ORIGINAL RESEARCH published: 16 October 2020 doi: 10.3389/fbioe.2020.588947 Inhibition of α-Synuclein Aggregation and Mature Fibril Disassembling With a Minimalistic Compound, ZPDm Samuel Peña-Díaz 1,2† , Jordi Pujols 1,2† , Francisca Pinheiro 1,2 , Jaime Santos 1,2 , Irantzu Pallarés 1,2 , Susanna Navarro 1,2 , María Conde-Gimenez 3 , Jesús García 4 , Xavier Salvatella 4,5 , Esther Dalfó 6,7 , Javier Sancho 3 and Salvador Ventura 1,2,5* 1 Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department of Biochemistry and Molecular and Cell Biology, Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, and Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain, 4 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain, 5 ICREA, Barcelona, Spain, 6 Medicine, M2, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 7 Faculty of Medicine, University of Vic-Central University of Catalonia (UVic-UCC), Barcelona, Spain Edited by: Jesus R. Requena, University of Santiago Synucleinopathies are a group of disorders characterized by the accumulation of de Compostela, Spain α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is Reviewed by: challenging because of the disordered nature of the protein and the stochastic nature Eva Zerovnik, Institut Jožef Stefan (IJS), Slovenia of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic Joaquín Castilla, intervention in these pathologies. A high-throughput screening initiative allowed us CIC bioGUNE, Spain to discover ZPDm, the smallest active molecule in a library of more than 14.000 *Correspondence: Salvador Ventura compounds. Although the ZPDm structure is highly related to that of the previously Salvador.Ventura@uab.cat; described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action salvador.ventura@uab.es are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α- † These authors have contributed Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein equally to this work misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong Specialty section: potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis This article was submitted to elegans model of Parkinson’s Disease, prove that these in vitro properties are translated Biosafety and Biosecurity, a section of the journal into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm Frontiers in Bioengineering and treated animals. Together with previous data, the present work illustrates how different Biotechnology chemical groups on top of a common molecular scaffold can result in divergent but Received: 29 July 2020 Accepted: 22 September 2020 complementary anti-amyloid activities. Published: 16 October 2020 Keywords: α-synuclein, protein aggregation, amyloid inhibitor, Parkinson’s disease, synucleinopathies, small Citation: molecules Peña-Díaz S, Pujols J, Pinheiro F, Santos J, Pallarés I, Navarro S, Conde-Gimenez M, García J, INTRODUCTION Salvatella X, Dalfó E, Sancho J and Ventura S (2020) Inhibition Parkinson’s disease (PD) is an incurable disorder that affects around 0.3% of the population of α-Synuclein Aggregation and Mature Fibril Disassembling With and more than 1% of people over 60 years of age (4% over 80 years), being the second a Minimalistic Compound, ZPDm. most prevalent neurodegenerative disease worldwide (Nussbaum and Ellis, 2003; Dexter and Front. Bioeng. Biotechnol. 8:588947. Jenner, 2013; Kalia and Lang, 2015). Together with Dementia with Lewy Bodies (DLB) doi: 10.3389/fbioe.2020.588947 and Multiple System Atrophy (MSA), PD is part of a group of human disorders known Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor as synucleinopathies (Spillantini et al., 1998a,b; Fanciulli SynuClean-D (SC-D) (Pujols et al., 2018) and ZPD-2 (Pena-Diaz and Wenning, 2015). Intracellular proteinaceous inclusions et al., 2019). In the present work, we present and characterize constitute the main culprit of neuronal damage and disease ZPDm (Figure 1), a novel molecule identified in the primary progression in the synucleinopathies, although the aggregates HTS screen. ZPDm was the smallest compound in the library accumulate in different cell types and affect distinct brain regions displaying a significant inhibitory potency at a substoichiometric depending on the disease (Fellner et al., 2011; Luk et al., concentration. ZPDm prevents the aggregation of wild type α- 2012). These abnormal protein deposits are mostly composed Syn and the familial A30P and H50Q variants. The molecule acts of aggregated α-synuclein (α-Syn). In the particular case of preferentially at the late stages of the polymerization reaction, PD, α-Syn aggregation occurs in the dopaminergic neurons of suggesting that it targets preferentially ordered aggregates. substantia nigra pars compacta. As a consequence, PD suffering Indeed, ZPDm is highly effective at disaggregating the mature patients display reduced dopamine levels, which results in the α-Syn fibrils of different strains. To the best of our knowledge, archetypic motor and non-motor symptoms of the disease it constitutes the minimal synthetic molecule that conjugates (Spillantini et al., 1997). Indeed, single-point mutations and inhibitory and α-Syn fibril disrupting activity in the same multiplications of the gene that encodes for α-Syn (SNCA) chemical scaffold. These in vitro activities are translated into (Singleton et al., 2003; Ibanez et al., 2004) have been related the