pubs.acs.org/chemneuro Letter Discovery of Neuroprotective Agents Based on a 5‑(4-Pyridinyl)- 1,2,4-triazole Scaffold Rosaria Gitto, Serena Vittorio, Federica Bucolo, Samuel Peña-Díaz, Rosalba Siracusa, Salvatore Cuzzocrea, Salvador Ventura, Rosanna Di Paola, and Laura De Luca* Cite This: ACS Chem. Neurosci. 2022, 13, 581−586 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Parkinson’s disease (PD) is characterized by the death of dopaminergic neurons. The common histopathological hallmark in PD patients is the formation of intracellular proteinaceous accumulations. The main constituent of these inclusions is alpha-synuclein (α-syn), an intrinsically disordered protein that in pathological conditions creates amyloid aggregates that lead to neurotoxicity and neurodegeneration. The main goal of our study was to optimize our previously identified α-syn aggregation inhibitors of 5-(4-pyridinyl)-1,2,4-triazole chemotype in terms of in vivo efficacy. Our efforts resulted in the identification of ethyl 2- ((4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate (15), which displayed the ability to prevent 1-methyl-4-phenyl-1,2,3,6- tetrahydropiridine-induced bradykinesia as well as to affect the levels of PD markers after the administration of the same neurotoxin. In addition to the in vivo evaluation, for the 5-(4-pyridinyl)-1,2,4-triazole-based compounds, we measured the prevention of the fibrillization process using light scattering and a ThT binding assay; these compounds have been shown to slightly reduce the α-syn aggregation. KEYWORDS: 5-(4-pyridinyl)-1,2,4-triazoles, synthesis, Parkinson’s disease, alpha synuclein, MPTP ■ INTRODUCTION A progressive loss of dopaminergic neurons in the substantia cytotoxicity through different mechanisms such as the increase of lipid membrane permeabilization, the mitochondrial damage, nigra pars compacta of the brain characterizes Parkinson’s and the oxidative stress.8 Moreover, the misfolded extracellular Disease (PD).1 Thus, the neurodegeneration results in a α-syn establishes a prion-like mechanism of propagation from significant reduction of dopamine (DA) in the synaptic neurons to glial cells.9 Finally, the misfolded α-syn acts as an terminals of the dorso-striatum, so that PD has recently been antigen, thus triggering an immune response closely associated considered as a synaptopathy. Although there is still much with neuroinflammatory events.10 debate about the main cause of PD, the analysis of the brain of The therapeutic treatment is exclusively focused on alleviating PD patients revealed a common factor: the formation of motor symptoms so that the restoring of DA levels represents intraneuronal inclusions, named Lewy bodies and Lewy the widespread approach by using the prodrug L-Dopa 1 (Figure neurites, formed by aggregates of a disordered protein called 1), which crosses the blood-brain barrier (BBB) and is converted alpha synuclein (α-syn). α-Syn is a small protein localized in the to DA.1,11 There are also therapeutics (see Figure 1) that target presynaptic terminal,2,3 whose sequence could be dissected in the dopamine receptor (e.g., ropinirole, 2) or inhibit mono- three distinctive domains: the N-terminal domain, central domain NAC, and C-terminal domain.4 α-Syn is physiologically Received: December 21, 2021 found as a soluble monomer, but after interaction with Accepted: February 11, 2022 phospholipids it adopts an α-helical structure.5 In the misfolded Published: February 18, 2022 state, the aggregation of α-syn into amyloid fibrils leads to neuronal pathological inclusions located both in the neuron soma and in axons.6,7 It is well-known that this process causes © 2022 American Chemical Society https://doi.org/10.1021/acschemneuro.1c00849 581 ACS Chem. Neurosci. 