Broad-spectrum suppression and disassembly of α-synuclein variant aggregates mediated by a ruthenium metallodrug via conserved metal coordination

Abstract

Inhibiting α-synuclein (α-Syn) aggregation is a promising therapeutic strategy for Parkinson's disease (PD); however, its structural disorder and the heterogeneity of aggregates across familial variants pose significant challenges. Here, we demonstrate that NAMI-A, a ruthenium-based compound, effectively inhibits aggregation of wild-type and pathogenic familial α-Syn variants. Biophysical and biochemical analyses revealed that NAMI-A binds α-Syn via coordination to conserved regions, preventing the structural transition to β-sheets and inhibiting aggregation. Notably, NAMI-A dismantles pre-formed aggregates of diverse structures from various pathogenic variants. Cellular assays confirmed that NAMI-A significantly reduces α-Syn-induced cytotoxicity in neuronal cells by attenuating ROS generation. This work establishes that metallo-agents can act as broad-spectrum therapeutic agents to overcome mutation-dependent limitations for PD treatment.

---

Introduction

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, affecting over 10 million people worldwide.^1,2 PD is pathologically characterized by the accumulation of misfolded α-synuclein (α-Syn) protein and the formation of toxic oligomers and fibrillar aggregates.³ These α-Syn aggregates form Lewy bodies that drive neuronal dysfunction and progressive loss of dopaminergic neurons in the substantia nigra.⁴ While current therapies temporarily alleviate symptoms, no therapeutic agents exist that inhibit α-Syn aggregation – the pathogenic driver of PD.

The intrinsically disordered nature of α-Syn is the key challenge for developing inhibitors that target α-Syn via structure-based drug design.^5,6 In addition to this challenge is the heterogeneity of α-Syn pathology. Familial mutations of α-Syn (e.g., V15A, H50Q, E46K, and T72M) typically accelerate aggregation and increase neurotoxicity.^7–11 These variants could also evade inhibitors optimized for wild-type (WT) α-Syn.¹² Consequently, the development of broad-spectrum aggregation inhibitors effective against both WT and pathogenic variants is essential.

We have recently discovered that metallo-agents can be promising candidates for targeting α-Syn.¹³ Unlike pure organic small molecules, which bind to specific binding pockets in target proteins via structure recognition, metallo-agents bind to proteins primarily through coordination to certain amino acid residues. This mechanism makes metallo-agents particularly suitable for disordered targets, such as α-Syn. NAMI-A, a ruthenium-based agent, was found to effectively inhibit α-Syn aggregation, thereby abolishing the α-Syn-mediated neuronal cytotoxicity and mitigating neurodegeneration and motor impairments in PD model rats.¹³ Given the special target-recognition mechanism of metallo-agents, we hypothesized that NAMI-A could act as a broad-spectrum α-Syn inhibitor.

In this work, we evaluated the inhibitory effect of NAMI-A on α-Syn aggregation using five disease-related familial variants. The interaction of NAMI-A with α-Syn variants was studied using biophysical, biochemical, and cellular assays. In addition to inhibiting protein aggregation, NAMI-A can also disassemble the fibrillar aggregates of the variants, even though they are in substantially different fibril structures.

Results and discussion

NAMI-A inhibits the aggregation of diverse familial α-Syn variants

WT α-Syn and familial variants (E46K, T72M, G51D, A53E and H50Q) were expressed and purified using our previous method.¹³ ESI-MS and gel electrophoresis results confirmed the high purity of the obtained proteins (Fig. S1).