ability to reduce significantly α-Syn aggregation in a well- to familial cases of PD with early-onset (Polymeropoulos et al., established Caenorhabditis elegans (C. elegans) model of PD. 1997), thus reinforcing the connection between α -Syn and PD. α-Syn is an intrinsically disordered protein highly expressed in the brain and associated with vesicle trafficking in healthy MATERIALS AND METHODS conditions (Bendor et al., 2013). In pathological situations, α-Syn aggregates into oligomers and amyloid fibrils that compromise Protein Expression and Purification cellular homeostasis, exert toxicity and ultimately lead to WT α-Syn and its variants (H50Q and A30P) were expressed and neuronal death (Serpell et al., 2000). Remarkably, diffusible purified as previously described (Pujols et al., 2017); the obtained aggregated species can be internalized by healthy neighboring protein was lyophilised and kept at -80◦ C until its use. neurons, where they seed the aggregation of soluble α-Syn molecules, a mechanism that has been compared with the In vitro Aggregation of α-Syn templated conformational conversion occurring in prion diseases Lyophilised α-Syn was carefully resuspended in sterile PBS (Hansen et al., 2011). As it occurs in prions, α-Syn assemblies 1X and filtered through 0.22 µm membrane to discard small can present diverse structural arrangements, forming strains (Li aggregates. Aggregation was performed at 70 µM of α-Syn (WT, et al., 2018) that differ in their aggregation properties (Bousset H50Q or A30P) in a sealed 96-well plate, in a total volume of et al., 2013), and target distinct brain regions and cell types 150 µL. 40 µM Th-T in PBS 1X, a 1/8” diameter Teflon polyball (Lau et al., 2020). (Polysciences Europe GmbH, Eppelheim, Germany) and 100 µM Many of the current therapeutic approaches for PD aim to ZPDm, (trifluoromethyl)benzene or DMSO (in control samples) reduce the neuronal load of aggregated α-Syn, either by targeting the α-Syn polypeptide directly or through indirect approaches such as the stimulation of degradation pathways (Spencer et al., 2009; Decressac et al., 2013; Xilouri et al., 2013) and gene silencing (Faustini et al., 2018; Kantor et al., 2018; Lassot et al., 2018; Zharikov et al., 2019). Among them, the identification of small compounds that might act as chemical chaperones blocking the aggregation and propagation of α-Syn or, in the best-case scenario, dismantling α-Syn mature aggregates into non-toxic species is receiving increasing attention. However, the disordered nature of α-Syn, together with the multiplicity of conformationally different species that populate the aggregation process, imposes significant difficulties for the rational design of effective α-Syn binders. For this reason, high-throughput screening (HTS) of large chemical libraries has become a significant focus of research in the hunt for disease-modifying lead compounds (Silva et al., 2011). To analyze the inhibitory potential of a chemical library with more than 14,000 chemically diverse structures, we optimized a robust HTS screening protocol (Pujols et al., 2017) based on Thioflavin-T (Th-T) fluorescence, light-scattering measurements FIGURE 1 | ZPDm, a ZPD-2 minimal structure. Chemical structures of (A) 2-nitro-4-(trifluoromethyl)phenyl vinyl sulfone, named ZPDm, and (B) ZPD-2, and Transmission Electron Microscopy (TEM). This pipeline both constituted by a hydrophobic core formed by aromatic rings and polar was used to discover and characterize small molecules that projections. act as potent inhibitors of α-Syn amyloid formation, such as Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor were also added to each well. The plate was incubated at 37◦ C and (Peabody, MA, United States) operating at an accelerating voltage 100 rpm fixed in an orbital shaker Max-Q 4000 (Thermo Fisher of 120 kV. A minimum of 30 fields were screened per sample, in Scientific, Waltham, Massachusetts, United States). Every 2 h, Th- order to collect representative images. T fluorescence was measured in a Victor3.0 Multilabel Reader (PerkinElmer, Waltham, Massachusetts, United States), exciting through a 430–450 nm filter and collecting emission signal with a Protein Misfolding Cyclic Amplification 480–510 filter. Each assay was done in triplicate and the values of (PMCA) the kinetic fitted according to the following equation: PMCA protocol was performed as previously described (Herva et al., 2014). Briefly, α-Syn was resuspended in Conversion 1 Buffer (PBS 1X, 1% Triton X-100, 150 mM NaCl) to a final ∝= 1 − (1) kb (eka t−1 ) + 1 concentration of 90 µM and supplemented with Complete Protease Inhibitor Mixture (Roche Applied Science, Penzberg, where kb and ka constitute the homogeneous nucleation Germany). 