Comparison of Î±-synuclein fibril inhibition by four different amyloid

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Comparison of #-synuclein fibril inhibition by four different amyloid inhibitors Narendra Nath Jha, Rakesh Kumar, Rajlaxmi Panigrahi, Ambuja Navalkar, Dhiman Ghosh, Shruti Sahay, Mrityunjoy Mondal, Ashutosh Kumar, and Samir K. Maji ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00261 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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ACS Chemical Neuroscience

Comparison of α-synuclein fibril inhibition by four different amyloid inhibitors Narendra Nath Jha, Rakesh Kumar, Rajlaxmi Panigrahi, Ambuja Navalkar, Dhiman Ghosh, Shruti Sahay, Mritunjoy Mondal, Ashutosh Kumar and Samir. K. Maji*. Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400 076, India.

ABSTRACT: Aggregation of α-synuclein (α-Syn) into toxic oligomers and fibrils leads to Parkinson’s disease (PD) pathogenesis. Molecules that can inhibit the fibrillization and oligomerization of α-Syn have potential therapeutic value. Here, we studied four selective amyloid inhibitors: Dopamine (Dopa), Amphotericin-B (Amph), Epigallocatechingallate (EGCG) and Quinacrinedihydrochloride (Quin) for their effect on oligomerization, fibrillization and preformed fibrils of α-Syn. The aggregation kinetics of α-Syn using ThT fluorescence and conformational transition by circular dichroism (CD) in the presence and absence of these four compounds suggest that, except Quin, remaining three molecules inhibit α-Syn aggregation in concentration dependent manner. In consistence with the aggregation kinetics data, the morphological study of aggregates formed in the presence of these compounds showed corresponding decrease in fibrillar size. The analysis of cell viability using MTT assay showed reduction in toxicity of αSyn aggregates formed in the presence of these compounds, which also correlates with reduction of exposed hydrophobic surface as studied by ANS binding. Additionally, these inhibitors except Quin demonstrated reduction in size as well as the toxicity of oligomeric/fibrillar aggregates of α-Syn. The residue specific interaction to low molecular weight (LMW) species of α-Syn by 2D NMR study revealed that, the region and extent of binding are different for all these molecules. Furthermore, fibril-binding data using SPR suggested that there is no direct relationship between the binding affinity and fibril inhibition by these compounds. The present study suggests that sequence based interaction of small molecules to soluble α-Syn might dictate their inhibition or modulation capacity, which might be helpful in designing modulators of α-Syn aggregation. KEYWORDS: α-synuclein, Parkinson’s disease, Aggregation, Inhibitor, Binding, Toxicity.

INTRODUCTION α-Syn is a small protein (~14 kDa) expressed at high levels in nervous tissue.(1, 2) Abnormal aggregation of α-Syn into cytoplasmic inclusions of Lewy bodies (LBs) and Lewy neuritis (LNs) in specific regions of the brain is the key pathogenic event involved in several neurodegenerative diseases like Parkinson’s disease (PD), multiple system atrophy (MSA) and Lewy body disease (LBD); collectively known as synucleinopathies.(3, 4) Pertaining to its association with disease pathogenesis, α-Syn aggregation has been extensively studied.(4-8) These studies revealed that α-Syn is a natively unfolded protein and has the ability to self-assemble into highly ordered fibrillar aggregates (amyloids) in vitro, closely resembling the α-Syn aggregates found in the LBs extracted from the diseased patients’ brains.(4, 8) Furthermore, it was shown that α-Syn aggregation is not a simple one-step process; it is rather complex involving the structural transformation of the unfolded monomeric protein into several partially folded oligomeric intermediates to the fibrillar aggregates.(7, 9, 10) The fibrillar aggregates of α-Syn in the brain have also been shown to be involved in the progress of disease pathology. Later on, α-Syn oligomers were found to be potent neurotoxic species and proposed to be responsible for neuronal injury and death in PD.(6, 9, 11) Therefore, inhibiting the aggregation of α-Syn into oligomeric and fibrillar species could be one of the key steps in controlling the development of synucleinopathies. A num-

ber of research works have been performed with small molecules of various groups (synthetic and natural polyphenols, lipid formulations and peptides) that can modulate α-Syn aggregation and its toxicity.(12-18) Here, we studied the mechanism of action of some selective amyloid inhibitors (Amph: Amphotericin-B, Dopa: Dopamine, EGCG: Epigallocatechin gallate and Quin: Quinacrinedihydrochloride) for their effectiveness against oligomerization and fibrillization of α-Syn (Figure1A). Amphotericin B is a broad spectrum antibiotic, which is most commonly used for its antifungal activity.(19, 20) It also acts as an immune stimulator and hence posses antiviral, antiprotozoal and antimicrobial activity.(20-22) Recently, antiprion like activities of Amph have been shown in the cell model, but its mechanism of action is unclear.(23) Furthermore, it has been shown that Amph inhibit amyloid formation by Aβ peptides(24) and α-Syn,(25) however, its effects on conformational transition of soluble toxic oligomers of Aβ peptides and α-Syn have not been reported so far. Dopamine is a neurotransmitter that controls motor activities in humans. It has been shown that Dopa inhibits fibril formation and promotes formation of SDS-resistant nonfibrillar α-Syn oligomers.(26-28) The inhibitory effect of dopamine arises due to its oxidative ligation to α-Syn species and thereby preventing conversion of protofibrils to mature fibrils of α-Syn.(28-30) Subsequently, it has been observed that, in pri-

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mary neuronal culture, dopamine induces conformational changes of α-Syn in such a way that the N- and C- termini come close to each other.(31) EGCG is a polyphenolic molecule, which exhibits inhibitory activity on fibrillation of Aβ peptide and α-Syn protein responsible for AD and PD, respectively.(32, 33) EGCG modulates α-Syn fibrils formation pathway by binding directly to natively unfolded α-Syn and stimulates assembly of nontoxic, offpathway oligomers.(34) Recently, it has been shown that EGCG converts large, mature Aβ and α-Syn fibrils into smaller, nontoxic amorphous aggregate;(33, 34) however, its role in disassembly of these amyloid fibrils into monomers or small diffusible oligomers remains unclear. Quinacrine is widely used as an antimalarial, antiprotozoal and anthelmintics medicine.(35, 36) Quin has been shown to exhibit inhibitory effects on prion replication in cells,(36, 37) and also inhibit Aβ fibrillation.(38) Despite its inhibitory effects in the cells, Quin did not show inhibitory activity against prion disorders in mice.(37, 39) Although, the mechanism of inhibition by Quin is not clear, however, it has been shown that addition of multiple copies of acridine moieties on the derived molecule of quinacrine enhances its inhibitory effects against Aβ fibril formation.(38) It follows that EGCG, Dopa, Amph, and Quin are wellknown amyloid inhibitors, among which EGCG, Dopa, and Amph have been studied in terms of their effects on α-Syn aggregation, whereas, effect of Quin on α-Syn aggregation has not been studied so far. Further, as mentioned earlier, α-Syn aggregation is a complex process, hence; the effect of these well known amyloid inhibitors on the different steps of α-Syn aggregation, viz. oligomerization and fibrillization, as well as their associated toxicity needs to be studied in detail. Therefore, in the present study, we selected these known α-Syn aggregation inhibitors (EGCG, Dopa, Amph) as well as other amyloid inhibitor (Quin) and tested their comparative effectiveness (at different concentrations) against α-Syn oligomerization, fibrillization, and its associated toxicity. Our study revealed that Dopa and EGCG at high concentrations slow down the fibrillization kinetics of α-Syn. Amph at all concentrations delayed the fibrillization of α-Syn while Quin has no significant effect on the kinetics of α-Syn aggregation. All the inhibitors studied here, except Quin, reduce the toxicity of αSyn aggregates in concentration dependent manner. Furthermore, the morphology of α-Syn aggregates formed in the presence of highest molar ratio of Amph, Dopa and EGCG showed small fibrillar length. Interestingly, these molecules also showed reduction in toxicity of α-Syn oligomers and preformed fibrils. Our NMR study revealed that the differences in inhibition of α-Syn could be due to selective binding of these inhibitors with the LMW α-Syn. RESULTS AND DISCUSSION Aggregation kinetics of α-Syn in the presence of inhibitors. We studied the aggregation kinetics of α-Syn in the presence and absence of the four different amyloid inhibitors (Amph, Dopa, EGCG and Quin) by using ThT fluorescence assay. For monitoring aggregation kinetics, 300 µM LMW of α-Syn (in 20 mM Gly-NaOH buffer pH 7.4, 0.01% sodium azide) was incubated at 37 °C, with slight agitation in absence and presence of these four inhibitors in different molar ratios (inhibitor:protein molar ratio were of 0.1, 0.25, 0.5, 1.0 and 2.0). The fibrillization kinetics was monitored by ThT fluores-

