ABSTRACT The formation of ordered amyloid fibrils by proteins and polypeptides is associated with human disorders. A recent extension of the amyloidogenic building block family includes

several small metabolites, which form assemblies with structural and functional similarities to well-established amyloids. Here we investigate whether generic amyloid polyphenolic inhibitors

can also restrict the formation of metabolite fibrils. We reveal that epigallocatechin gallate and tannic acid inhibit amyloid-like fibrillation of adenine, phenylalanine, and tyrosine.

Moreover, the compounds reduce the cytotoxicity triggered by these assemblies. In contrast, acetylsalicylic acid, used as a control does not have an inhibitory effect. The compounds’

inhibition process and dynamics of association. SIMILAR CONTENT BEING VIEWED BY OTHERS GALLIC ACID OXIDATION PRODUCTS ALTER THE FORMATION PATHWAY OF INSULIN AMYLOID FIBRILS Article Open

access 02 September 2020 TUNING THE RATE OF AGGREGATION OF HIAPP INTO AMYLOID USING SMALL-MOLECULE MODULATORS OF ASSEMBLY Article Open access 24 February 2022 STRUCTURAL BASIS FOR THE

INHIBITION OF IAPP FIBRIL FORMATION BY THE CO-CHAPERONIN PREFOLDIN Article Open access 02 May 2022 INTRODUCTION Amyloid aggregates are well-ordered β-sheet-rich supramolecular structures,

formed by the self-assembly of a wide class of proteins and polypeptides, which do not exhibit simple structural or functional similarities1,2,3,4,5. This group of proteins and polypeptides

is associated with a family of amyloid diseases, related to intra- and extra-cellular protein-misfolding processes, which result in the formation of deposits and plaques and lead to cell

death. There are currently > 30 known amyloid-associated disorders, including common neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and prion

disorders4,6,7,8,9,10. Amyloid assemblies possess a common set of biological, chemical, physical, and ultrastructural characteristics, including a twisted and elongated fibrillar morphology

with a diameter of 5–20 nm3,11,12. The formed fibrils are known to interact with amyloid-specific dyes, such as thioflavin-T (ThT), owing to their β-sheet-rich secondary structure, resulting

in a typical fluorescence signature that can be monitored in a time-dependent manner13,14. Although several potential therapeutic approaches for ameliorating amyloidogenic diseases

currently exist, one of the more promising approaches allows the inhibition of amyloid formation by small-molecule inhibitors. This approach is supported by the propensity of small molecules

to specifically interact with amyloid assemblies at the molecular level and, thus, inhibit the self-assembly process15,16,17,18. Polyphenols, an important group of chemical entities found

to be effective in the inhibition of amyloid structures formation, are composed of one or more small aromatic phenolic rings that specifically and efficiently inhibit amyloid aggregation.

High concentrations of natural polyphenols can be found in a wide variety of plants, where they function in protection against diseases and UV light. This family of molecules, which

represent the first generation of amyloid-based potential therapeutic agents, cause a dramatic reduction in amyloidogenic-related cell death, as well as efficient inhibition of amyloid

self-assembly in vitro. Moreover, dietary polyphenols have shown beneficial health-promoting effects in chronic and neurodegenerative diseases19,20,21. By employing a reductionist approach

aimed to identify the minimal assembly units required for the formation of ordered amyloid aggregates, it was demonstrated that penta- and tetra- peptides can form well-ordered amyloid

assemblies, which were also found to be cytotoxic via an apoptotic cell death pathway22,23. Interestingly, in aqueous solution, the diphenylalanine dipeptide (FF), the core recognition motif

of the β-amyloid polypeptide, was also found to self-assemble into ordered nanotubular assemblies24,25, which show similarities to amyloids in terms of their mechanical rigidity, formation

of reactive oxygen species and luminescence properties26,27,28,29. Thus, minimal building blocks assemblies, composed of peptides as short as dipeptides, reflect some of the fundamental

physical properties of full-length amyloid proteins and polypeptides (Fig. 1a). Further studies, presented by our group and extended by others, have shown that even the single phenylalanine

amino acid can self-assemble to form well-ordered supramolecular amyloid-like assemblies (Fig. 1a), possessing the common biophysical, biochemical, and ultrastructural amyloid

properties30,31,32,33,34. Moreover, these assemblies display a cytotoxic effect on a neuronal cell model, which can be depleted by antibodies specifically raised against the assemblies30,32.