60 µL of this solution were loaded into 200 µL rate constant and the autocatalytic rate constant, respectively PCR tubes containing 1.0 mm silica beads (Biospec Products, (Crespo et al., 2016). Bartlesville, OK, United States). α-Syn was then exposed to 24 h ZPDm was added at different concentrations in the titration cycles of 30 s sonication and 30 min of incubation at 37◦ C, using assays (200, 150, 100, 75, and 50 µM). In time-dependent assays, 7 a Misonix 4000 sonicator, at 70% power. The incubated sample independent aggregation reactions were prepared simultaneously was recovered after each 24 h cycle and 1 µL was added to a new and incubated as aforementioned in a 96-well plate as triplicates. PCR tube containing fresh α-Syn at 90 µM. In the case of ZPDm 100 µM of ZPDm were added at different time points after the treated samples, the compound was added to the fresh sample in reaction had begun (4, 8, 12, 16, 20, and 24 h). In all cases α- each step to a final concentration of 128 µM, which corresponds Syn concentration was constant at 70 µM. An equivalent volume to the 0.7:1 α-Syn:ZPDm ratio of the previous aggregation assays. of DMSO was added to the control sample at the beginning Untreated samples were prepared adding the same concentration of the reaction. of DMSO (0.26%) present in the treated mixtures. This process For the study of the disaggregation assays, α-Syn 70 µM was was repeated for 5 days. All the reactions were made in triplicate. incubated in a 96-well plate as previously described for 2 days Ten microliter of aggregated samples at the end of each and Th-T fluorescence measured. Then, ZPDm was added to a cycle were diluted 1:10 with 90 µL of PBS 1X, 40 µM Th- final concentration of 100 µM. The plate was incubated for an T. Th-T fluorescence emission was measured in a Cary Eclipse additional 24 h and Th-T fluorescence was measured. Fluorescence Spectrophotometer (Agilent, Santa Clara, CA, Strains were generated as previously described (Bousset et al., United States), by exciting the samples at 445 nm and collecting 2013; Peelaerts et al., 2015; Li et al., 2018; Carija et al., 2019). the emission signal between 460 and 600 nm. Briefly, α-Syn was resuspended in PBS 1X and dialysed for 24 h in a 1:1,000 (v/v) ratio with either buffer B (50 mM Tris-HCl pH 7.0) or buffer C (50 mM Tris-HCl pH 7.0, 150 Proteinase K Digestion mM NaCl). Dialysed samples were filtered through 0.22 µm 18 µL of PMCA-aggregated α-Syn were incubated with 6 µL of membranes and incubated at 70 µM for 2 days as described Proteinase K (5 µg/mL as final concentration) for 30 min at 37◦ C. above. ZPDm was added to a final concentration of 100 µM and Then, 8 µL of loading buffer containing 1% β-mercaptoethanol plates incubated for additional 24 h. Then, Th-T fluorescence was added and the sample was incubated 10 min at 95◦ C for was measured. PK inactivation. Finally, 7 µL of the samples were loaded into a Tricine-SDS-PAGE gel. Unstained Protein Standard markers Light-Scattering (Thermo Fisher Scientific, Waltham, MA, United States) were 80 µL of end-point aggregates were collected, placed used as a reference. Gels were stained with Blue safe. into a quartz cuvette and analyzed in a Cary Eclipse Fluorescence Spectrophotometer (Agilent, Santa Clara, CA, Nuclear Magnetic Resonance (NMR) United States) by exciting at 300 and 340 nm and 90o collecting 15 N-labeled human WT α-Syn was expressed in E. coli BL21 between 280 and 360 nm. DE3 strains. Cells were grown in LB medium until the optical density (OD) at 600 nm reached a level of 0.6. Cultures were Transmission Electron Microscopy (TEM) then centrifuged at 3,000 rpm for 15 min and the obtained End-point α-Syn aggregates were collected and Diluted 1/10 (v/v) pellets resuspended in 1 L of minimal medium: 768 mL of miliQ in PBS 1X. Diluted samples were gently sonicated for 5 min and 5 water with 1 mL of ampicillin 100 mg/mL, 100 µL CaCl2 1M, µL of the resultant sample were placed on a carbon-coated copper 2 mL MgSO4 2 M, 20 mL glucose 20%, 10 mL vitamins 100x grid for 5 min. Using a filter paper, the grids were dried to remove (Sigma-Aldrich, Darmstadt, Germany), 200 mL salts M9 and the excess of sample and washed twice with miliQ water. Finally, 1 g 15 NH4 (Cambridge Isotope Laboratories, Inc., Tewksbury, 5 µL of 2% (w/v) uranyl acetate were added and left incubate for MA, United States). Cells were incubated for 1 h at 250 rpm 2 min. As previously indicated, the excess of uranyl acetate was and 37◦ C. Finally, 1 mM IPTG was added to induce protein removed, and grids were left to air-dry for 10 min. Images were expression for 4 h. Protein was purified as previously described obtained using a Transmission Electron Microscopy Jeol 1400 (Pujols et al., 2017). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor Caenorhabditis Elegans Assays Microscopy and Imaging Maintenance Animals were placed in a 1 mM solution of sodium azide and Animals synchronization was carried out by bleaching and mounted with a coverslip on a 4% agarose pad. Animals were overnight hatching in M9 (3 g/L KH2 PO4 , 6 g/L Na2 HPO4 , visualized with a Nikon Eclipse E800 epifluorescence microscope. 5 g/L NaCl, 1 M MgSO4 ) buffer. Thus, nematodes were cultured The system acquires a series of frames at specific Z-axis at 20◦ C on growth media plates (NGM) containing 1 mM position (focal plane) using a Z-axis motor device. Animals were CaCl2 , 1 mM MgSO4 , 5 µg/mL cholesterol, 250 M KH2 PO4 examined at 100 × magnification to examine α-Syn induced DA pH 6.0, 17 g/L Agar, 3 g/L NaCl. Plates were previously seeded cell death and at 40× to examine α-Syn apparent aggregate. with E. coli OP50 strain. Nematodes were maintained using standard protocols (Brenner, 1974). Statistical Analysis All graphs were generated with GraphPad Prism 6.0 software Strains (GraphPad Software Inc., La Jolla, CA, United States). Data were Strain NL5901, unc-119(ed3) III; pkIs2386 [Punc-54::α-SYN::YFP; analyzed by two-way ANOVA Tukey test using SPSS software