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cence assay. ThT is a dye, which binds to the β-sheet secondary structure of protein and hence is used to monitor the amyloid aggregation kinetics.(18, 40, 41) Since polyphenolic and conjugated compounds may interfere the ThT fluorescence reading, we have used the same concentration of ThT and the respective compounds in buffer and buffer+ThT as controls. The absorbance spectra and the corresponding fluorescence spectra of all the four inhibitors at highest concentration (20 µM, corresponding to 1:2 molar ratio of α-Syn:inhibitors for ThT fluorescence measurement) in the absence and presence of ThT (10 µM) are shown in figure S1. From the absorbance spectra we found multiple absorbance peaks in each of these compounds. Therefore, we monitored the compound fluorescence in the absence and presence of ThT by exciting at each of the absorptions maxima. The fluorescence spectra of Amph, Dopa and EGCG showed negligible fluorescence in the region of ThT absorbance/fluorescence. However, we observed a significant fluorescence of Quin in the spectral region of ThT (Figure S1). Therefore, during the aggregation kinetics study, we measured the ThT fluorescence of all these control samples kept in separate wells of the 96 well plate and the fluorescence intensity at 482 nm was subtracted from the ThT fluorescence intensity of the respective samples at each time point. Change in ThT fluorescence intensity at 482 nm with respect to time points was plotted and fitted into sigmoidal growth curve. The aggregation kinetics monitored by ThT generally follows sigmoidal growth curve with three different phases: 1st phase corresponding to lag phase, where protein molecules slowly self-associate to form aggregation competent nuclei. The 2nd phase is rapid growth phase where aggregation prone nucleus grows to mature fibrils. The end phase is stationary phase, where fibrils are in equilibrium with monomers.(7, 18) Similar to sigmoidal growth curve of amyloid aggregation, α-Syn aggregation monitored by using ThT binding data showed that at the beginning of aggregation α-Syn did not bind ThT significantly. However with time, α-Syn exhibit increase in ThT fluorescence, which subsequently becomes plateau, showing characteristics of nucleation-dependent polymerization (Figure 1B). From the ThT fluorescence plot, we also measured the lag time of α-Syn aggregation in absence and presence of different molar ratio of all the four molecules by using the equations 1 and 2, mentioned in the experimental section. Lag time of αSyn aggregation in the absence of these four molecules was 39±6 h (Figure 1C, upper panel). Furthermore, ThT fluorescence binding data of α-Syn aggregation in presence of all the molar ratio of Amph did not show any significant increase in intensity during entire aggregation period (Figure 1B). However, a slight increase in ThT fluorescence intensity at the end of aggregation kinetics was observed (180 h). This data suggest that Amph in all concentrations strongly inhibit α-Syn aggregation. In contrast to Amph, α-Syn in presence of lower molar ratio of Dopa (0.1 and 0.25), showed a sigmoidal curve of ThT fluorescence intensity with time, however, their saturation intensity was lesser than that for α-Syn only. Lag times of α-Syn aggregation in presence of 0.1 and 0.25 molar ratios of Dopa were of 45±6 h and 56±7 h, respectively (Figure 1C upper panel). Interestingly at higher molar concentrations, (0.5, 1.0 and 2.0), Dopa inhibited α-Syn aggregation. Similar observation as that of Dopa was obtained for α-Syn aggregation in presence of EGCG, where, in presence of lower molar ratio (0.1 and 0.25); α-Syn was able to aggregates and bind ThT having lesser value of saturation intensity as that of α-Syn only (Figure 1C, lower panel). The lag time in the presence of


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0.1 and 0.25 molar ratio of EGCG were of 58±5 h and 81±6 h, respectively (Figure 1C, upper panel). On the other hand, at higher molar ratio of EGCG, α-Syn aggregation was reduced significantly as evidenced by small value of ThT fluorescence intensity. The α-Syn aggregation in presence of all the used molar ratio of Quin was not affected. The lag times of α-Syn aggregation in the presence of Quin were similar to as that of only α-Syn (Figure 1C, upper panel). Secondary structure and morphology of α-Syn in the presence of different molar ratio of inhibitors. We also monitored the secondary structure of α-Syn in the absence and presence of inhibitors at the initial and final time points of aggregation. To do so, 15 µM of α-Syn in absence and presence of varying concentrations of inhibitors were taken in quartz cuvette and the spectra were acquired in the range of 198 nm - 260 nm.(42) At the beginning of the aggregation kinetics, α-Syn only and α-Syn in presence of all molar ratios of inhibitors showed single negative minimum near ~198 nm in CD spectra, indicating the presence of random coil structure in all the samples (Figure 2A). At the end of aggregation, the CD spectra of α-Syn only showed single negative minima ~218 nm characteristics of β-sheet structure (Figure 2B). α-Syn in the presence of all concentrations of Amph did not show βsheet structure at the end of aggregation kinetics; this is further consistent with the ThT binding data (Figure 1B). CD spectra of α-Syn in the presence of lower concentration of both Dopa and EGCG showed β-sheet structure, while at higher concentrations of Dopa and EGCG; α-Syn did not show β-sheet structure in CD (Figure 2B). α-Syn incubated in the presence of Quin at all molar ratio shows presence of β-sheet structure, indicating that Quin in this concentration range doesn’t have any significant inhibitory effect on the secondary structure transition of α-Syn (Figure 2B). Since, we did not observe the concentration dependent effect of Quin on secondary structure of α-Syn aggregates in CD; therefore, we measured FTIR spectra of α-Syn aggregates formed in the absence and presence of the various molar ratio of Quin (Figure S2). FTIR is one of the commonly used techniques to measure the protein secondary structures and amyloids.(7, 17, 43-45) The FTIR absorbance spectra of 10 µl of 300 µM of α-Syn only and in the presence of various molar ratio of quinacrine were acquired in the region of 1500 cm-1 to 1800 cm-1 and then deconvoluted in the region corresponding to the amide-I region (1600 cm-1 to 1700 cm-1). FTIR spectra of α-Syn aggregates formed in the presence of various molar ratio of Quin showed prominent peaks in the range of 1620 cm-1 to 1639 cm-1 and a small peak after 1685 cm-1 indicating the presence of antiparallel β-sheet structure (Figure S2). Additionally, some peaks were also observed at ~1662 cm-1 and ~1675 cm-1 indicative of the presence of helical and β-turn structures, respectively (Figure S2). At the end of aggregation kinetics, we also performed transmission electron microscopy to monitor the morphology of α-Syn aggregates formed in the presence of lowest (0.1) and highest (2.0) molar ratios of inhibitors. The α-Syn kinetics endproduct formed in the presence of both lowest and highest molar ratio of Amph showed mostly amorphous aggregates (Figure 2C). α-Syn in the presence of Dopa and EGCG at molar ratio of 0.1 showed mostly small and intermediate length fibrils as compared to α-Syn fibrils alone (Figure 2C), while at the molar ratio of 2.0, protofibrillar and oligomeric species were observed (Figure 2C, bottom panel). Quin showed very less effect on the length of α-Syn fibrils formed at the end of aggregation kinetics (Figure 2C).