Aggregation of WT α-Syn and variants was monitored via the Thioflavin T (ThT) assay. The increased fluorescence clearly showed the time-dependent fibrillization of these proteins at different rates (Fig. 1A). The ThT fluorescence was not affected by NAMI-A (Fig. S2), confirming the protein fibrillization-induced fluorescence increase. The sigmoidal profile indicated a typical nucleation-dependent polymerization process.¹⁴ Pathogenic variants exhibited different aggregation rates relative to wild-type α-Syn; H50Q showed the most rapid fibrillization and G51D showed the slowest kinetics, aligning with the literature results.^15–18

Fig. 1 NAMI-A inhibits the aggregation of familial α-Syn variants. (A) Aggregation profiles obtained using the ThT fluorescence assay. α-Syn or its variants (70 μM) were incubated at 37 °C in the presence (blue lines) or absence (black lines) of NAMI-A (350 μM). (B) ThT fluorescence after 70 h of incubation. Data are presented as the mean ± s.d. from 3 independent experiments. Statistical significance was determined by Student's t-tests (*P < 0.05, **P < 0.01, and ***P < 0.001), with the untreated samples as references. (C) Gel electrophoresis analysis of proteins retained in the supernatant after 36 h of incubation at 37 °C. Data are presented as the mean ± s.d. from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test (***P < 0.001; n.s., not significant), relative to the unincubated samples as references. Raw gel electrophoresis images are available in the SI. (D) Representative TEM images of familial α-Syn variants. Samples were prepared by incubation of α-Syn variants (70 μM) at 37 °C for 36 h in the absence (top) or presence (bottom) of NAMI-A (350 μM). Scale bar: 2 μm.

NAMI-A potently suppressed aggregation across all α-Syn variants, reducing the final ThT fluorescence to <15% of untreated controls (Fig. 1A and B). Although H50Q exhibited reduced susceptibility to inhibition, NAMI-A significantly prolonged its lag phase by >200% (Fig. 1A).

Sedimentation assays independently validated the inhibitory effect of NAMI-A. Gel electrophoresis analysis showed that the amount of supernatant protein clearly reduced after 36 h of incubation, confirming the aggregation of all α-Syn variants, while NAMI-A effectively inhibited the protein aggregation (Fig. 1C). Consistent with these findings, TEM imaging showed that NAMI-A treatment clearly prevented the formation of all these fibrils (Fig. 1D). Collectively, these results demonstrate that NAMI-A effectively prevents aggregation of both WT and its disease-associated familial variants.

NAMI-A inhibits the β-sheet transition of familial α-Syn variants

Given the broad-spectrum inhibitory effects of NAMI-A, we investigated the mechanism of NAMI-A's action. NAMI-A quenched the intrinsic fluorescence of α-Syn variants at 310 nm (Fig. 2A), which is consistent with the action of WT α-Syn,¹³ indicating the direct binding of NAMI-A to these variants. The concentration-dependent fluorescence quench suggested comparable binding affinity across α-Syn variants (Fig. 2B and S3). The kinetic profiles further confirmed their similar binding activity (Fig. S4).

Fig. 2 Effect of NAMI-A on the secondary structures of WT and α-Syn variants. (A) The normalized fluorescence spectra of WT and variants (G51D and H50Q) upon incubation with NAMI-A. The proteins (20 μM) were incubated with NAMI-A (0–1000 μM) at 37 °C for 2 h in 20 mM phosphate buffer (pH 7.4). The curves show the mean fluorescence intensities ± s.d. from 3 independent samples. (B) Stern–Volmer plot of fluorescence quenching. The log[(F₀ − F)/F] was plotted against log[NAMI-A] (0–200 μM) for linear fitting. Data are presented as the mean ± s.d. from 3 independent experiments. (C) Far-UV CD spectra of α-Syn variants (20 μM) with/without NAMI-A (100 μM) at 37 °C.

Since WT α-Syn aggregation involves a structural transition from disordered to β-sheets,¹⁹ we monitored the conformational alteration of α-Syn familial variants using far-UV CD spectroscopy. All untreated α-Syn variants converted to a β-sheet-rich conformation after 36 h of incubation, as evidenced by the decreased negative ellipticity at 200 nm and the increased negative ellipticity at 220 nm (Fig. 2C). NAMI-A treatment blocked this structural transition in all variants. Collectively, these results demonstrate that NAMI-A directly binds to α-Syn variants with similar affinities, preventing their structural transition from disordered states to a β-sheet-rich conformation.