unc-119(+)] was obtained from the C. elegans Genetic Center version 20.0 (IBM Analytics, Armonk, NY, United States) and (CGC). For the α-Syn induced dopaminergic degeneration t-test using GraphPad software version 6.0 (GraphPad Software analysis, strain UA196 (Harrington et al., 2012), gifted generously Inc., La Jolla, CA, United States). All data are shown as means by the laboratory of Dr. Guy Caldwell (Department of and standard error of mean (SEM). P < 0.05 was considered Biological Science, The University of Alabama, Tuscaloosa, statistically significant. In the graphs ∗ , ∗∗ , and ∗∗∗ indicate United States), was used; [sid-1(pk3321]; baIn33 [Pdat-1::sid-1, p < 0.05, p < 0.01, and p < 0.001, respectively. Pmyo-2::mCherry]; baIn11 [Pdat-1::α-SYN; Pdat-1::GFP]). In the main text, this strain was named Pdat-1::GFP; Pdat-1::α -SYN. ZPDm Administration RESULTS After cooling, the autoclaved NGM agar medium (1 mM CaCl2 , 1 mM MgSO4 , 5 µg/mL cholesterol, 250 M KH2 PO4 pH 6.0, ZPDm Inhibits α-Synuclein Aggregation 17 g/L Agar, 3 g/L NaCl) was enriched with 100 µM of a stock in vitro solution of 4 mM ZPDm in 0.2% DMSO to a final concentration ZPDm is a molecule that was initially identified as a positive of 10 µM. After 2 days, plates were seeded with 250 µL of hit in the HTS performed by our lab back in 2017 on top of E. coli OP50 with 10 µM of ZPDm. Nematodes were placed the HitFinderTM chemical library from Maybridge (Pujols et al., on the plates at larval stages L4 and exposed either to ZPDm 2017). Although they were found independently, structurally, or DMSO (controls) for 7 days. Daily transfer was done to ZPDm is a minimalistic version of ZPD-2 (Pena-Diaz et al., 2019) avoid cross progeny. with a reduced MW, LogP, and TPSA, which, in principle, would increase its drug-likeness (Figure 1 and Table 1). Still, ZPDm Aggregate Quantification implements the generic physicochemical properties common to The number of cellular inclusions was quantified as previously most of the published inhibitors of α-Syn aggregation, namely, described (van Ham et al., 2008; Munoz-Lobato et al., a planar hydrophobic core formed by aromatic rings that 2014). Briefly, NL5901 (Punc-54::α-SYN::YFP) worms were age- interact with apolar exposed regions in α-syn assemblies. This synchronized and left overnight to hatch. Nematodes in phase core is frequently coated with polar projections that interfere L1 were cultured and grown into individual NGM plates seeded with hydrophobic packing and disrupt intermolecular hydrogen with E. coli OP50. When animals reached L4 developmental stage, they were transferred onto either ZPDm treated plates or DMSO treated plates (negative control). Every day, animals TABLE 1 | SwissADME predicted properties of ZPDm and ZPD-2. were transferred into a new plate to avoid cross contamination. ZPDm ZPD-2 At stage L4+7, the aggregates in the anterior part of every single animal were counted. For each experiment, thirty 7- Molecular weight (g/mol) 281.21 424.4 days old nematodes per treatment were analyzed using a Heavy atoms 18 29 Nikon Eclipse E800 epifluorescence microscope equipped with Aromatic heavy atoms 6 12 an Endow GFP HYQ filter cube (Chroma Technology Corp., H-bond acceptors 7 8 Bellows Falls, Vermont United States) and each experiment was H-bond donors 0 1 carried out in triplicate. Inclusions could be described as discrete TPSA (Å2 ) 88.34 140.66 bright structures, with edges distinguishable from surrounding Log PO/W 2.42 3.9 fluorescence. ImageJ software was used for measuring the Solubility (mg/mL) 9.28E-02 1.94E-03 number of cellular aggregates considering the area dimensions. GI absorption High Low For the quantification of α-syn aggregates in C. elegans one Drug-like (Lipinski) 0 violations 0 violations single image was taken from each animal. However, every image Drug-like (Veber) 0 violations 1 violations contained among 30–45 stacks (1 µm) that allowed to detect Drug-like (Egan) 0 violations 2 violations aggregates that are at different positions. Leadlikeness 0 violations 2 violations Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor bonds; they difficult elongation and, eventually, might promote the synthesis of the more complex ZPD-2 molecule. Considering fibril disassembly (Pujols et al., 2020). A benzene ring constitutes these structural differences, we performed a set of orthogonal the hydrophobic moiety of ZPDm, connected to three polar experiments to contrast if, as ZPD-2, ZPDm might turn to be a groups—vinyl sulfone, nitro, and trifluoromethyl – in carbon lead compound with significant α-Syn anti-aggregational activity. positions 1, 2, and 4, respectively. Remarkably, the ZPD- The incubation of 70 µM α-Syn with 100 µM ZPDm 2 chemical structure displays the same functional groups in inhibited the protein aggregation, decreasing the final Th- carbons 1, 2, and 4 of the primary phenyl ring. However, the T fluorescence signal by 60% compared to control untreated vinyl sulfone is substituted by a sulfide group that extends the samples (Figure 2A). Fitting of the kinetic data to a typical molecule to incorporate additional aromatic moieties, which we sigmoidal nucleation-polymerization reaction revealed that previously assumed to be critical for its activity (Pena-Diaz et al., ZPDm diminishes the primary nucleation rate constant by 2020). Thus, ZPDm can be considered as a building block