Toxicity and exposed hydrophobic surface of α-Syn aggregates formed in the presence of inhibitors. To study the effect of these molecules on the toxicity of α-Syn aggregates that formed after aggregation kinetics, we performed MTT reduction assay.(46) MTT assay is one of the commonly used techniques for assaying amyloid toxicity. For MTT assay, αSyn was allowed to aggregates in the presence and absence of different molar ratios of these four molecules. At the end of aggregation kinetics, aggregates were centrifuged from the solution and the concentrations of aggregates were measured by subtracting supernatant concentration from the concentration of protein solution kept for incubation. 10 µM of all these aggregated samples were added to SH-SY5Y cells and incubated for 24 h, after which the assay was performed on incubated cells. The α-Syn aggregates formed alone showed maximum toxicity (minimum cell viability) in comparison to αSyn aggregates formed in presence of these inhibitors (Figure 3A). α-Syn aggregates formed in the presence of Amph showed decrease in toxicity at all the concentration of Amph, however, the extent of decrease was less at 0.1 molar ratio, whereas α-Syn aggregates formed in the presence of 0.1 molar ratio of Dopa and EGCG showed similar extent of toxicity as that of α-Syn alone incubated sample. However, at higher molar ratio of Dopa and EGCG, α-Syn showed gradual increase in cell viability with corresponding increase in their concentrations (Figure 3A). In case of Quin, only at 1.0 and 2.0 molar ratio showed decrease in cytotoxicity of the α-Syn fibrils, while at all other molar ratio (0.1, 0.25 and 0.5), the change in cytotoxicity was insignificant (Figure 3A). It has been shown that exposed hydrophobic surface of the amyloid oligomers is responsible for its toxicity(47) which is further supported by several studies with small molecule modulators of α-Syn aggregation.(16, 17) Therefore, we also measured the hydrophobic surface exposure of α-Syn by ANS fluorescence (Figure 3B). ANS is a dye, which is commonly used to probe the solventexposed hydrophobic surface of the protein.(48, 49) α-Syn with Amph, Dopa and EGCG showed concentration-dependent decrease in ANS fluorescence intensity and the extent of decrease was maximum in presence of Amph with respect to Dopa and EGCG (Figure 3B). At lower molar ratio (0.1) of Dopa and EGCG, ANS fluorescence intensity was similar to that of α-Syn only. Quin at the higher molar ratio (1.0 and 2.0) showed the decrease in ANS fluorescence intensity, while at other molar ratios (0.1, 0.25 and 0.5), showed the similar value of ANS fluorescence intensity as that of α-Syn only (Figure 3B). Overall we observed concentration dependent decrease in toxicity, which could be due to reduction in exposed hydrophobic surface area of the α-Syn aggregates as observed by reduction in the ANS fluorescence intensity. Effect of inhibitors on α-Syn oligomerization. We studied the efficacy of these inhibitors on the oligomerization of α-Syn (early step of aggregation kinetics). For this study, 300 µM of LMW was mixed with an equimolar ratio of inhibitors (300 µM) and incubated at 37 °C for 12 h. To see the effect of these inhibitors on the secondary structure of α-Syn oligomers, CD spectra were acquired (Figure 4A). The α-Syn oligomers formed in the absence and presence of these inhibitors showed random coil-like structure characterized by a single minimum near 200 nm (Figure 4A). We further measured the ThT fluorescence of these oligomers and found maximum fluorescence intensity for α-Syn only and slightly less ThT fluorescence intensity was observed in presence of Amph, Dopa and EGCG (Figure 4B). α-Syn in the presence of Quin showed a similar



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value of ThT fluorescence intensity as that of α-Syn only (Figure 4B). Subsequently, we measured the exposed hydrophobic surface of α-Syn only and α-Syn in the presence of these inhibitors by ANS fluorescence assay. α-Syn only in presence of Quin showed similar extent of ANS fluorescence intensity; while α-Syn incubated in presence of Amph, Dopa and EGCG showed slightly lesser ANS fluorescence intensity (Figure 4C). This change in exposed hydrophobic surface further resulted into altered toxicity of α-Syn on SH-SY5Y cells measured by MTT reduction assay (Figure 4D) and LDH release assay(50) (Figure 4E). Cumulative results of both the assays showed reduced toxicity of α-Syn oligomers formed in the presence of Amph, Dopa and EGCG, whereas Quin did not show this reduction effect (Figure 4D and 4E). Next, we monitored the effect of these inhibitors on the morphology of α-Syn oligomers by using TEM. α-Syn only and in the presence of Quin showed oligomers of similar morphology; while in presence of Dopa and EGCG the oligomers were of smaller size. α-Syn incubated in presence of Amph showed small amorphous aggregates (Figure 4F). The cumulative results of inhibitors on α-Syn oligomerization indicate that, except Quin all of these molecules showed inhibitory effect. Effect of inhibitors on α-Syn fibrils. We further studied the effect of these inhibitors on the preformed α-Syn fibrils. For this study, LMW α-Syn was incubated for fibrillation. The fibril formation was confirmed by the presence of a negative minimum at ~218 nm in CD spectra characteristics of the βsheet secondary structure as well as strong ThT binding as evidenced by high ThT fluorescence intensity. The solution containing α-Syn fibrils was centrifuged at 14,000×g for 1h and the supernatant concentration was measured by UV absorbance at 280 nm. The fibril concentration was measured by subtraction of supernatant concentration from the protein solution kept for incubation. The final concentration of α-Syn fibril was kept constant as 300 µM and the inhibitors were mixed in such a way that the final ratios of inhibitors were 0.1, 0.25, 0.50, 1.0 and 2.0. The eppendorf tubes containing α-Syn fibrils in the presence of inhibitors were incubated in an EchoTherm model RT11 vertical rotor at ~50 rpm, inside a 37 °C incubator. At regular time intervals, an aliquot of samples was taken out and the ThT fluorescence was measured. The ThT plots of α-Syn fibrils incubated in presence of these inhibitors showed decrease in fluorescence intensity with time, however, a negligible decrease in ThT fluorescence intensity was observed in case of α-Syn only (Figure 5). The α-Syn incubated with Amph showed gradual decrease in ThT with time in concentration dependent manner (Figure 5A). Similarly, α-Syn fibrils incubated with Dopa and EGCG showed decrease in ThT fluorescence with time (Figure 5B and 5C). However, this decrease in ThT fluorescence in the case of Dopa and EGCG was less with respect to Amph. Quin also showed decrease in ThT fluorescence, but this decrease in intensity was minimal (Figure 5D). Further, we examined the effect of these inhibitors on exposed hydrophobic surface of preformed α-Syn fibrils by ANS fluorescence. α-Syn fibrils incubated in presence of higher molar ratio of Amph, Dopa and EGCG showed decrease in ANS fluorescence, whereas no such decrease in fluorescence was observed at the lower molar ratios (Figure 6A). Quin did not show any significant change in ANS fluorescence. As, solvent exposed hydrophobicity of amyloids are related with their toxicity(16, 49), therefore, we measured the toxicity of α-Syn fibrils incubated in the presence of these inhibitors by MTT reduction assay (Figure 6B). The cell via-