2022, 13, 581−586 ACS Chemical Neuroscience pubs.acs.org/chemneuro Letter Figure 1. Chemical structures of well-known therapeutics for PD. Figure 2. Small molecules as α-syn aggregation inhibitors. Figure 3. Newly studied compounds 12−15 bearing the 5-(4-pyridinyl)-1,2,4-triazol-4-amine core. amine oxidase B (MAO B) (e.g., selegiline, 3) and catechol-O- blocking the fibril formation. Concerning nonpolyphenol methyltransferase (COMT) (e.g., tolcapone, 4).12,13 Sadly, the derivatives, inhibitory effects on the aggregation of α-syn were side effects of the above-mentioned therapeutics have been found for mannitol (7) and several terpenoids as well as turned to the exploration of new targets. alkaloids.16,17 Collecting structure-affinity relationship informa- On the basis of these considerations, the inhibition of α-syn tion, it was observed that the presence of aromatic rings and/or aggregation might be considered a valuable approach to research planar structures could represent an important requirement for new agents that might be able to prevent the disease progression inhibitory activity.18 in place of relieving the symptoms. A number of promising small molecule inhibitors from Recently, it has been suggested that a therapeutic strategy synthetic sources have been discovered using innovative might be achieved inhibiting or reversing the α-syn aggregation approaches, including high-throughput screening. SynuClean- by using peptides or small molecule able to interact with α-syn D (8), ZPD-2 (9), and anle318b (10) were demonstrated to protein through distinct modalities and to prevent the formation inhibit the α-syn aggregation and to prevent their propagation in of oligomers and subsequently amyloid fibrils.14 Currently two PD.19−21 main classes of small molecules have been identified through in On the basis of the knowledge that the aggregation-prone vivo screening assays; they are polyphenol and nonpolyphenol NAC domain of α-syn is crucial for the conformational shift of compounds (see Figure 2) from natural and synthetic sources. the protein to β-sheets, potential α-syn aggregation inhibitors Among polyphenol compounds, it has been demonstrated that were developed as ligands of the target residues 69−72. caffeic acid (5) possesses an antifibrillating ability in a dose- Accordingly, we focused our attention to identify of newer α- dependent manner against the aggregation of α-syn induced by syn aggregation inhibitors oriented toward the NAC domain of treatment with escitalopram. Indeed, by analyzing the structure α-syn.22 From a set of 4-amino-5−(4-pyridinyl)-4H-1,2,4- of phenolic compounds, it was observed that the presence of the triazole-derived compounds, we identified the 3-(cinnamylsul- catechol moiety leads to an antiamyloidogenic activity.15 The fanyl)-5-(4-pyridinyl)-1,2,4-triazol-4-amine (11) that proved to flavone baicalein (6) proved to prevent the α-syn cytotoxicity reduce α-syn aggregation in an in vitro assay. Docking studies and inhibit and stabilize the oligomerization of α-syn, thus suggested the binding into a specific site placed between N- 582 https://doi.org/10.1021/acschemneuro.1c00849 ACS Chem. Neurosci. 