for eightfold (kb = 0.0034), relative to the control reaction FIGURE 2 | In vitro analysis of the capacity of ZPDm to inhibit α-Syn aggregation. (A) α-Syn aggregation kinetics in the absence (black) and presence (red) of 100 µM of ZPDm followed by Th-T fluorescence. (B) Light-scattering measurements at 300 and 340 nm, in the absence (white) and presence (red) of ZPDm. (C) H50Q and A30P α-Syn variants aggregation in the absence (white) and presence (blue) of ZPDm. (D) Inhibition of α-Syn aggregation with different concentrations of ZPDm. (E,F) Representative TEM images in the absence (E) and presence (F) of ZPDm. Th-T fluorescence is plotted as normalized means. Final points were obtained at 48 h. Error bars are represented as SE of mean values; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. ZPDm prevents the aggregation of WT, A30P, and H50Q α-Syn variants in vitro, even at substoichiometric ratios. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor (kb = 0.0275), at the expenses of a slightly higher autocatalytic rate constant, with ka of 0.449 and 0.323 h−1 for ZPDm treated and untreated samples, respectively. This results in t0 and t1/2 being increased by 4 and 2 h, respectively, in the compound’s presence. Light-scattering measurements at 300 and 340 nm at the end of the reaction reported a decrease of 81 and 78% in the dispersed light (Figure 2B) in the presence of ZPDm, respectively, consistent with a reduction of the total aggregated material. The visual inspection of α-Syn samples by TEM corroborated a reduction in the number of amyloid fibrils per field in the presence of ZPDm (Figure 2F), compared to untreated samples (Figure 2E). A titration assay in which we incubated 70 µM α-Syn in the presence of decreasing amounts of ZPDm indicated a dose-dependent inhibition, with a statistically significant activity at a substoichiometric concentration of 50 µM, at which ZPDm still reduces the Th-T signal at the end of the reaction by 35% (Figure 2D). We further examined if ZPDm was able to prevent the aggregation of two mutants of α-Syn, H50Q, and A30P, which have been associated with familial PD (Kruger et al., 1998; Appel- Cresswell et al., 2013). The incubation of these α-Syn variants with ZPDm reduced Th-T fluorescence at the end of the reaction by 81 and 71% for H50Q and A30P, respectively (Figure 2C). FIGURE 3 | Inhibitory activity of ZPDm in PMCA assays. (A,B) Bis/Tris SDS-PAGE gels of PMCA samples in the absence (A) and presence (B) of ZPDm, ZPD-2, and SC-D and other positive hits in ZPDm, before (–) and after (+) PK digestion. (C) Th-T fluorescence of different the library, share a common property, the presence of a PMCA cycles in samples treated (red) and untreated (white) with ZPDm. Th-T trifluoromethyl group connected to an aromatic ring. We fluorescence is plotted as normalized means. Error bars are represented as hypothesized that perhaps we were in front of the minimal SE of mean values; ∗∗ p < 0.01 and ∗∗∗ p < 0.001. ZPDm anti-aggregation inhibitory unit, which might be very useful for future Structure activity results in delayed formation of PK-resistant and Th-T positive amyloid structures. Activity Relationship (SAR) studies. Therefore, we synthesized the (trifluoromethyl)benzene moiety and assessed its anti- aggregational potential (Supplementary Figure S1). Both kinetic absent until the 3rd cycle, and they never reach the levels of data using Th-T and light scattering measurements converged to the control samples, although certain adaptation of misfolded α- indicate that this molecule is devoid of any activity, suggesting Syn to ZPDm seems to occur in cycles 4th and 5th (Figure 3B). that it might be necessary, but not sufficient to endorse ZPDm The Th-T fluorescence signal is negligible in the two first PMCA with the above-described anti-aggregation properties. cycles and significantly lower than that of control samples at any considered cycle (Figure 3C). ZPDm Prevents α-Syn Aggregation in Protein Misfolding Cyclic Amplification ZPDm Exhibits Amyloid Disaggregation Assays Activity in vitro We used protein-misfolding cyclic amplification (PMCA) to ZPD-2 does not interact with soluble and monomeric α-Syn and, test the inhibitory capacity of ZPDm under continuous seeding therefore, is not expected to interfere with the protein’s functional conditions. Based on the nucleation-dependent polymerization state. Nuclear Magnetic Resonance 1 H-15 N HSQC spectra of N15 model for prion replication, PMCA is a technique that forces labeled α-Syn in the presence and absence of ZPDm, indicates aggregation to happen by seeding soluble α-Syn with preformed that this is also the case for this smaller molecule since we fibrils (Jung et al., 2017). After the first round of fibril elongation, could not identify any perturbations in chemical shifts or peak aggregates are sonicated and used as seeds for the second cycle of intensities in the spectra (Supplementary Figure S2). PMCA. A representative sample from each cycle is then treated To address the time window in which ZPDm remains active, with proteinase K (PK) and analyzed by SDS-PAGE, to evidence we set up an experiment in which a constant amount of ZPDm fibril formation, since in contrast to soluble α-Syn, the fibrils was added to different aggregation reactions at different time are significantly resistant to proteolysis. Using this