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bility measured by MTT assay showed Amph, Dopa and EGCG were not able to modulate the toxicity of α-Syn fibrils at the lower molar ratio, however, at higher molar ratio, loss in cytotoxicity ( enhanced cell viability) was observed (Figure 6B). Quin did not show the reduction in cell viability of α-Syn fibrils at all the molar ratio (Figure 6B). Subsequently, we examined the morphology of α-Syn fibrils incubated in presence of highest molar ratio (2.0) of inhibitors by transmission electron microscopy. The TEM images showed the presence of very small and broken fibrils in Amph containing sample (Figure 6C). α-Syn fibrils incubated with Dopa and EGCG also showed reduced fibrillar length, but the effect was less as compared to Amph (Figure 6C). Additionally, incubation with EGCG leads to the appearance of laterally associated short fibrils. In the presence of Quin, the fibrillar morphology was similar to that of α-Syn fibrils in absence of inhibitors (Figure 6C). Interactions of inhibitors with the low molecular weight species of α-Syn. In the present study, we found strong inhibitory effect of Amph followed by Dopa, EGCG while a minimal inhibitory effect of Quin on α-Syn aggregation. Based on these data, we propose that the different extent of modulation/inhibition of α-Syn aggregation by these molecules depends on the mode/extent of binding to α-Syn in soluble form. To delineate the binding mode and residue specific interaction by these molecules, we performed two-dimensional NMR studies of α-Syn in the presence of various molar ratios of these four inhibitor molecules.(16, 45, 51, 52) 300 µM LMW α-Syn was titrated against different concentrations of these small molecules (60 µM to 2.4 mM) and 1H-15N HSQC was recorded for each titration. Significant shifts in the amide cross peaks were observed for the LMW α-Syn in the presence of twice the concentration (600 µM) of Dopa, EGCG and Quin (Figure 7). Though no shift in the amide peaks was observed, a significant decrease in intensity was seen in the peaks of LMW αSyn in presence of Amph (Figure 7). As the concentration of Amph was increased during titration, a gradual decrease in the relative intensity was observed for the residues in the Nterminal and non-amyloidogenic component (NAC) region (Figure 8). The inhibition of α-Syn aggregation even at small concentration of Amph is due to the strong interaction between these region and Amph (Figure 8A). The intensity of the peaks of residues in N-terminal region from 1 to 50 shows a drastic decrease in the peak intensity relative to that of the LMW α-Syn only while residues from 51 to 113 exhibited slight decrease in it. The residues from 113-140 however did not show any significant perturbation or change in intensity indicating that C-terminus of α-Syn does not play any role in this interaction. Interestingly, as shown in figure 8, significant perturbation of the amide cross peaks were observed in the initial region (V3, F4, M5, K6, L8 and S9) and end region (V48, H50, G51 and V52) of N-terminal of LMW α-Syn in presence of 2.0 molar equivalence of Dopa (Figure 8B). A long stretch of residues in the C-terminal of α-Syn (L100A140) showed significant shift during the interactions indicating the involvement of these residues. The shift in the peaks of the C-terminals indicates a weak interaction. Also, disappearance of G51 peak at low concentration of Dopa indicates the strong interaction of this residue with Dopa leading to the perturbation in the nearby residues such as V48, H50 and V52. EGCG showed non-specific interaction with entire sequence of LMW α-Syn (Figure 8C). All the residues showed perturbation in the peaks; the residues from M1-V52 and A89-E137


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showed greater perturbation as compared to that of NAC region. Titration of Quin against LMW α-Syn showed perturbation in the residues in C-terminal of LMW α-Syn (Figure 8D). Quin interacts with LMW α-Syn through L110–A140 residues. Even at very high concentration of Quin (2.4 mM, 8.0 molar equivalence of LMW α-Syn), no other residues of LMW α-Syn showed any interaction. Therefore the N-terminal and the NAC region are free to aggregate and form the β-sheet structure in the presence of even higher concentration of Quin. This explains the inability of the Quin in the inhibition of αSyn aggregation. The NMR data suggest that the extent and mode of binding between the LMW α-Syn and small molecules may dictate the rate of aggregation. Interactions of inhibitors with the preformed fibrils of αSyn. To delineate the relationship of inhibitors binding to fibrils and their effect on α-Syn aggregation, we studied the binding affinity of these inhibitors to the preformed fibrils of α-Syn by using surface plasmon resonance (SPR) spectroscopy.(53) For this study, α-Syn fibrils were immobilized on CM5 chip and the inhibitors were allowed to pass on it in the concentration range of 0.5 µM to 100 µM. The sensorgram of each sample showed increase in response unit with corresponding increase in concentrations of inhibitors. These sensorgram showed distinct pattern of association and dissociation for different inhibitors to the immobilized α-Syn fibrils (Figure 9, top panel). The rate of dissociation in case of Amph and EGCG showed gradual decrease in its response unit with time indicating strong binding with Kd values of 13.5±2.6 and 4.25±0.7 µM, respectively (Figures 9A, 9C and 9E). The response curve of Dopa and Quin do not follow a classical type binding pattern of SPR sensorgram (as observed for Amph and EGCG) and showed steep increase in response unit as well as very fast dissociation indicating non-specific binding with the immobilized fibrils of α-Syn.(54) Moreover, between Dopa and Quin, Dopa sensorgram displayed less increase in RU in contrast to Quin sensorgram, which showed higher increase in RU with concentration. This indicates non-specific binding for both inhibitors, but weak binding for Dopa and strong binding for Quin with preformed α-Syn fibrils (Figure 9B and 9D). The difference in extent of non-specific binding of Dopa and Quin with α-Syn fibril could be due to their molecular size and difference in their hydrophobicity, which eventually dictates their interaction with amyloids. The Quin is more hydrophobic and less hydrophilic compared to Dopa, therefore expected to interact more strongly compared to Dopa with the preformed fibrils (Figure 9B and 9D). Overall, the SPR binding data therefore suggest that there is no apparent correlation between inhibitors interaction with preformed α-Syn fibrils for their aggregation inhibition. CONCLUSION Our study on selective amyloid inhibitors for their effectiveness against oligomerization and fibrillization of α-Syn revealed that Dopa and EGCG at high concentrations slow down the fibrillization kinetics. Amph at all the concentrations delayed the fibrillization of α-Syn, while Quin has no significant effect on the aggregation kinetics of α-Syn. We observed decrease in cytotoxicity of α-Syn aggregates in the presence of higher concentration of these small molecules which could be due to the reduction of exposed hydroponic surface. Furthermore, the α-Syn aggregates formed in the presence of the highest concentration of Amph, Dopa and EGCG showed

small fibrillar length. These molecules (except Quin) also showed inhibitory effect on α-Syn oligomerization. Additionally, Amph, Dopa and EGCG showed reduction in fibrillar length of the preformed α-Syn fibrils. Furthermore, the differences in inhibitory effect on the α-Syn aggregation was found to be due to sequence specific binding of the inhibitor molecules with the soluble form of α-Syn. This notion of sequence based binding of inhibitor molecules to the soluble α-Syn could be executed in designing of therapeutic molecules having inhibitory role in α-Syn aggregation and hence for the treatment of PD. METHODS Chemicals and reagent. All the four small molecule inhibitors (Amphotericin-B, Dopamine, Epigallocatechingallate and Quinacrine) used in this study were purchased from Sigma (St. Louis, MO, USA). Other chemicals used in this study were either purchased from Sigma or from other companies with analytical grade quality. Double distilled and deionised water was obtained from a Milli-Q system (Millipore Corp., Bedford, MA, USA). Protein expression and purification. Wild type (wt) α-Syn protein was expressed and isolated from Escherichia coli BL21 (DE3) strain as described earlier.(16, 55) The purified protein was lyophilized and stored at -20 °C for further use. Preparation of low molecular weight (LMW). Lyophilized α-Syn protein was dissolved in 20 mM Gly-NaOH buffer (pH 7.4, 0.01 % NaN3) at a concentration of 30 mg/ml. Since, the α-Syn is acidic in nature, the pH of the resulting solution was low (~ 6.0) and protein was sparingly soluble in the buffer. In order to dissolve it, few µl of 5M NaOH solution was added to the solution and mixed until the protein got dissolved completely and a clear solution was obtained. Then final pH of the protein solution was adjusted to 7.4 by gradually adding a few µl of 2M HCl. Thereafter, the α-Syn solution was dialyzed overnight using 12 kDa dialysis membranes (Sigma) against 20 mM Gly-NaOH buffer at 4 °C, to remove salt and small protein fragments if any. The overnight dialyzed α-Syn solution was then passed through 100 kDa MWCO filter (centricon YM-100 filter, Millipore, USA) and the resulting flow through solution contained Low Molecular Weight (LMW) species of α-Syn less than 100 kDa. The concentration of the LMW was measured by UV absorbance at 280 nm, using molar absorptivity value of α-Syn as 5960 M-1 cm-1. Aggregation kinetics study. LMW species of α-Syn was used as the starting material for the aggregation studies. For studying the effect of inhibitors on the aggregation kinetics of α-Syn, the LMW form of α-Syn was incubated with 5 different concentrations of each of the 4 inhibitors (EGCG, Dopa, Quin, Amph). Stock solution of all these inhibitors was prepared with concentration of 5 mM in 20 mM Gly-NaOH buffer (pH 7.4, 0.01% NaN3). This stock of 5 mM was further diluted in the same buffer accordingly. For each inhibitor, 5 different samples were prepared where the final concentrations of inhibitors were 30 µM, 75 µM, 150 µM, 300 µM and 600 µM, whereas, the final concentration of α-Syn LMW was fixed to 300 µM in each sample. The concentration of each inhibitor was measured by w/v ratio. The inhibitor: protein molar ratio in each of the 5 samples was thus, 0.1, 0.25, 0.5, 1.0 and 2.0. All the samples were prepared in 1.5 ml eppendorf tubes and the final volume of each sample was 500 µl. As a protein control, sample containing 500 µl of only α-Syn LMW of 300 µM