2022, 13, 581−586 ACS Chemical Neuroscience pubs.acs.org/chemneuro Letter Figure 4. Effects of 15 on TH expression in the midbrain of MPTP-treated mice. The immunohistochemical analysis demonstrated, compared with the Sham mice (A,A′), a pronounced loss of TH-positive cells (B,B′). Animals treated with 15 showed an increase in TH expression (C,C′). Data are expressed as the percentage of TH-positive pixels and are the means ± SEM of five mice per group. (D) ***p < 0.001 vs Sham; ###p < 0.001 vs MPTP. Figure 5. Effects of compound 15 on α-syn expression in midbrain of MPTP-treated mice. The immunohistochemical investigation discovered, compared with Sham animals (A,A′), a positive stain for α-syn (B,B′). Compound 15 treatment significantly reduced the positive stain for α-syn in the SN (C,C′). (D) ***p < 0.001 vs Sham; ###p < 0.001 vs MPTP. Data are expressed as the percentage of α-syn-positive pixels and are the mean ± SEM of five mice per group. terminal and NAC domains. As a continuation of these (i) a salt bridge contact, (ii) an unusual π-anion interaction, and promising achievements, we now report the exploitation of a (iii) several hydrophobic and van der Waals interactions (see pyridinyl-triazole scaffold of hit compound 11 for further in vivo Figure 3). On the basis of this pharmacophore pattern, we and in vitro investigations. Therefore, the new designed designed new compounds with the aim to achieve further compounds were screened as α-syn aggregation inhibitors. structural data for this series of compounds. Specifically, we Moreover, we performed in vivo studies by means of the chose to maintain the pyridinyl-triazol-4-amine core and experimental protocol of 1-methyl-4-phenyl-1,2,3,6-tetrahydro- explored the eastern region of prototype 11 by the removal of piridine (MPTP)-induced degeneration of dopaminergic the cinnamyl fragment and the insertion of small fragments. In neurons. detail, the new compounds were prepared to study the effect of ■ RESULT AND DISCUSSION Design and Synthesis of New 4-Amino-5-(4-pyridinyl)- introducing small substituents able to increase H-bond donor/ acceptor contacts as well as hydrophobic interactions in the subpocket lined by crucial NAC domain residues. Therefore, we 4H-1,2,4-triazole-Derived Compounds. Our proposed investigated the α-syn aggregation inhibitory effects of the pharmacophore for α-syn aggregation inhibition consisted of starting material 4-amino-5-(4-pyridinyl)-4H-1,2,4-triazole-3- 583 https://doi.org/10.1021/acschemneuro.1c00849 ACS Chem. Neurosci. 2022, 13, 581−586 ACS Chemical Neuroscience pubs.acs.org/chemneuro Letter Figure 6. In vitro analysis of the capacity of the different compounds to inhibit α-syn aggregation. (A) Aggregation kinetics of α-syn in the absence (black) or presence of 100 μM of 12 (yellow), 13 (violet), 14 (red), or 15 (brown) compared to previously described molecules SC-D (blue) and C-5 (green). Normalized intensity of Th-T fluorescence is plotted as a function of time. (B) Light-scattering measurements at 300 nm of end-point aggregates in the absence (black) or presence of 100 μM of 12 (yellow), 13 (violet), 14 (red), or 15 (brown) compared to previously described molecules SC-D (blue) and C-5 (green). Th-T fluorescence and light-scattering is plotted as normalized means; error bars are represented as the SE of mean values. *p < 0.05 and ***p < 0.001. thiol (12) and its corresponding three derived analogue aggregated α-syn levels in the solution (Figure 6B), with compounds 13−15 reported in Figure 3. decrements of 44%, 31%, 18%, and 18%, respectively. The The synthetic route for compounds 13−15 was based on the impacts of compounds 14 and 15 are similar to that of C-5 well-established reaction of the starting material 12 with an (21%).22 The discrepancy between the influence of