NAMI-A forms conserved adducts with familial α-Syn variants

The adducts of NAMI-A binding to WT α-Syn and two variants with distinct aggregation rates (fast aggregation H50Q and slow aggregation G51D) were analyzed using ESI-MS. Compared to the spectrum of the untreated protein, new peaks with increased m/z appeared in the spectra of NAMI-A adducts (Fig. 3A). Mass assignment identified similar adducts across variants, including [α-Syn + Ru], [α-Syn + Ru(lm)] and [α-Syn + Ru(lm)(H₂O)] (Fig. 3B). Isotopic distribution of these peaks matched their theoretical isotopic patterns (Fig. 3C and Table S1), confirming the assignment. These results indicate that mutations of α-Syn do not disrupt NAMI-A binding.

Fig. 3 ESI-MS analysis of the NAMI-A adducts of α-Syn variants. (A) ESI-MS spectra of WT α-Syn, G51D and H50Q variants (100 μM) before (top) and after (bottom) incubation with 500 μM NAMI-A. The selected m/z region shows +8 charged peaks. The reaction was performed in 20 mM phosphate buffer for 2 h at 37 °C and the unreacted NAMI-A was removed by ultrafiltration. (B) Assignment of ESI-MS peaks in (A). Detailed information is shown in Table S1. (C) Expanded view of the +8 charged peak-selected adducts. The theoretical isotopic patterns are shown for comparison.

Conserved NAMI-A binding sites across α-Syn variants

¹H–¹⁵N HSQC NMR was applied to analyze the NAMI-A binding to H50Q (fast aggregation) and G51D (slow aggregation) variants. The appearance of peaks within the ¹H chemical shift range of 7.2–8.4 ppm confirmed intrinsically disordered states (Fig. 4A, blue).²⁰ After NAMI-A incubation, substantial peak attenuations were observed on specific residues (M5, L8, H50, T72, D119, D121, N122, S129, E130, and Q134) (Fig. 4A, red), indicating direct coordination or proximal binding of these sites. Quantitative analysis of the peak intensity revealed that NAMI-A binding significantly affected four specific regions: the N-terminal, the preNAC segment (E46–A53), the hydrophobic NACore (A69–G73) and the C-terminal region (E110–E135) in both WT and variants (Fig. 4B). These findings confirm that the NAMI-A binding sites remain unaffected by pathogenic mutations.

Fig. 4 ¹H–¹⁵N HSQC NMR analysis of the reaction of WT α-Syn and variants with NAMI-A. (A) Overlay of the ¹H–¹⁵N HSQC NMR spectra of WT, G51D, and H50Q α-Syn (0.2 mM) recorded before (blue) and after (red) incubation with NAMI-A (1.0 mM) for 2 h at 37 °C in 20 mM phosphate buffer (pH 7.4). (B) Relative intensity changes of the peaks in A. The colors denote WT (blue), G51D (orange), and H50Q (purple) α-Syn.

NAMI-A attenuates the cytotoxicity of familial α-Syn variants

We next assessed the ability of NAMI-A to promote the degradation of pre-formed aggregates of α-Syn variants. TEM imaging showed near-complete disassembly of fibrils, while ThT assays confirmed a reduction in oligomers (Fig. S5). These results indicated that NAMI-A effectively disrupts the aggregates of α-Syn variants.

The cytotoxicity of aggregates of α-Syn variants was evaluated in SH-SY5Y cells. MTT assays showed that all aggregates inhibited cell proliferation (Fig. 5A). NAMI-A treatment significantly mitigated this toxicity (Fig. 5B). This protective effect was further confirmed by a live/dead cell staining assay; the result clearly showed that the NAMI-A treatment reduced the proportion of dead cells induced by the aggregates of α-Syn variants (Fig. S6). Moreover, we measured ROS levels in the cells treated with the aggregates of α-Syn variants, as α-Syn aggregation would trigger oxidative stress and promote Lewy body formation and pathology.^21–25 Fluorescence imaging clearly showed that the aggregates of WT α-Syn or variants significantly increased the ROS levels in SH-SY5Y cells (Fig. 5C and D). Treatment of NAMI-A nearly completely suppressed the ROS elevation caused by all α-Syn variants (Fig. 5C and D), confirming that ROS reduction contributes to the cytoprotective effects of NAMI-A.