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concentration was also studied. For inhibitors controls, identical concentrations of only compounds in 20 mM Gly-NaOH buffer (pH 7.4, 0.01% NaN3) were also incubated separately. The eppendorf tubes containing these samples were placed into an Echo-Therm model RT11 vertical rotor (Torrey Pines Scientific, USA) with a speed corresponding to ~50 rpm, inside a 37 °C incubator. The aggregation kinetics of these samples was then monitored as an increase in ThT fluorescence with time. Aliquots at regular time intervals were taken from the samples and diluted in buffer to the final concentration of 10 µM. ThT fluorescence of 10 µM α-Syn in the absence and presence of various concentration of inhibitors was measured in 96 well plates (non-treated, black, clear flat bottom, Corning, catalogue# 3631) by using SpectraMax M2e Multimode Microplate Reader (Molecular Devices, USA). The parameters used for ThT fluorescence were, excitation wavelength of 450 nm, auto cutoff of 475 nm, emission wavelength of 482 nm and 6 flash/read was used for each well. Here we used the same concentration of ThT and the respective compounds in buffer as well as buffer+ThT as controls. During the aggregation kinetics study, we measured the ThT fluorescence of all these control samples kept in separate wells of the 96 well plate at each time point. The ThT fluorescence intensity of control samples at 482 nm was subtracted from the ThT fluorescence intensity of the respective samples containing α-Syn at each time point. The experiment was performed in triplicate wells and three times independently. At the end of aggregation kinetics, average value of ThT fluorescence intensity at 482 nm were plotted with respect to corresponding time points and fitted into sigmoidal growth curve. We also calculated the lag time of α-Syn aggregation for those samples, which showed saturation in ThT fluorescence intensity at the end of aggregation kinetics. The lag time (tlag) was calculated from the ThT fluorescence spectra by using the following equations;(56) = + ( − )/( 1 + (/) )…………… (1) = / − 2/………………… (2) here, “y” is the intensity of ThT fluorescence at time “t” while “y0” is the fluorescence intensity at initial time point. Circular dichroism (CD) spectroscopy. 10 µl of protein solution from stocks kept for incubation was diluted to 200 µl in 20 mM Gly-NaOH buffer (pH 7.4 with 0.01% sodium azide). The solution was placed into a 0.1 cm path-length quartz cell (Hellma, Forest Hills, NY). Spectra were acquired at 25 °C using JASCO-810 instrument (JASCO, Easton, MD 21601, USA). Spectra were recorded at the scan speed of 100 nm/min, over the wavelength range of 198-260 nm; data pitch 1 nm, bandwidth 1 nm, and response time of 1 sec. The raw data were processed by subtraction of buffer spectra followed by smoothing in accordance of the manufacturer’s instructions. Fourier transformed infra red (FTIR) spectroscopy. For FTIR experiments, the KBr pellet was made by compressing KBr powder in a hydraulic pressure pump at the pressure of ~5 ton. The pellet was kept under IR lamp and 10 µl of 300 µM α-Syn fibrils only and α-Syn fibrils formed in the presence of various molar ratio of Quin were spotted and dried. For blank correction, 10 µl of the Gly-NaOH buffer (pH 7.4, 0.01% NaN3) was spotted on another KBr pellet. The FTIR spectra in the range of 1800-1500 cm-1 were recorded at a resolution of 4 cm-1 in Bruker Vertex-80 instrument connected with DTGS detector. The resultant spectrum was recorded as an average of 32 scans for each sample. For secondary structural analysis of amide-I band (1600-1700 cm-1), the Fourier self-deconvoluted

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spectra were subjected to Lorentzian curve fitting using OPUS-65 software. The peaks in the fitted spectra were assigned in accordance with the available report.(17, 43, 44) Transmission electron microscopy (TEM). For TEM imaging, 10 µl of 40 µM α-Syn aggregates formed in the presence of lowest and highest concentrations of inhibitors were spotted on formvar coated copper grid and stained with 1.0% uranyl formate. After air-drying of samples, the images were taken at 6,800 and 8,000 magnifications using a 200 kV transmission electron microscope (PHILIPS, CM-200, Netherlands). ANS (8-Anilinonaphthalene-1-sulfonate) fluorescence. For ANS fluorescence study, 2 µl of ANS from 5 mM stock was added to 200 µl of 10 µM of α-Syn and mixed into a quartz cuvette of 1 cm path-length. Then the samples were excited at 370 nm and the ANS emission spectra were recorded from 400 nm to 600 nm using a spectrofluorometer (FluoroMax-4, HORIBA Jobin Yvon). Both excitation and emission slit widths for ANS fluorescence was kept at 5 nm. The ANS fluorescence intensity at 470 nm from three independent set of experiments was plotted for each sample. MTT reduction assay. MTT reduction assay was performed on neuroblastoma cell line “SH-SY5Y”, cultured in DMEM (Dulbecco’s modified Eagle's medium) supplemented with 10% FBS, at 37 °C and 5% CO2. Cells were seeded at a density of ~10,000 cells/well in 96-well plate. After 16 h of incubation, the spent media was removed and 100 µl of fresh media containing 10 µM of α-Syn only and α-Syn in the presence of various concentrations of these inhibitors were added. After 24 h of incubation with samples, 10 µl of 5 mg/ml MTT solution was added to each well and then further incubated for 4 h. At the end, 100 µl of the lysis solution (50% dimethylformamide and 20% SDS, pH 4.8) was added and kept for overnight incubation. The absorption values at 570 nm and 690 nm were determined with plate reader (M2e, ThermoFisher Scientific). The absorption spectrum of formazan (reduced MTT) falls in between 450 nm to 650 nm. Therefore, absorption at wavelength above 650 nm could be used as reference wavelength for the absorption of various components present in the solution. Here, we used 690 nm as background absorption wavelength. Therefore absorbance at 690 nm (background absorption, eliminating the effect of scattering by the cell debris) was subtracted from the 570 nm for each well and the final value was converted to % cell viability by considering 100% viability for the buffer control. The average value with standard error of mean (SEM) of % cell viability from triplicate wells of three independent experiments was plotted for each sample. Lactate dehydrogenase (LDH) release assay. The LDH release assay was performed on SH-SY5Y cells in 96 well plates. For LDH release assay, 10 µM of α-Syn oligomers/fibrils formed/incubated in absence and presence of various concentrations of inhibitors were added to SH-SY5Y cells and incubated for 24 h (5% CO2 at 37 °C). The extent of LDH released to the medium was measured by LDH toxicological kit (TOX- 7, Sigma) according to the protocol provided with the kit. The absorbance at 490 nm and 690 nm were measured using SpectraMax M2e microplate reader (Molecular Devices, USA). The difference in absorbance of 490 nm and 690 nm (background absorption, eliminating the effect of scattering by the cell debris) was plotted as % LDH release, assuming 100% release in case of TritonX-100 treated cells. The average value