compounds opportune alkyl halide in alkaline medium as previously 12 and 13 in Th-T and light-scattering signals suggests that the reported by us.22 The synthesized compounds were structurally aggregates formed in the presence of these molecules exhibit a characterized by means of spectroscopic measurements, and all higher affinity for Th-T, likely being richer in intermolecular β- data are reported in the Methods section. sheet. In Vivo Studies. Among the new synthesized compounds, Overall, these studies indicated that the 5-(4-pyridinyl)-4H- we selected ethyl 2-((4-amino-5-(pyridin-4-yl)-4H-1,2,4-tria- 1,2,4-triazole core could be a new chemical template to design zol-3-yl)thio)acetate (15) to ascertain its protective effect by neuroprotective agents in PD. In detail, the immunohistochem- measuring specific markers of PD. We measured the capacity of ical assays revealed that compound 15 proved to enhance levels compound 15 to prevent the neurodegeneration produced by of TH and DAT in the midbrain of mice treated with neurotoxin the neurotoxin MPTP investigating the levels of tyrosine MPTP; further, compound 15 was able to reduce the α-syn hydroxylase (TH) and α-syn in the midbrain. expression in the same test. Moreover, compound 15 By an immunohistochemical analysis, the expression of TH- ameliorated a motor deficit in MPTP-treated mice (see DAT positive neurons was significantly reduced 8 d after MPTP and the behavioral analysis in the Supporting Information). To administration (Figure 4B,B′,D) compared to the results of the provide evidence for the role of α-syn aggregation inhibition in Sham group (Figure 4A,A′,D). Treatment with compound 15 mediating the in vivo effects, we performed in vitro studies that incremented the levels of this protein (Figure 4C,C′,D). revealed that the studied 5-(4-pyridinyl)-4H-1,2,4-triazole- In reverse, we detected an important immunoreactivity in based compounds might affect fibrillization. Taken together, MPTP-damaged mice (Figure 5B,B′,D) compared with Sham these data might indicate a plausible association between the α- animals (Figure 5A,A′,D). Rather, the compound 15 treatment syn aggregation inhibition and the in vivo effects toward MPTP- appreciably reduced α-syn expression in the midbrain after induced toxicity in mice. MPTP intoxication (Figure 5C,C′,D). In Vitro Studies. On the basis of the promising in vivo effects measured for compound 15, we moved our attention to the ■ METHODS Chemistry. Reagents and solvents were purchased from commercial evaluation of the ability to reduce α-syn aggregation in vitro. suppliers (Merck KGaA and Thermofisher Scientific Inc.) and were Then, the four pyridinyl-triazole derivatives 12−15 were tested used without further purification. Melting points of synthesized by the same protocol used for the identification of potent α-syn compounds were recorded on a Buchi B-545 apparatus (BUCHI aggregation inhibitors like SynuClean-D (SC-D) that was used Labortechnik AG) and are uncorrected. The purity of compounds was evaluated by combustion analysis measurements (C, H, N) on a Carlo as a reference molecule together the previously reported22 Erba Elemental Analyzer (Model 1106); the collected data confirmed a parent compound C-5 in which a benzyl moiety (R = CH2Ph) is purity of at least 95%. A thin-layer chromatography (TLC) analysis was linked to a sulfur atom of compound 12. We monitored the performed on fluorescent silica gel 60 F254 and visualized using UV kinetics of aggregation of 70 μM α-syn in the absence or light (λ = 254 nm/366 nm) or staining with iodine