protocol, PK- intervals after the reaction has begun (Figure 4A). To our resistant species could be observed already in the 1st cycle of surprise, the respective Th-T signals indicated that the anti- PMCA in untreated samples, with a maximum of PK resistance amyloid activity increased as the reaction progressed, which is at the 4th cycle (Figure 3A). Th-T fluorescence measurements in stark contrast with the behavior of ZPD-2, which was mostly of the same samples indicated that this protection correlates active when added at the early stages of the reaction and inactive with the presence of amyloid-like assemblies (Figure 3C). In when added at the plateau phase (Pena-Diaz et al., 2019). The contrast, in the presence of ZPDm, PK-resistant species are time-dependent activity profile of ZPDm can only be explained Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor if this compound recognizes the Th-T positive aggregated species display poor pharmacokinetics, which should be significantly and exerts an intense fibril disruption activity. To confirm this optimized before they become drugs, small molecules are still the extent, α-Syn mature fibrils were incubated for 24 h with ZPDm. preferred option for the treatment of the diseases caused by the The Th-T fluorescence analysis revealed that treated samples aggregation of proteins within the brain. suffered a signal reduction of 74% (Figure 4B). This data was The screening of large chemical libraries in the search for α- supported by TEM images, which illustrated the disruption of Syn aggregation inhibitors has provided potent molecules like large fibrillar clusters into shorter fibrils or amorphous aggregates anle138b (Wagner et al., 2013), BIOD303 (Moree et al., 2015), (Figures 4C,D). SynuClean-D (Pujols et al., 2018), 582032 (Toth et al., 2019) or We assessed if this amyloid-disrupting activity was the collection of compounds recently reported by Kurnik et al. independent of the conformational properties of the mature (2018). All these molecules display two or more aromatic rings in fibrils, by aggregating α-Syn in 50 mM Tris–HCl pH 7.0 in their structures, a property that is shared by active polyphenols the absence or presence of 150 mM NaCl, which generates like curcumin (Pandey et al., 2008), EGCG (Bieschke et al., two different strains, known as strain B and C, respectively 2010), and baicalein (Jiang et al., 2010), repurposed inhibitors (Bousset et al., 2013; Carija et al., 2019). As shown in Figure 4, like Fasudil (Tatenhorst et al., 2016), and LMTM (Schwab et al., the addition of ZPDm to the mature fibrils of these strains 2017) or compounds generated by rational design like NPT100- promoted a significant decrease in the amount of amyloid-like 18A (Wrasidlo et al., 2016). Usually, the aromatic rings form a material as monitored both by Th-T fluorescence and TEM planar hydrophobic core that is thought to interact with apolar 24 h after the addition of the molecule (Figure 5). These data exposed regions in α-Syn or its assemblies. However, despite the suggest that in contrast to ZPD-2, insensitive to preformed presence of multiple aromatic groups is recurrent in natural α- amyloid fibrils, ZPDm is endorsed with a generic and potent Syn aggregation inhibitors and many of the reported screening disaggregation activity. efforts result in the identification of this kind of molecules, several natural compounds exhibiting a single aromatic ring have been ZPDm Decreases the Formation of α-Syn shown to act as α-Syn aggregation modulators (Supplementary Aggregates in a C. elegans Model of PD Figure S3), including scyllo-inositol, gallic acid, dopamine, We decided to test if the ZPDm in vitro activity can be translated safranal and caffeic acid (Herrera et al., 2008; Di Giovanni in vivo to a simple animal model of PD. To do so, we employed et al., 2010; Liu et al., 2014; Ibrahim and McLaurin, 2016; the well-described strain NL5901 of C. elegans. In this strain, α- Save et al., 2019). Syn is fused to Yellow Fluorescence Protein (YFP) and expressed We recently used a robust high-throughput screening pipeline under the control of the unc-54 promoter, transgene pkIs2386 to uncover molecules able to modulate α-Syn fibrillation (Pujols [Punc-54::α-SYN::YFP] (Hamamichi et al., 2008; van Ham et al., et al., 2017). Among the active compounds, we searched for a 2008), generating protein inclusions in body wall muscle cells. small compound bearing a single aromatic ring. We identified ZPDm was administered in the food at 10 µM final concentration ZPDm, which, interestingly enough, is a minimal version of ZPD- to animals at the L4 stage, and they were examined 7 days later, 2, a potent inhibitor identified in the same library (Pena-Diaz 9 days after hatching (L4+7). These aged worms are intended to et al., 2019), with half of its heavy aromatic atoms. This opened an mimic aged PD patients. We used epifluorescence microscopy opportunity to approach a comparative SAR for these molecules. to visualize the fluorescent aggregates. The images demonstrated ZPDm reduces the in vitro aggregation of WT α-Syn and the that ZPDm reduced the formation of muscular inclusions by protein’s A30P and H50Q familial variants in a 60%, or higher, 43% (Figure 6A), with an average of 20.2 ± 2.13 apparent α- at a 0.7:1 (protein: ZPDm) ratio. This activity was orthogonally Syn aggregates in treated worms (Figure 