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with SEM of % LDH release from triplicate wells of three independent experiments was plotted for each sample. Nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra were recorded on a Bruker Ascend 750 MHz spectrometer with 5 mm TXI probe at 15 °C in Gly-NaOH buffer (pH 7.4). Two-dimensional 1H-15N correlation heteronuclear single quantum coherence (HSQC) spectroscopy was recorded to study the interaction of small molecules: Amph, Dopa, EGCG and Quin with LMW α-Syn. 300 µM of LMW α-Syn was titrated against different concentrations of these small molecules (60 µM to 2400 µM). 20 mM stock concentration of small molecules in 20 mM Gly-NaOH was used here in order to minimize the change in volume of the sample due to addition of compounds. The data thus obtained were volume corrected and processed by using Topspin 3.2 software followed by analysis by using CcpNMR (Collaborative Computational Project for NMR) software. Peaks were assigned using the assignments of α-Syn published by 1H-15N chemical shift values and by reduced dimensionality experiments.(51, 52) Chemical shift perturbation (CSP) was calculated by using CSP = δH + (0.1δN) ), δH and δN are difference in the chemical shift of proton and nitrogen respectively. Surface plasmon resonance (SPR) spectroscopy. The kinetics of interaction between these small molecule inhibitors to α-Syn fibrils were determined by surface plasmon resonance (SPR) spectroscopy (BIAcore T200, GE Healthcare, USA). 2 mg/ml of α-Syn fibrils in 10 mM sodium acetate buffer pH 4.5 was immobilized on CM5 sensor chip (immobilization level ~ 1350 RU). All the inhibitors in the concentration range of 0.5 µM to 100 µM were injected at the flow rate of 30 µl/min (10 mM phosphate buffer, pH 7.4). For each sample the contact time and dissociation time were 90 sec and 600 sec respectively. The response units of reference surface and the blank run (without any analyte) were subtracted from response unit of samples. The resultant response unit for each sample was fitted in steady-state two-step model and the respective kd values were determined.(53) Statistical analysis. For statistical significance calculation, one-way ANOVA and Student-Newman-Keuls Multiple Comparison post hoc test was used, *P≤0.05, **P≤0.01, ***P≤0.001; NS P>0.05.

ASSOCIATED CONTENT The supporting information contains figure S1 and figure S2. Figure S1 shows absorbance and fluorescence spectra of 20 µM of all these inhibitors. Figures S2 illustrate the secondary structure of α-Syn aggregates formed in absence and presence of Quin, determined by FTIR. It is attached as a separate file.

AUTHOR INFORMATION Corresponding Author Dr. Samir K. Maji Associate Professor Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai 400 076, India Tel: +(91-22) 2576-7774 Fax: +(91-22) 2572 3480 Email: [email protected]. Author Contributions

SKM and NNJ designed the research. All the authors except SKM performed the experiments. NNJ and SKM wrote the manuscript. The final version of the manuscript was approved by all the authors. There is no conflict of interest among the authors.

ACKNOWLEDGMENT Authors would like to thank Department of Biotechnology, India, for financial support with grant no “BT/PR14344Med/30/501/2010”. Authors wish to acknowledge SAIF (IIT Bombay) for transmission electron microscopy and IRCC (IIT Bombay) for NMR and SPR spectroscopy facilities. Narendra Nath Jha is thankful to council of scientific and industrial research (CSIR), India, for research fellowship (Government of India).

REFERENCES 1.

Wakabayashi, K., Yoshimoto, M., Tsuji, S., and Takahashi, H. (1998) α-synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy, Neurosci Lett. 249, 180182. 2. Lucking, C. B., and Brice, A. (2000) α-synuclein and Parkinson's disease, Cell Mol. Life Sci. 57, 1894-1908. 3. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) α-synuclein in Lewy bodies, Nature 388, 839-840. 4. Baba, M., Nakajo, S., Tu, P. H., Tomita, T., Nakaya, K., Lee, V. M., Trojanowski, J. Q., and Iwatsubo, T. (1998) Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies, Am J Pathol. 152, 879-884. 5. El-Agnaf, O. M., Salem, S. A., Paleologou, K. E., Cooper, L. J., Fullwood, N. J., Gibson, M. J., Curran, M. D., Court, J. A., Mann, D. M., Ikeda, S., Cookson, M. R., Hardy, J., and Allsop, D. (2003) α-synuclein implicated in Parkinson's disease is present in extracellular biological fluids, including human plasma, FASEB J 17, 1945-1947. 6. Karpinar, D. P., Balija, M. B., Kugler, S., Opazo, F., RezaeiGhaleh, Wender, N., Kim, H., Taschenberger, G., Falkenburger, B. H., Heise, H., Kumar, A., Riedel, D., Fichtner, L., Voigt, A., Braus, G. H., Giller, K., Becker, S., Herzig, A., Baldus, M., Jackle, H., Eimer, S., Schulz, J. B., Griesinger, C., and Zweckstetter, M. (2009) Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models, EMBO J 28, 3256-3268. 7. Ghosh, D., Singh, P. K., Sahay, S., Jha, N. N., Jacob, R. S., Sen, S., Kumar, A., Riek, R., and Maji, S. K. (2015) Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation, Sci. Rep. 5, 9228. 8. Cookson, M. R. (2005) The biochemistry of Parkinson's disease, Annu Rev Biochem. 74, 29-52. 9. Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson, R. E., and Lansbury, P. T., Jr. (2000) Acceleration of oligomerization, not fibrillization, is a shared property of both α-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy, Proc. Natl. Acad. Sci. U.S.A. 97, 571-576. 10. Uversky, V. N., Li, J., and Fink, A. L. (2001) Evidence for a partially folded intermediate in α-synuclein fibril formation, J Biol Chem. 276, 10737-10744. 11. Winner, B., Jappelli, R., Maji, S. K., Desplats, P. A., Boyer, L., Aigner, S., Hetzer, C., Loher, T., Vilar, M., Campioni, S., Tzitzilonis, C., Soragni, A., Jessberger, S., Mira, H., Consiglio, A., Pham, E., Masliah, E., Gage, F. H., and Riek, R. (2011) In vivo demonstration that α-synuclein oligomers are toxic, Proc. Natl. Acad. Sci. U.S.A. 108, 4194-4199. 12. Madine, J., Doig, A. J., and Middleton, D. A. (2008) Design of an N-methylated peptide inhibitor of α-synuclein aggregation guided by solid-state NMR, J Am Chem Soc. 130, 7873-7881.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