vapor. All 1H and presence of 100 μM of the studied derivatives by following the 13 C NMR spectra were recorded in deuterated dimethyl sulfoxide increase in thioflavin-T (Th-T) fluorescence (Figure 6A). (DMSO-d6) with a Varian Gemini 500 spectrometer (Varian Inc.). The Compounds 12 and 13 slightly accelerated the reaction, whereas chemical shifts are given in δ (ppm), and coupling constants (J) are the inhibitory effect of compound 15 was indistinguishable from given in hertz (Hz). The exchangeable proton atoms were detected by D2O. C-5 (8% reduction in Th-T signal), and compound 14 General Procedure for the Synthesis of Pyridinyl-Triazole performed slightly better (15% reduction in Th-T signal). Derivatives (13−15). The starting material 4-amino-5-(4-pyridinyl)- Light-scattering measurements at 300 nm at the end of the 4H-1,2,4-triazole-3-thiol (12) (1 equiv) was dissolved in MeOH (5 reaction indicated that 12, 13, 14, and 15 reduced the mL) and NaOH (1 equiv). Then, the suitable alkyl bromide derivative 584 https://doi.org/10.1021/acschemneuro.1c00849 ACS Chem. Neurosci. 2022, 13, 581−586 ACS Chemical Neuroscience ■ pubs.acs.org/chemneuro Letter (1 equiv) was added to the mixture, and it was stirred at room AUTHOR INFORMATION temperature. After the mixture was stirred for 120 min, the resulting crude product was filtered, dried, and recrystallized from EtOH to give Corresponding Author desired final derivatives 13−15. The CAS numbers for 13−15 have Laura De Luca − Department of Chemical, Biological, been already assigned as reported below for each compound in the Pharmaceutical and Environmental Sciences, University of Supporting Information. The structural characterization as well as the Messina, I-98125 Messina, Italy; orcid.org/0000-0003- physicochemical properties were generally in agreement with previous 0614-5713; Email: laura.deluca@unime.it data.23−25 In Vivo Studies. We performed experiments on neurodegenerative Authors diseases with compound 15 at a dose of 10 mg/kg after a preliminary Rosaria Gitto − Department of Chemical, Biological, dose−response study conducted in our laboratories26,27 as described in Pharmaceutical and Environmental Sciences, University of more detail in the Supporting Information. Messina, I-98125 Messina, Italy Experimental Groups. The mice were arbitrarily allocated into four Serena Vittorio − Department of Chemical, Biological, groups: Pharmaceutical and Environmental Sciences, University of Group 1. Sham+Veh = Vehicle solution (saline) was administered intraperitoneal during the first day, like the MPTP protocol (N = 10). Messina, I-98125 Messina, Italy Group 2. Sham+15 = Same as the Sham+Veh group, but 15 (10 mg/ Federica Bucolo − Department of Chemical, Biological, kg body weight, soluble in saline, orally) was administered starting 24 h Pharmaceutical and Environmental Sciences, University of after the first vehicle solution injection and continuing through seven Messina, I-98125 Messina, Italy additional days after the last administration of saline (N = 10). Samuel Peña-Díaz − Institut de Biotecnologia i Biomedicina, Group 3. MPTP+Veh = MPTP solution was administered as Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain; described for the administration of saline (N = 10). Departament de Bioquimica i Biologia Molecular, Universitat Group 4. MPTP+15 = Same as the MPTP+Veh group, but 15 (at a Autonoma de Barcelona, 08193 Bellaterra, Spain dose of 10 mg/kg body weight, orally) was administered starting 24 h Rosalba Siracusa − Department of Chemical, Biological, after the first MPTP administration