Fig. 5 NAMI-A attenuates α-Syn-induced cytotoxicity in SH-SY5Y cells. (A) Cytotoxicity of WT, G51D, and H50Q α-Syn oligomers. Viability was assessed by the MTT assay on SH-SY5Y cells after treatment with the indicated oligomers (0.1, 1, 2, and 5 μM). (B) NAMI-A rescues cell viability suppressed by oligomers. SH-SY5Y cells were incubated with the indicated oligomers (1 μM) pre-incubated with or without NAMI-A (5 μM). (C) Representative fluorescence microscopy images of intracellular ROS levels in SH-SY5Y cells. The ROS was stained with a DCF probe (green) and the nuclei were stained with DAPI (blue). The cells were treated for 12 h with α-Syn oligomers (1 μM) pre-incubated with NAMI-A (5 μM). PBS-treated cells served as controls. (D) ROS levels measured in the cells in (C). Data are presented as fold changes relative to the control group. Data in (A), (B), and (D) are presented as the mean ± s.d. of n = 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. Symbols denote *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., not significant.

The pathological aggregation of α-Syn is a key factor in the development of PD;²⁶ hence, inhibiting α-Syn aggregation represents a promising therapeutic strategy.^27,28 Current inhibitors primarily target WT α-Syn by binding to specific residues (e.g., V48, H50, G51 and V52);²⁹ which could lose efficacy against pathogenic variants (e.g., A30P and H50Q).³⁰ Here, we found that NAMI-A inhibited early nucleation in diverse variants, preventing aggregate formation, as demonstrated by the ThT assay, gel electrophoresis, and TEM imaging. NAMI-A primarily targeted the conserved NAC and C-terminal regions, generated conserved adducts, and prevented disorder-to-β-sheet transitions. It has been found that residues favor metal coordination and play important roles in the binding of metallo-agents.^31,32 Our study elucidates that NAMI-A coordinates to the conserved metal-binding residues across various familial variants, underscoring the fundamental strength of metallodrugs: their target recognition relies on coordination chemistry rather than predefined protein folds (typically required for organic inhibitors), making them ideal for intrinsically disordered targets like α-Syn and other amyloidogenic proteins. This finding demonstrates NAMI-A's unique ability to overcome mutation-dependent limitations in α-Syn aggregation, highlighting its advantage in drug design for heterogeneous pathologies.

In addition to targeting soluble α-Syn monomers, disruption of pre-formed aggregates is another strategy for PD treatment; however, various familial variants form distinct aggregate structures,^33–35 and this structural heterogeneity challenges conventional structure-based drug design.^36–38 Indeed, these α-Syn familial variants with different fibril structures are often associated with severe symptoms, including early-onset and rapidly progressive disease.^39,40 The discovery that NAMI-A exhibits broad-spectrum inhibition of the aggregation of familial α-Syn variants and promotes the disassembly of their aggregates offers a promising approach for the development of effective anti-PD agents.

Conclusions

In summary, this study demonstrates that the ruthenium complex NAMI-A effectively inhibits the pathological aggregation of α-synuclein (α-Syn) across diverse familial variants, overcoming the limitation of conventional structure-based inhibitors. NAMI-A binds to α-Syn through metal coordination to conserved regions (N-terminal, NACore, and C-terminal regions), which prevents the disorder-to-β-sheet structural transition in all tested variants, thereby potently suppressing their aggregation. Notably, NAMI-A disassembles pre-formed fibrils, highlighting its dual therapeutic potential. Cellular assays confirmed that NAMI-A significantly suppresses ROS overproduction and reduces cytotoxicity induced by wild-type and disease-associated α-Syn variants to neuronal cells. Given the association of familial variants with early-onset and aggressive PD pathology, the ability of NAMI-A to counteract polymorphic aggregates positions it as a promising broad-spectrum therapeutic agent. This work further underscores the significance of metallo-agents in targeting intrinsically disordered proteins.