13. Ono, K., and Yamada, M. (2006) Antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for αsynuclein fibrils in vitro, J Neurochem. 97, 105-115. 14. Braga, C. A., Follmer, C., Palhano, F. L., Khattar, E., Freitas, M. S., Romao, L., Di Giovanni, S., Lashuel, H. A., Silva, J. L., and Foguel, D. (2011) The anti-Parkinsonian drug selegiline delays the nucleation phase of α-synuclein aggregation leading to the formation of nontoxic species, J Mol Biol. 405, 254-273. 15. Meng, X., Munishkina, L., Fink, A., and Uversky, V. N. (2010) Effects of Various Flavonoids on the α-Synuclein Fibrillation Process, Parkinsons Dis. 2010, 650794. 16. Singh, P. K., Kotia, V., Ghosh, D., Mohite, G. M., Kumar, A., and Maji, S. K. (2013) Curcumin modulates α-synuclein aggregation and toxicity, ACS Chem Neurosci. 4, 393-407. 17. Jha, N. N., Ghosh, D., Das, S., Anoop, A., Jacob, R. S., Singh, P. K., Ayyagari, N., Namboothiri, I. N., and Maji, S. K. (2016) Effect of curcumin analogs on α-synuclein aggregation and cytotoxicity, Sci Rep. 6, 28511. 18. Ghosh, D., Dutta, P., Chakraborty, C., Singh, P. K., Anoop, A., Jha, N. N., Jacob, R. S., Mondal, M., Mankar, S., Das, S., Malik, S., and Maji, S. K. (2014) Complexation of amyloid fibrils with charged conjugated polymers, Langmuir 30, 3775-3786. 19. Matsumoto, T., Kaku, M., Furuya, N., Usui, T., Kohno, S., Tomono, K., Tateda, K., Hirakata, Y., and Yamaguchi, K. (1993) Amphotericin B-induced resistance to Pseudomonas aeruginosa infection in mice, J Antibiot. 46, 777-784. 20. Hartsel, S., and Bolard, J. (1996) Amphotericin B: new life for an old drug, Trends Pharmacol Sci. 17, 445-449. 21. Konopka, K., Guo, L. S., and Duzgunes, N. (1999) Anti-HIV activity of amphotericin B-cholesteryl sulfate colloidal dispersion in vitro, Antiviral Res. 42, 197-209. 22. Maesaki, S. (2002) Drug delivery system of anti-fungal and parasitic agents, Curr Pharm Des. 8, 433-440. 23. Mange, A., Milhavet, O., McMahon, H. E., Casanova, D., and Lehmann, S. (2000) Effect of amphotericin B on wild-type and mutated prion proteins in cultured cells: putative mechanism of action in transmissible spongiform encephalopathies, J Neurochem. 74, 754-762. 24. Smith, N. W., Annunziata, O., and Dzyuba, S. V. (2009) Amphotericin B interactions with soluble oligomers of amyloid Aβ1-42 peptide, Bioorg Med Chem. 17, 2366-2370. 25. Masuda, M., Suzuki, N., Taniguchi, S., Oikawa, T., Nonaka, T., Iwatsubo, T., Hisanaga, S., Goedert, M., and Hasegawa, M. (2006) Small molecule inhibitors of α-synuclein filament assembly, Biochemistry 45, 6085-6094. 26. Leong, S. L., Cappai, R., Barnham, K. J., and Pham, C. L. (2009) Modulation of α-synuclein aggregation by dopamine: a review, Neurochem Res. 34, 1838-1846. 27. Cappai, R., Leck, S. L., Tew, D. J., Williamson, N. A., Smith, D. P., Galatis, D., Sharples, R. A., Curtain, C. C., Ali, F. E., Cherny, R. A., Culvenor, J. G., Bottomley, S. P., Masters, C. L., Barnham, K. J., and Hill, A. F. (2005) Dopamine promotes αsynuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway, FASEB J 19, 1377-1379. 28. Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Kinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adduct, Science 294, 1346-1349. 29. Lee, H. J., Baek, S. M., Ho, D. H., Suk, J. E., Cho, E. D., and Lee, S. J. (2011) Dopamine promotes formation and secretion of non-fibrillar α-synuclein oligomers, Exp Mol Med. 43, 216222. 30. Nakaso, K., Tajima, N., Ito, S., Teraoka, M., Yamashita, A., Horikoshi, Y., Kikuchi, D., Mochida, S., Nakashima, K., and Matsura, T. (2013) Dopamine-mediated oxidation of methionine 127 in α-synuclein causes cytotoxicity and oligomerization of αsynuclein, PLoS One 8, e55068. 31. Herrera, F. E., Chesi, A., Paleologou, K. E., Schmid, A., Munoz, A., Vendruscolo, M., Gustincich, S., Lashuel, H. A., and Carloni, P. (2008) Inhibition of α-Synuclein Fibrillization by

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43. 44. 45.

46.

47.

48.

49.

Page 8 of 20

Dopamine Is Mediated by Interactions with Five C-Terminal Residues and with E83 in the NAC Region, PLoS One 3, e3394 ]. Grelle, G., Otto, A., Lorenz, M., Frank, R., Wanker, E., and Bieschke, J. (2011) Black tea theaflavins inhibit formation of toxic amyloid-β and α-synuclein fibrils, Biochemistry 50, 1062410636. Bieschke, J., Russ, J., Friedrich, R., Ehrnhoefer, D., Wobst, H., Neugebauer, K., and Wanker, E. E. (2010) EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity, Proc. Natl. Acad. Sci. U.S.A. 107, 7710-7715. Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R., Engemann, S., Pastore, A., and Wanker, E. E. (2008) EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers, Nat Struct Mol Biol. 15, 558-566. Baird, J. K. (2011) Resistance to chloroquine unhinges vivax malaria therapeutics, Antimicrob Agents Chemother. 55, 18271830. Klingenstein, R., Lober S Fau - Kujala, P., Kujala, P., Godsave, S., Leliveld, S., Gmeiner, P., Peters, P., and Korth, C. (2006) Tricyclic antidepressants, quinacrine and a novel, synthetic chimera thereof clear prions by destabilizing detergent-resistant membrane compartments, J Neurochem. 98, 748-759. May, B. C., Fafarman, A. T., Hong, S. B., Rogers, M., Deady, L. W., Prusiner, S. B., and Cohen, F. E. (2003) Potent inhibition of scrapie prion replication in cultured cells by bis-acridines, Proc. Natl. Acad. Sci. U.S.A. 100, 3416-3421. Dolphin, G. T., Chierici, S., Ouberai, M., Dumy, P., and Garcia, J. (2008) A multimeric quinacrine conjugate as a potential inhibitor of Alzheimer's β-amyloid fibril formation, Chembiochem. 9, 952-963. Collins, S. J., Lewis, V., Brazier, M., Hill, A. F., Fletcher, A., and Masters, C. L. (2002) Quinacrine does not prolong survival in a murine Creutzfeldt-Jakob disease model, Ann Neurol. 52, 503-506. LeVine, H., 3rd. (1999) Quantification of β-sheet amyloid fibril structures with thioflavin T, Methods Enzymol. 309, 274284. Sahay, S., Ghosh, D., Dwivedi, S., Anoop, A., Mohite, G. M., Kombrabail, M., Krishnamoorthy, G., and Maji, S. K. (2015) Familial Parkinson disease-associated mutations alter the sitespecific microenvironment and dynamics of α-synuclein, J Biol Chem. 290, 7804-7822. Brahms, S., and Brahms, J. (1980) Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism, J Mol Biol. 138, 149-178. Barth, A. (2007) Infrared spectroscopy of proteins, Biochim Biophys Acta. 1767, 1073-1101. Hiramatsu, H., and Kitagawa, T. (2005) FT-IR approaches on amyloid fibril structure, Biochim Biophys Acta. 1753, 100-107. Ghosh, D., Mondal M Fau - Mohite, G. M., Mohite Gm Fau Singh, P. K., Singh Pk Fau - Ranjan, P., Ranjan P Fau - Anoop, A., Anoop A Fau - Ghosh, S., Ghosh S Fau - Jha, N. N., Jha Nn Fau - Kumar, A., Kumar A Fau - Maji, S. K., and Maji, S. K. (2013) The Parkinson's disease-associated H50Q mutation accelerates α-Synuclein, Biochemistry 52, 6925-6927 Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J Immunol Methods 65, 55-63. Campioni, S., Mannini, B., Zampagni, M., Pensalfini, A., Parrini, C., Evangelisti, E., Relini, A., Stefani, M., Dobson, C. M., Cecchi, C., and Chiti, F. (2010) A causative link between the structure of aberrant protein oligomers and their toxicity, Nat Chem Biol. 6, 140-147. Cardamone, M., and Puri, N. K. (1992) Spectrofluorimetric assessment of the surface hydrophobicity of proteins, Biochem J 282 ( Pt 2), 589-593. B, B., Kumita, J. R., Barros, T. P., Esbjorner, E. K., Luheshi, L. M., Crowther, D. C., Wilson, M. R., Dobson, C. M., Favrin, G.,


Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

50.

