1H), 6.85 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 3.82 (s, 3H), 3.03 (s, 6H). MS-ESI: m/z calculated for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Exact Mass: 268.12, found 269.1 [M + H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were analyzed using GraphPad Prism (v10.0.2). Group comparisons were performed by\u003cem\u003e\u0026nbsp;t\u003c/em\u003e-test or one/two-way ANOVA with Tukey or Dunnett\u0026apos;s post hoc test as indicated. Data is presented as mean \u0026plusmn; SD unless otherwise noted. Significance thresholds were defined as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001; ns indicates not significant (p \u0026ge; 0.05).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe thank Prof. Junying Yuan (IRCBC, CAS) for her generous help on this work, Chinese Academy of Sciences for the continued support, Dr. Yifan Ge for her help in imaging experiments, and National Facility for Protein Science in Shanghai, Ms. Ting Li (IRCBC, CAS), and Dr. Jiang Bian (SJTU) for their help in animal studies. This work was supported by National Natural Science Foundation of China (22425704 to C.L., 82188101 to C.L. and L.T.), Shanghai Basic Research Pioneer Project (L.T., C.L., Y.Z. and Z.H.), Shanghai Municipal Science and Technology Major Project (L.T., C.L., Y.Z. and Z.H.), Shanghai Leading Talent Program of Eastern Talent Plan (Y.L.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB1060000 to L.T., and C.L.), and the Shanghai Key Laboratory of Aging Studies (19DZ2260400 to C.L.). Dr. Cong Liu is SANS Exploration Scholar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e L.T. and Y.L.\u0026nbsp;conceived the project. L.T., Q.H. and K.S. designed and synthesized the compounds. C.L., Y.L., Y.C., S.Z. and Y.T. designed and performed the \u0026alpha;-syn fibril propagation, binding, and colocalization assays. Y.C., C.Q., Q.H. and H.X. conducted the animal studies. S.Z., K.L. and D.L. performed cryo-EM sample preparation, data collection and visualization. Y.C., Y-X.L. and Y.Z. conducted the proteomic studies. Y.C., S.W., X.L., and B.S. designed and performed the optical tweezers analysis. W.Z. and Z.H. designed and performed the tau-aggregation cellular assays. L.T., C.L. and Y.L. supervised the project. L.T., C.L., Y.C., Q.H. and S.Z. wrote the manuscript; All the authors contributed to discussion of the manuscript and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e L.T., Q.H., Y.C., C.L., and Y.L. are inventors on patent applications relating to this work, owned by SIOC. All other authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession no. EMD-64224 for HQY1027-bound \u0026alpha;-syn fibrils, The corresponding atomic models have been deposited in the PDB under accession no. 9UJQ for HQY1027-bound \u0026alpha;-syn fibrils.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoss, C. A. \u0026amp; Poirier, M. A. Protein aggregation and neurodegenerative disease. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e10 Suppl\u003c/strong\u003e, S10-17 (2004). https://doi.org/10.1038/nm1066\u003c/li\u003e\n\u003cli\u003eSoto, C. \u0026amp; Pritzkow, S. Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. \u003cem\u003eNat Neurosci\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1332-1340 (2018). https://doi.org/10.1038/s41593-018-0235-9\u003c/li\u003e\n\u003cli\u003evan Dyck, C. 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However, most reported anti-fibrillogenesis agents show limited efficacy in cells or in vivo, and their mechanisms are often poorly defined. Here, we report the unexpected finding that Pittsburgh compound B (PiB), the first clinically used amyloid tracer, selectively suppresses α-syn aggregation. Using an optimized PiB analog, HQY1027, we demonstrate that these compounds function as chemical chaperones. By directly binding polymorphic α-syn fibrils, they remodel the fibril surface and enhance Hsp40 recognition of fibril cores, thereby suppressing fibril formation both in vitro and in cells. HQY1027 is highly effective in neuronal models and significantly reduced α-syn aggregate propagation and motor deficits in a PD mouse model. Our findings establish a chemical chaperone strategy for targeting pathogenic α-syn and identify HQY1027 as a promising therapeutic lead for synucleinopathies. These results further highlight the potential of phenotype-based discovery and optimization of fibril-binding ligands to target intractable protein aggregates in neurodegenerative disorders.","manuscriptTitle":"Discovery of amyloid binders as chemical chaperone to block pathological α-synuclein aggregation in synucleinopathies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 08:32:29","doi":"10.21203/rs.3.rs-6562222/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"def180bb-ceba-4270-b814-c3a6c73c1e28","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65330691,"name":"Biological sciences/Drug discovery"},{"id":65330692,"name":"Physical sciences/Chemistry/Chemical biology"}],"tags":[],"updatedAt":"2026-04-04T14:32:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 08:32:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6562222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6562222","identity":"rs-6562222","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}