Author contributions

Y. L. conceived the idea and supervised the project with the assistance of K. C. S. W. and K. C. designed the experiments. S. W. performed the experiments, collected the data and analyzed the results with assistance from W. W., L. S., S. Y. and W. W. S. W. and K. C. wrote the initial manuscript. Y. L. wrote the final manuscript. All authors made contributions to the project.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data that support the findings of this study are available in the article and its supplementary information (SI). Supplementary information: experimental details, protein characterization, fluorescence quenching assays, transmission electron microscopy images, ESI-MS peak assignments, and raw gel electrophoresis images. See DOI: https://doi.org/10.1039/d5qi01863c.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (22377116, 22177109, 22407001, 52021002, and 22207101), the National Key R&D Program of China (2020YFA0710700), the Plans for Major Provincial Science & Technology Projects (202303a07020004), and the USTC Research Funds of the Double First-Class Initiative (YD2060002502 and YD9100002033).

References

1. B. R. Bloem, M. S. Okun and C. Klein, Parkinson's disease, Lancet, 2021, 397, 2284–2303.
2. D. Su, Y. Cui, C. He, P. Yin, R. Bai, J. Zhu, J. S. Lam, J. Zhang, R. Yan and X. Zheng, Projections for prevalence of Parkinson's disease and its driving factors in 195 countries and territories to 2050: modelling study of Global Burden of Disease Study 2021, BMJ, 2025, 388, 080952.
3. L. X. Yi, E. K. Tan and Z. D. Zhou, The α-synuclein seeding amplification assay for Parkinson's disease, Int. J. Mol. Sci., 2025, 26, 389.
4. Y. Yang, Y. Shi, M. Schweighauser, X. Zhang, A. Kotecha, A. G. Murzin, H. J. Garringer, P. W. Cullinane, Y. Saito and T. Foroud, Structures of α-synuclein filaments from human brains with Lewy pathology, Nature, 2022, 610, 791–795.
5. G. Zanotti, Intrinsic disorder and flexibility in proteins: A challenge for structural biology and drug design, Crystallogr. Rev., 2023, 29, 48–75.
6. S. Saurabh, K. Nadendla, S. S. Purohit, P. M. Sivakumar and S. Cetinel, Fuzzy drug targets: disordered proteins in the drug-discovery realm, ACS Omega, 2023, 8, 9729–9747.
7. R. A. Fredenburg, C. Rospigliosi, R. K. Meray, J. C. Kessler, H. A. Lashuel, D. Eliezer and P. T. Lansbury, The impact of the E46K mutation on the properties of α-synuclein in its monomeric and oligomeric states, Biochemistry, 2007, 46, 7107–7118.
8. O. Khalaf, B. Fauvet, A. Oueslati, I. Dikiy, A.-L. Mahul-Mellier, F. S. Ruggeri, M. K. Mbefo, F. Vercruysse, G. Dietler and S.-J. Lee, The H50Q mutation enhances α-synuclein aggregation, secretion, and toxicity, J. Biol. Chem., 2014, 289, 21856–21876.
9. C. Fevga, Y. Park, E. Lohmann, A. J. Kievit, G. J. Breedveld, F. Ferraro, L. de Boer, R. van Minkelen, H. Hanagasi and A. Boon, A new alpha-synuclein missense variant (Thr72Met) in two Turkish families with Parkinson's disease, Parkinsonism Relat. Disord., 2021, 89, 63–72.