and continuing through seven additional days after the last injection of MPTP (N = 10). Pharmaceutical and Environmental Sciences, University of Immunohistochemical Localization of Tyrosine Hydroxylase (TH) Messina, I-98125 Messina, Italy and α-Synuclein. We used the immunohistochemical techniques as Salvatore Cuzzocrea − Department of Chemical, Biological, previously described.28 The antibodies that were incubated overnight Pharmaceutical and Environmental Sciences, University of on the brain sections were anti-TH (Millipore, 1:500 in phosphate- Messina, I-98125 Messina, Italy buffered saline (PBS), v/v, AB152) and anti-α-syn (Santa Cruz Salvador Ventura − Institut de Biotecnologia i Biomedicina, Biotechnology, 1:50 in PBS, v/v, LB509 sc-58480). To demonstrate the Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain; specificity of the antibodies, the brain sections of five mice for each Departament de Bioquimica i Biologia Molecular, Universitat group were treated either with a primary or only with a secondary antibody. The images were captured by a Zeiss microscope and Axio Autonoma de Barcelona, 08193 Bellaterra, Spain; ICREA, Vision software. The ImageJ IHC profiler plug-in was used for a 08010 Barcelona, Spain densitometric analysis. When this is selected, it automatically traces a Rosanna Di Paola − Department of Chemical, Biological, histogram profile of the deconstructed diaminobenzidine image Pharmaceutical and Environmental Sciences, University of showing a corresponding score log. The histogram profile corresponds Messina, I-98125 Messina, Italy to the positive pixel intensity value obtained from the computer Complete contact information is available at: software. Immunohistochemical analyses were performed by experi- enced people who did not know the treatment. https://pubs.acs.org/10.1021/acschemneuro.1c00849 In Vitro Studies. We performed α-synuclein aggregation and inhibition in vitro assays. Human wt α-syn was expressed and purified as Author Contributions previously indicated29 and kept lyophilized at −80 °C until use. Before R.G., S.V., and L.D.L conceived the idea and designed the use, the protein was gently resuspended in sterile PBS 1X and filtered manuscript; S.V., F.B., R.S., and S.P.-D. conducted the through a 0.22 μm membrane to remove small aggregates. The experiments. S.V., F.B, R.S., and S.Ve wrote the manuscript, inhibitory capacity of the compounds was assessed as previously with editing by R.G., R.D.P., S.C., and L.D.L. All authors have described.20,22,29,30 Briefly, 70 μM of soluble α-syn was placed in a read and agreed to the published version of the manuscript. sealed 96-well plate containing 40 μM Th-T in PBS 1X, a 1/8 in. diameter Teflon polyball (Polysciences Europe GmbH) and 100 μM of Notes The authors declare no competing financial interest. ■ the different molecules or DMSO as control samples. Each well contained a final volume of 150 μL. Samples were then incubated at 37 °C and 100 rpm in an orbital shaker Max-Q 4000 (ThermoScientific). ACKNOWLEDGMENTS Th-T fluorescence emission was measured every 2 h in a TECAN The authors thank the “Programma Operativo Nazionale SPARK-1 plate reader (Tecan Trading AG) by exciting through a 430− 450 nm filter and collecting with a 480−510 nm filter. Light-scattering Ricerca & Innovazione 2014-2020, Azione I.1 “Dottorati measurements were performed in a Cary Eclipse Fluorescence Innovativi con caratterizzazione industriale” for financial Spectrophotometer (Agilent). 