6C) compared with the confirmed by light-scattering and TEM. Moreover, the inhibitory 35.2 ± 3.04 observed in control samples (Figure 6B). activity of ZPDm reduced the number of PK-resistant and Th- T positive species in PMCA assays, thus interfering with α-Syn templated seeding and/or aggregates amplification (Herva et al., DISCUSSION 2014). It should be explored whether adaptation of α-Syn to ZPDm at late PMCA stages might translate in some resistance The identification of small compounds that may abrogate the to the molecule during aggregates propagation. process of protein aggregation in neurodegenerative disorders Solution NMR measurements indicated that ZPDm was not is attracting increasing interest, both in academia and industry interacting with soluble α-Syn monomers, and, therefore, it (Pujols et al., 2020). is not expected to impact the physiological function of the The lack of structural information about the intermediate protein. Moreover, the addition of ZPDm at different time species that populate the reaction, and the intrinsically disordered points of the aggregation reaction suggested that ZPDm is nature of many of the proteins behind these diseases, has made it mainly active at the latest stages of the aggregation and indeed, challenging to use of structure-guided drug design for amyloid further analysis demonstrated that the molecule is capable of inhibitors. Only recently, the high-resolution structures of the disassembling mature α-Syn amyloid fibrils generated under fibrils formed by proteins connected to different amyloidosis have different solution conditions, conceptually similar to the α- allowed the rational design of peptides that interfere with the Syn strains observed in different synucleinopathies (Bousset growth or seeding of the fibrils (Seidler et al., 2018; Saelices et al., et al., 2013; Peelaerts et al., 2015). Importantly, these features 2019; Sangwan et al., 2020). However, because peptides usually translate into a significant reduction in the number of Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor FIGURE 4 | Disaggregational activity of ZPDm. (A) Th-T fluorescence of α-Syn end-point aggregates after the addition of ZPDm at different time points during the aggregation kinetics. (B) Th-T fluorescence assay before and 24 h after the addition of ZPDm to mature α-Syn fibrils. (C,D) Representative TEM images in the absence (C) and presence (D) of ZPDm. Th-T fluorescence is plotted as normalized means. Error bars are represented as SE of mean values; *p < 0.05 and ***p < 0.001. ZPDm inhibitory capacity increases with the reaction progress, indicating that the compound may interact with aggregated structures and disentangle them. apparent aggregates in the muscular cell wall of a C. elegans would allow interactions with hydrophobic regions, and the model of PD when the compound is added in the food polar groups might disrupt the abundant short inter-strand at a concentration of 10 uM; whether this in vivo anti- hydrogen bonds that contribute to the amyloid structure’s aggregational effect results in animal phenotypic benefits should sustainment. Indeed, despite their different size, the number be further explored. of hydrogen bonds acceptors in ZPD-2 and ZPDm is fairly The above-described results illustrate how despite ZPD-2 and similar (Table 1). Despite speculative, the preferential affinity for ZPDm share a significant part of their chemical structure and early-stage aggregates exhibited by ZPD-2 could be explained both are effective α-Syn aggregation inhibitors, their mechanism by its extended aromatic core and higher Log PO/W , which of action differs significantly, with ZPD-2 acting preferentially at might facilitate interactions with exposed hydrophobic patches the early stages of the fibrillation and becoming inactive once in oligomers and small aggregates. In contrast, the compact the polymerization has advanced significantly, being devoid of structure of ZPDm might allow targeting defined binding detectable fibril disrupting activity. pockets at the ends of amyloid fibrils, interfering with In contrast, ZPDm is more effective at later stages and fibril elongation, and eventually disrupting pre-formed non- behaves as a robust disaggregating agent. The fact that covalent interactions. (trifluoromethyl)benzene is an inactive molecule indicates that From a pharmacokinetic point of view, ZPDm is predicted to the bulk of the structure shared by ZPD-2 and ZPDm acts be more soluble than ZPD-2, to exhibit a higher gastrointestinal as a building block and that the particular chemistry and absorption and better drug-likeness (Table 1). ZPDm is also spatial disposition of the groups that decorate this moiety predicted to be a better lead compound from a medicinal are responsible for the different mode of action of these chemistry perspective than ZPD-2 (Table 1). compounds. For instance, the (trifluoromethyl)benzene contains To the best of our knowledge, the only other active molecule the aromatic ring common to the vast majority of small with a single aromatic ring derived from the screening of a active compounds. However, it lacks a strong hydrogen bond large chemical library is the compound 576755 (Toth et al., donor/acceptor, which is another characteristic common to 2019), which in addition to a benzene ring, displays the many of these molecules (Supplementary Figure S3). Therefore, expected hydrogen bonds acceptors/donors (Supplementary our data suggest that these are the minimum requirements Figure S3). 