10. M. Avenali, S. Cerri, I. Palmieri, G. Ongari, R. Stiuso, G. Buongarzone, C. Tassorelli, T. Biagini, M. Valente and C. Cereda, Functional Study of SNCA p.V15A Variant: Further Linking α-Synuclein and Glucocerebrosidase, Mov. Disord., 2024, 39, 1060–1065.
11. C. R. Bodner, A. S. Maltsev, C. M. Dobson and A. Bax, Differential phospholipid binding of α-synuclein variants implicated in Parkinson's disease revealed by solution NMR spectroscopy, Biochemistry, 2010, 49, 862–871.
12. X. Meng, L. A. Munishkina, A. L. Fink and V. N. Uversky, Molecular mechanisms underlying the flavonoid-induced inhibition of α-synuclein fibrillation, Biochemistry, 2009, 48, 8206–8224.
13. K. Cao, Y. Zhu, Z. Hou, M. Liu, Y. Yang, H. Hu, Y. Dai, Y. Wang, S. Yuan and G. Huang, α–Synuclein as a target for metallo–anti–neurodegenerative agents, Angew. Chem., Int. Ed., 2023, 62, e202215360.
14. W. Han, M. Wei, F. Xu and Z. Niu, Aggregation and phase separation of α-synuclein in Parkinson's disease, Chem. Commun., 2024, 60, 6581–6590.
15. M. B. Fares, N. Ait-Bouziad, I. Dikiy, M. K. Mbefo, A. Jovičić, A. Kiely, J. L. Holton, S. J. Lee, A. D. Gitler and D. Eliezer, The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells, Hum. Mol. Genet., 2014, 23, 4491–4509.
16. D. Ghosh, M. Mondal, G. M. Mohite, P. K. Singh, P. Ranjan, A. Anoop, S. Ghosh, N. N. Jha, A. Kumar and S. K. Maji, The Parkinson's disease-associated H50Q mutation accelerates α-Synuclein aggregation in vitro, Biochemistry, 2013, 52, 6925–6927.
17. E. A. Greenbaum, C. L. Graves, A. J. Mishizen-Eberz, M. A. Lupoli, D. R. Lynch, S. W. Englander, P. H. Axelsen and B. I. Giasson, The E46K mutation in α-synuclein increases amyloid fibril formation, J. Biol. Chem., 2005, 280, 7800–7807.
18. D. Ghosh, S. Sahay, P. Ranjan, S. Salot, G. M. Mohite, P. K. Singh, S. Dwivedi, E. Carvalho, R. Banerjee and A. Kumar, The newly discovered Parkinson's disease associated Finnish mutation (A53E) attenuates α-synuclein aggregation and membrane binding, Biochemistry, 2014, 53, 6419–6421.
19. P. Kumari, D. Ghosh, A. Vanas, Y. Fleischmann, T. Wiegand, G. Jeschke, R. Riek and C. Eichmann, Structural insights into α-synuclein monomer–fibril interactions, Proc. Natl. Acad. Sci. U. S. A., 2021, 118, e2012171118.
20. S. Ray, N. Singh, R. Kumar, K. Patel, S. Pandey, D. Datta, J. Mahato, R. Panigrahi, A. Navalkar and S. Mehra, α-Synuclein aggregation nucleates through liquid–liquid phase separation, Nat. Chem., 2020, 12, 705–716.
21. J. Dorszewska, M. Kowalska, M. Prendecki, T. Piekut, J. Kozłowska and W. Kozubski, Oxidative stress factors in Parkinson's disease, Neural Regener. Res., 2021, 16, 1383–1391.
22. E. O. Olufunmilayo, M. B. Gerke-Duncan and R. D. Holsinger, Oxidative stress and antioxidants in neurodegenerative disorders, Antioxidants, 2023, 12, 517.
23. F. Galli, D. Bartolini and C. Ronco, Oxidative stress, defective proteostasis and immunometabolic complications in critically ill patients, Eur. J. Clin. Invest., 2024, 54, e14229.
24. S. Sahoo, A. A. Padhy, V. Kumari and P. Mishra, Role of ubiquitin–proteasome and autophagy-lysosome pathways in α-synuclein aggregate clearance, Mol. Neurobiol., 2022, 59, 5379–5407.