51.

52.

53.

54.

55.

56.

ACS Chemical Neuroscience and Yerbury, J. J. (2010) ANS binding reveals common features of cytotoxic amyloid species, ACS Chem Biol. 5, 735-740. Behl, C., Davis, J. B., Lesley, R., and Schubert, D. (1994) Hydrogen peroxide mediates amyloid-β protein toxicity, Cell 77, 817-827. Reddy, J. G., and Hosur, R. V. (2014) A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins, J Biomol NMR 59, 199-210. Ranjan, P., Ghosh, D., Yarramala, D. S., Das, S., Maji, S. K., and Kumar, A. (2017) Differential copper binding to α-synuclein and its disease-associated mutants affect the aggregation and amyloid formation, Biochim Biophys Acta. 1861, 365-374. Ghosh, D., Sahay, S., Ranjan, P., Salot, S., Mohite, G. M., Singh, P. K., Dwivedi, S., Carvalho, E., Banerjee, R., Kumar, A., and Maji, S. K. (2014) The newly Discovered Parkinson’s Disease Associated Finnish Mutation (A53E) Attenuates αSynuclein Aggregation and Membrane Binding, Biochemistry 53, 6419-6421. Kamatari, Y. O., Hayano, Y., Yamaguchi, K., Hosokawa-Muto, J., and Kuwata, K. (2013) Characterizing antiprion compounds based on their binding properties to prion proteins: implications as medical chaperones, Protein Sci. 22, 22-34. Volles, M. J., and Lansbury, P. T., Jr. (2007) Relationships between the sequence of α-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity, J Mol Biol. 366, 1510-1522. Willander, H., Presto, J., Askarieh, G., Biverstal, H., Frohm, B., Knight, S. D., Johansson, J., and Linse, S. (2012) BRICHOS Domains Efficiently Delay Fibrillation of Amyloid β-Peptide, J Biol Chem. 287, 31608-31617.



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Figure 1. Chemical structures of inhibitors and kinetics of α-Syn aggregation in the presence of various molar ratios of these inhibitors. (A) Chemical structures of all the four inhibitors (Amphotericin B, Dopamine, EGCG and Quinacrine) used in this study. (B) ThT fluorescence as a function of incubation time for α-Syn only and α-Syn in the presence of various concentrations of inhibitors. (C) Lag time (upper panel) and maximum ThT fluorescence intensity (lower panel) of α-Syn in the presence and absence of various concentrations of inhibitors. Figure 2. Secondary structure and morphology of α-Syn aggregates formed in presence of various concentrations of inhibitors. CD spectra of α-Syn only and α-Syn in presence of various molar ratios of Amph, Dopa, EGCG and Quin at the beginning (A); and end (B) of aggregation kinetics. (C) Electron microscopic images of α-Syn amyloid fibrils formed in presence of lowest (0.1) and highest (2) molar ratios of Amph, Dopa, EGCG and Quin at the end of aggregation kinetics. Scale: 200 nm. Figure 3. Toxicity and exposed hydrophobic surface area of α-Syn aggregates. (A) MTT assay of α-Syn aggregates formed in the absence and presence of various molar ratios of inhibitors. Bar graph of MTT represents mean±SE from triplicate wells of three independent sets of experiments. (B) ANS fluorescence of α-Syn aggregates at the end of aggregation kinetics. Bar graph of ANS fluorescence represents mean±SE from three independent sets of experiments. Figure 4. Efficacy of inhibitors on α-Syn oligomerization. (A) The secondary structure of α-Syn oligomers formed in the presence of equal concentration of inhibitors. (B) ThT fluorescence of α-Syn oligomers formed in presence of equimolar ratio of inhibitors. (C) ANS fluorescence of α-Syn oligomers formed in presence of equimolar ratio of inhibitors. Bar graph of ThT and ANS fluorescence represents mean±SE from three independent sets of experiments. (D) MTT cell viability assay of SH-SY5Y in presence of αSyn oligomers formed with an equimolar ratio of Amph, Dopa, EGCG and Quin. (E) LDH release assay for α-Syn oligomers formed in presence of equimolar ratio of inhibitors. The bar diagram of MTT and LDH assay represents mean±SE from triplicate wells of three independent set of experiments. (F) Morphology of α-Syn oligomers formed in the presence of equimolar ratio of inhibitors. Scale bar = 200 nm. Figure 5. ThT fluorescence of preformed α-Syn fibrils incubated with different molar ratio of inhibitors. ThT fluorescence monitored at regular time interval showed decrease in intensity with corresponding increase in molar ratio of Amph(A), Dopa (B) and EGCG (C), while Quin (D) showed minimal effect. Figure 6. Hydrophobic surface exposure, toxicity and morphology of preformed α-Syn fibrils in absence and presence of inhibitors. (A) Exposed hydrophobic surface of preformed α-Syn fibrils incubated for 4 days in the presence of different molar ratio of the inhibitors was measured by ANS fluorescence. (B) Toxicity of preformed α-Syn fibrils incubated for 4 days in presence of different molar ratio of the inhibitors was measured by MTT reduction assay. (C) Electron micrograph of preformed α-Syn fibrils after days of incubation in presence of 1:2 molar ratio of the inhibitors. The extent of reduction in fibrillar length was highest for Amph, followed by Dopa and EGCG, while Quin showed no effect. Scale bar = 200 nm. Figure 7. 1H-15N HSQC NMR spectra of 300 µM of LMW α-Syn (red), in presence of 600 µM of Amph (navy blue), 600 µM of Dopa (blue), 600 µM of EGCG (green) and 600 µM of Quin (purple). The HSQC spectra of α-Syn with Amph showed decrease in intensity of some of the peaks while in presence of Dopa, EGCG and Quin shift in peak positions were observed. Figure 8. (A) Intensity (I/I0) profile of cross amide peaks obtained from 1H-15N HSQC spectra of LMW α-Syn in the presence of different equivalence of Amph (0.2: pink; 0.4:green; 1.0: blue and 2.0: red). Chemical Shift Perturbation (CSP) of residues of LMW α-Syn in presence of different equivalence of Dopa (0.2: pink; 0.4: green; 1.0: blue and 2.0: red) (B); in presence of different equivalence of EGCG (0.2: pink; 0.4: green; 1.0: blue and 2.0: red) (C); and in presence of different equivalence of Quin (0.2: pink; 0.4:green; 1.0: blue and 2.0: red) (D). Figure 9. SPR sensorgram showing interaction of Amph, Dopa, EGCG and Quin with preformed α-Syn fibrils. (A) Amph showing small response unit with slow rate of dissociation, (B) Dopa showing weak interaction with abrupt association and dissociation, (C) EGCG showing strong binding with slow rate of dissociation and (D) Quin showing very high response unit with abrupt dissociation indicating nonspecific binding with preformed α-Syn fibrils. The lower panel represents the corresponding saturation plot for each inhibitor. (E) Dissociation constant (kd) values of different inhibitors for their interaction with the preformed α-Syn fibrils.


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For Table of Contents Use Only

Comparison of α-synuclein fibril inhibition by four different amyloid inhibitors Narendra Nath Jha, Rakesh Kumar, Rajlaxmi Panigrahi, Ambuja Navalkar, Dhiman Ghosh, Shruti Sahay, Mritunjoy Mondal, Ashutosh Kumar and Samir. K. Maji*. Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400 076, India.


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Comparison of Î±-synuclein fibril inhibition by four different amyloid

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