576755 is a potent aggregation inhibitor that acts for an active α-Syn aggregation inhibitor. The aromatic rings at the oligomerization stage both in vitro and in cells. However, Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor FIGURE 5 | Disaggregational effect of ZPDm in preformed fibrils of two different strains. (A) Strain B aggregates disaggregation in the presence (red) and absence (white) of ZPDm as monitored by Th-T fluorescence. (B,C) Representative TEM images of untreated (B) and ZPDm treated (C) samples. (D) Strain C aggregates disaggregation in presence (red) and absence (white) of ZPDm as monitored by Th-T fluorescence. (E,F) Representative TEM images of untreated (E) and ZPDm treated (F) samples. Data are shown as means, and error bars are shown as the SE of means; ***p < 0.001. The disaggregational ability of ZPDm is also observed in two morphologically different α-Syn strains. FIGURE 6 | Inhibition of α-Syn inclusions formation in a C. elegans model. (A) Quantification of α-Syn muscle inclusions per area in NL5901 worms in the absence (white) and presence of ZPDm (red). (B,C) Representative images of apparent α-Syn muscle aggregates obtained by epifluorescence microscopy of NL5901 worms treated without (B) and with ZPDm (C). (Scale bars, 10 µm). Between 40 and 50 animals were analyzed per condition. Aggregates are indicated by white arrows. Data are shown as means, and error bars are shown as the SE of means; **p < 0.01. NL5901 C. elegans strain forms visible accumulations of aggregated α-Syn that are reduced when ZPDm is administered. it was identified in a screening for compounds that interact Overall, here we describe a new small molecule with the with monomeric α-Syn, and therefore, is not expected to potential to be converted into a lead compound and, perhaps have fibril disrupting activity. Indeed, it did not impact fibril more importantly, together with previous data, envision a way transmission (Toth et al., 2019), consistent with its activity and to design minimal aromatic molecules with different α-Syn anti- being complementary to that of ZPDm, a fibril anti-propagating aggregational activities rationally. Because ZPD-2 and ZPDm agent in PMCA assays. function on the same target but have complementary activity, it Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 October 2020 | Volume 8 | Article 588947 Peña-Díaz et al. A Small α-Synuclein Aggregation Inhibitor will be interesting to test if a combination of them can have a ACKNOWLEDGMENTS synergic effect that overpasses the individual molecules’ potential. We thank the Infraestructura Científica y Técnica Singular NMR facility at Centres Científics i Tecnològics de la DATA AVAILABILITY STATEMENT Universitat de Barcelona for help with NMR, the Servei de Microscòpia at Universitat Autònoma de Barcelona for All datasets presented in this study are included in the their help with TEM, and Anna Villar-Pique for help with article/Supplementary Material. plasmid construction. AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL SV designed the research. SP-D, JP, FP, JST, SN, MC-G, JSC, The Supplementary Material for this article can be found JG, and ED performed the research. SP-D, JP, IP, XS, JSC, online at: https://www.frontiersin.org/articles/10.3389/fbioe. ED, and SV analyzed the data. SP-D, JP, and SV wrote the 2020.588947/full#supplementary-material manuscript. All authors contributed to the article and approved the submitted version. Supplementary Figure 1 | Inhibitory capacity of (trifluoromethyl)benzene. (A) Chemical structures of (trifluoromethyl)benzene, (left) and ZPDm (right). (B) α-Syn aggregation kinetics in the absence (black) and presence (green) of 100 µM of FUNDING (trifluoromethyl)benzene followed by Th-T fluorescence. (C) Light-scattering measurements at 300 and 340 nm, in the absence (white) and presence (green) of (trifluoromethyl)benzene. SV was supported by the Ministerio de Economía y Competitividad (MINECO) (BIO2016-78310-R), the ICREA Supplementary Figure 2 | NMR analysis of ZPDm lack ofinteraction with (ICREA-Academia 2015), and the Fundación La Marató de monomeric α-Syn. 1 H-15 N HSQC NMR spectra of 15N-labeled α-Syn (70 µM) in the presence (A) and in the absence (B) ofZPDm (100 µM). The superposition of TV3 (Ref. 20144330). JSC was supported by the MINECO the two NMR spectra is shown in (C). (BFU2016-78232-P) and the Gobierno de Aragón (E45_17R). ED was supported by the Instituto de Salud Carlos III Supplementary Figure 3 | Chemical structures of different α-Syn aggregation inhibitors with asingle aromatic ring. Chemical structures of (A) scyllo-inositol, (B) (PH613883/ERDF/ESF). 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The use, distribution or reproduction in other forums is permitted, provided 1371/journal.pgen.1000027 the original author(s) and the copyright owner(s) are credited and that the original Wagner, J., Ryazanov, S., Leonov, A., Levin, J., Shi, S., Schmidt, F., et al. publication in this journal is cited, in accordance with accepted academic practice. No (2013). Anle138b: a novel oligomer modulator for disease-modifying therapy use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 12 October 2020 | Volume 8 | Article 588947