80 μL of end-point aggregates was support in form of a PhD scholarship “DOT1314952” granted to S.V. and F.B. ■ inserted into a quartz cuvette and excited at 300 nm; the 90° emission was collected between 280 and 320 nm. ■ * ASSOCIATED CONTENT sı Supporting Information REFERENCES (1) Chakraborty, A.; Brauer, S.; Diwan, A. Possible therapies of Parkinson’s disease: A review. J. Clin. Neurosci. 2020, 75, 1−4. The Supporting Information is available free of charge at (2) Cardinale, A.; Calabrese, V.; de Iure, A.; Picconi, B. Alpha- https://pubs.acs.org/doi/10.1021/acschemneuro.1c00849. Synuclein as a Prominent Actor in the Inflammatory Synaptopathy of 1 Parkinson’s Disease. Int. J. Mol. Sci. 2021, 22 (12), 6517. H NMR spectra and physicochemical properties for (3) Save, S. S.; Rachineni, K.; Hosur, R. V.; Choudhary, S. Natural synthesized compounds 13−15. Supplementary data of in compound safranal driven inhibition and dis-aggregation of α-synuclein vivo studies (PDF) fibrils. Int. J. Biol. Macromol. 2019, 141, 585−595. 585 https://doi.org/10.1021/acschemneuro.1c00849 ACS Chem. Neurosci. 2022, 13, 581−586 ACS Chemical Neuroscience pubs.acs.org/chemneuro Letter (4) Savitt, D.; Jankovic, J. Targeting α-Synuclein in Parkinson’s synuclein amyloid aggregation for the treatment of Parkinson’s disease. Disease: Progress Towards the Development of Disease-Modifying J. Enzyme Inhib. Medi. Chem. 2020, 35 (1), 1727−1735. Therapeutics. Drugs 2019, 79 (8), 797−810. (23) Bayrak, H.; Demirbas, A.; Demirbas, N.; Karaoglu, S. A. Synthesis (5) Afitska, K.; Priss, A.; Yushchenko, D. A.; Shvadchak, V. V. of some new 1,2,4-triazoles starting from isonicotinic acid hydrazide Structural Optimization of Inhibitors of α-Synuclein Fibril Growth: and evaluation of their antimicrobial activities. Eur. J. Med. Chem. 2009, Affinity to the Fibril End as a Crucial Factor. J. Mol. Biol. 2020, 432 (4), 44 (11), 4362−4366. 967−977. (24) Sung, K.; Lee, A. R. Synthesis of [(4,5-Disubstituted-4h-1,2,4- (6) Kyriukha, Y. A.; Afitska, K.; Kurochka, A. S.; Sachan, S.; Galkin, Triazol-3-Yl)Thio]Alkanoic Acids and Their Analogs as Possible M.; Yushchenko, D. A.; Shvadchak, V. V. alpha-Synuclein Dimers as Antiinflammatory Agents. J. Heterocyclic Chem. 1992, 29 (5), 1101− Potent Inhibitors of Fibrillization. J. Med. Chem. 2019, 62 (22), 10342− 1109. 10351. (25) Jiang, X.; Tang, G. Y.; Yang, J.; Ding, J. C.; Lin, H. W.; Xiang, X. L. (7) Serpell, L. C.; Berriman, J.; Jakes, R.; Goedert, M.; Crowther, R. A. Synthesis of some new acylhydrazone compounds containing the 1,2,4- Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid- triazole structure and their neuritogenic activities in Neuro-2a cells. Rsc. like cross-beta conformation. Proc. Natl. Acad. Sci. U. S. A 2000, 97 (9), Adv. 2020, 10 (32), 18927−18935. 4897−4902. (26) Paterniti, I.; Campolo, M.; Siracusa, R.; Cordaro, M.; Di Paola, (8) Chen, J.; Malone, B.; Llewellyn, E.; Grasso, M.; Shelton, P. M. M.; R.; Calabrese, V.; Navarra, M.; Cuzzocrea, S.; Esposito, E. Liver X Olinares, P. D. B.; Maruthi, K.; Eng, E. T.; Vatandaslar, H.; Chait, B. T.; receptors activation, through TO901317 binding, reduces neuro- et al. Structural Basis for Helicase-Polymerase Coupling in the SARS- inflammation in Parkinson’s disease. PLoS One 2017, 12 (4), No. e0174470. CoV-2 Replication-Transcription Complex. Cell 2020, 182 (6), 1560− (27) Crupi, R.; Impellizzeri, D.; Cordaro, M.; Siracusa, R.; Casili, G.; 1573e1513. Evangelista, M.; Cuzzocrea, S. N-palmitoylethanolamide Prevents (9) Brundin, P.; Melki, R. Prying into the Prion Hypothesis for Parkinsonian Phenotypes in Aged Mice. Mol. Neurobiol. 2018, 55 Parkinson’s Disease. J. Neurosci. 