25. F. Le Guerroué and R. J. Youle, Ubiquitin signaling in neurodegenerative diseases: an autophagy and proteasome perspective, Cell Death Differ., 2021, 28, 439–454.
26. S. Negi, N. Khurana and N. Duggal, The Misfolding Mystery: α-syn and the Pathogenesis of Parkinson's Disease, Neurochem. Int., 2024, 105760.
27. S. Peña-Díaz, J. García-Pardo and S. Ventura, Development of small molecules targeting α-synuclein aggregation: a promising strategy to treat Parkinson's disease, Pharmaceutics, 2023, 15, 839.
28. M. Galkin, A. Priss, Y. Kyriukha and V. Shvadchak, Navigating α–Synuclein Aggregation Inhibition: Methods, Mechanisms, and Molecular Targets, Chem. Rec., 2024, 24, e202300282.
29. N. N. Jha, R. Kumar, R. Panigrahi, A. Navalkar, D. Ghosh, S. Sahay, M. Mondal, A. Kumar and S. K. Maji, Comparison of α-synuclein fibril inhibition by four different amyloid inhibitors, ACS Chem. Neurosci., 2017, 8, 2722–2733.
30. J. Pujols, S. Peña-Díaz, D. F. Lázaro, F. Peccati, F. Pinheiro, D. González, A. Carija, S. Navarro, M. Conde-Giménez and J. García, Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons, Proc. Natl. Acad. Sci. U. S. A., 2018, 115, 10481–10486.
31. B. Pan, Y. Yang, H. Liu, Y. Li and X. Su, Coordination of platinum to α-synuclein inhibits filamentous aggregation in solution, ChemBioChem, 2019, 20, 1953–1958.
32. B. Pan, H. Liu, W. Meng and X. Su, Pt(IV) complex selectively oxidizes alpha-synuclein methionine as disclosed by NMR, Inorg. Chem. Front., 2023, 10, 296–304.
33. K. Zhao, Y. Li, Z. Liu, H. Long, C. Zhao, F. Luo, Y. Sun, Y. Tao, X.-d. Su and D. Li, Parkinson's disease associated mutation E46K of α-synuclein triggers the formation of a distinct fibril structure, Nat. Commun., 2020, 11, 2643.
34. T. Ohgita, N. Namba, H. Kono, T. Shimanouchi and H. Saito, Mechanisms of enhanced aggregation and fibril formation of Parkinson's disease-related variants of α-synuclein, Sci. Rep., 2022, 12, 6770.
35. S. Sahay, D. Ghosh, P. K. Singh and S. K. Maji, Alteration of structure and aggregation of α-synuclein by familial Parkinson's disease associated mutations, Curr. Protein Pept. Sci., 2017, 18, 656–676.
36. S. Mehra, L. Gadhe, R. Bera, A. S. Sawner and S. K. Maji, Structural and functional insights into α-synuclein fibril polymorphism, Biomolecules, 2021, 11, 1419.
37. S. Arar, M. A. Haque and R. Kayed, Protein aggregation and neurodegenerative disease: Structural outlook for the novel therapeutics, Proteins: Struct., Funct., Bioinf., 2025, 93, 1314–1329.
38. S. Choudhary, M. Lopus and R. V. Hosur, Targeting disorders in unstructured and structured proteins in various diseases, Biophys. Chem., 2022, 281, 106742.
39. S. Erer, U. Egeli, M. Zarifoglu, G. Tezcan, G. Cecener, B. Tunca, S. Ak, E. Demirdogen, G. Kenangil and H. Kaleagası, Mutation analysis of the PARKIN, PINK1, DJ1, and SNCA genes in Turkish early-onset Parkinson's patients and genotype-phenotype correlations, Clin. Neurol. Neurosurg., 2016, 148, 147–153.
40. H. Long, W. Zheng, Y. Liu, Y. Sun, K. Zhao, Z. Liu, W. Xia, S. Lv, Z. Liu and D. Li, Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding, Proc. Natl. Acad. Sci. U. S. A., 2021, 118, e2012435118.

---