2017, 37 (41), 9808−9818. (11), 8455−8472. (10) Miraglia, F.; Ricci, A.; Rota, L.; Colla, E. Subcellular localization (28) Cordaro, M.; Siracusa, R.; Crupi, R.; Impellizzeri, D.; Peritore, A. of alpha-synuclein aggregates and their interaction with membranes. F.; D’Amico, R.; Gugliandolo, E.; Di Paola, R.; Cuzzocrea, S. 2- Neural Regen. Res. 2018, 13 (7), 1136−1144. Pentadecyl-2-Oxazoline Reduces Neuroinflammatory Environment in (11) Haddad, F.; Sawalha, M.; Khawaja, Y.; Najjar, A.; Karaman, R. the MPTP Model of Parkinson Disease. Mol. Neurobiol. 2018, 55 (12), Dopamine and Levodopa Prodrugs for the Treatment of Parkinson’s 9251−9266. Disease. Molecules 2018, 23 (1), 40. (29) Pujols, J.; Pena-Diaz, S.; Conde-Gimenez, M.; Pinheiro, F.; (12) Carrera, I.; Cacabelos, R. Current Drugs and Potential Future Navarro, S.; Sancho, J.; Ventura, S. High-Throughput Screening Neuroprotective Compounds for Parkinson’s Disease. Curr. Neuro- Methodology to Identify Alpha-Synuclein Aggregation Inhibitors. Int. J. pharmacol. 2019, 17 (3), 295−306. Mol. Sci. 2017, 18 (3), 478. (13) Stoker, T. B.; Torsney, K. M.; Barker, R. A. Emerging Treatment (30) Pena-Diaz, S.; Pujols, J.; Pinheiro, F.; Santos, J.; Pallares, I.; Approaches for Parkinson’s Disease. Front. Neurosci. 2018, 12, 693. Navarro, S.; Conde-Gimenez, M.; Garcia, J.; Salvatella, X.; Dalfo, E.; (14) Fields, C. R.; Bengoa-Vergniory, N.; Wade-Martins, R. Targeting et al. Inhibition of alpha-Synuclein Aggregation and Mature Fibril Alpha-Synuclein as a Therapy for Parkinson’s Disease. Front. Mol. Disassembling With a Minimalistic Compound, ZPDm. Front. Bioeng. Neurosci. 2019, 12, 299. Biotechnol. 2020, 8, 588947. (15) Korshavn, K. J.; Jang, M.; Kwak, Y. J.; Kochi, A.; Vertuani, S.; Bhunia, A.; Manfredini, S.; Ramamoorthy, A.; Lim, M. H. Reactivity of Metal-Free and Metal-Associated Amyloid-beta with Glycosylated Polyphenols and Their Esterified Derivatives. Sci. Rep. 2015, 5, 17842. (16) Javed, H.; Nagoor Meeran, M. F.; Azimullah, S.; Adem, A.; Sadek, B.; Ojha, S. K. Plant Extracts and Phytochemicals Targeting alpha- Synuclein Aggregation in Parkinson’s Disease Models. Front. Pharmacol. 2019, 9, 1555. (17) Ghanem, S. S.; Fayed, H. S.; Zhu, Q.; Lu, J. H.; Vaikath, N. N.; Ponraj, J.; Mansour, S.; El-Agnaf, O. M. A. Natural Alkaloid Compounds as Inhibitors for Alpha-Synuclein Seeded Fibril Formation and Toxicity. Molecules 2021, 26 (12), 3736. (18) Oliveri, V. Toward the discovery and development of effective modulators of α-synuclein amyloid aggregation. Eur. J. Med. Chem. 2019, 167, 10−36. (19) Peña-Díaz, S.; Pujols, J.; Conde-Giménez, M.; Č arija, A.; Dalfo, E.; García, J.; Navarro, S.; Pinheiro, F.; Santos, J.; Salvatella, X.; et al. ZPD-2, a Small Compound That Inhibits α-Synuclein Amyloid Aggregation and Its Seeded Polymerization. Front. Mol. Neurosci. 2019, 12, 306. (20) Wagner, J.; Ryazanov, S.; Leonov, A.; Levin, J.; Shi, S.; Schmidt, F.; Prix, C.; Pan-Montojo, F.; Bertsch, U.; Mitteregger-Kretzschmar, G.; et al. Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease. Acta Neuropathol. 2013, 125 (6), 795−813. (21) Pujols, J.; Peña-Díaz, S.; Lázaro, D. F.; Peccati, F.; Pinheiro, F.; González, D.; Carija, A.; Navarro, S.; Conde-Giménez, M.; García, J.; et al. Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons. Proc. Natl. Acad. Sci. U. S. A 2018, 115 (41), 10481−10486. (22) Vittorio, S.; Adornato, I.; Gitto, R.; Pena-Diaz, S.; Ventura, S.; De Luca, L. Rational design of small molecules able to inhibit alpha- 586 https://doi.org/10.1021/acschemneuro.1c00849 ACS Chem. Neurosci. 2022, 13, 581−586