Interestingly, in pathological conditions, a physiological accumulation of phenylalanine can be found in patients of the genetic phenylketonuria (PKU) inborn error of metabolism disorder35.

The presence of antibodies raised against the phenylalanine fibrils in a PKU mouse model, as well as the presence of phenylalanine aggregate deposits in PKU patients’ brains _post mortem_,

indicate a possible association between PKU and phenylalanine amyloid-like assemblies30. To further extend the new paradigm linking genetic metabolic disorders to amyloid diseases, we have

recently revealed the ability of other amino acids and small metabolites, which were found to accumulate in the course of different inborn error of metabolism disorders, to self-assemble

into amyloid-like fibrillary structures. These metabolite-based fibrils were shown to bind amyloid-specific dyes, further implying on the formation of β-sheet supramolecular structures, and,

similar to protein and peptide amyloids, to trigger cytotoxicity via an apoptotic cell death mechanism36,37. Each error of metabolism disorder is derived from a mutation in a single gene

encoding a metabolic enzyme, thereby resulting in the accumulation of metabolites corresponding to the substrates of the faulty enzymes. These accumulated substrates can be toxic to cells

and tissues, and thus cause severe abnormalities. Although these inherited disorders are relatively rare, collectively they constitute a very significant portion of pediatric genetic

diseases38,39. Up until recently, the pathology and course of toxicity have been poorly understood, leading to the lack of a viable course of treatment, relying mainly on very restrictive

diets. Given the amyloid-like properties of small metabolite assemblies and the ability of known amyloid inhibitors to inhibit the formation of phenylalanine32,33,34,40 and tyrosine33,41

assemblies, it is of great interest to examine whether chemical agents known to inhibit the assembly of protein and peptide-based amyloid structures can also restrict the formation of these

metabolite fibrils. Furthermore, owing to the similarity between the protein and metabolite assemblies, the later can serve as an attractive minimalistic model to probe the mechanism of

amyloid inhibition, which is yet to be fully deciphered. Here, we explore the inhibitory effect exerted on metabolite amyloid formation by two polyphenolic compounds, (-)-Epigallocatechin

gallate (EGCG) and tannic acid (TA). Both inhibitors were previously shown to efficiently inhibit the formation of various protein amyloids in vitro and in vivo, and display beneficial

preventive and therapeutic effects in neurodegenerative diseases42,43,44,45,46. We show that these two polyphenols successfully inhibit the self-assembly of adenine, phenylalanine, and

tyrosine into amyloid-like fibrils, which accumulate in adenine phosphoribosyltransferase deficiency, PKU and tyrosinemia metabolic disorders, respectively. We further present the ability of

generic polyphenol amyloid inhibitors to effectively reduce the formation of metabolite amyloid fibrils, as demonstrated by in vitro assays, molecular dynamics simulations and using a

neuronal cell culture model. Our recent extension of the generic amyloid hypothesis, linking amyloid formation and metabolite amyloids in inborn error of metabolism disorders, and the

inhibition of metabolite amyloids by natural small polyphenolic compounds as presented here, may lead to an innovative course of treatment for these less-explored metabolic disorders.

RESULTS SPECTROSCOPIC MONITORING OF METABOLITE AMYLOID ASSEMBLY INHIBITION We first examined the inhibitory potential of polyphenols in the context of metabolite amyloid aggregation. Two

polyphenols, EGCG and TA, which were previously shown to inhibit fibril formation by various amyloidogenic proteins and peptides, were selected and their inhibition of metabolites

aggregation was studied. EGCG was found to effectively inhibit fibril formation by the prion PrPsc protein42 and to reduce the toxicity of α-synuclein and Aβ polypeptide aggregates43,44,45.

Similarly, TA has been previously described as the most potent polyphenol inhibitor of prion PrPsc aggregation42, and was found to inhibit Aβ fibrillation46. Thus, the effect of the

inhibitors on the kinetics of amyloid fibril formation by adenine, phenylalanine, and tyrosine was explored. For this purpose, we used ThT, a common fluorescent, amyloid-specific dye, often

used as a reporter of the amyloid self-assembly process, showing a strong increase in its fluorescent intensity upon binding to β-sheet-rich supramolecular structures. Thus, this

fluorometric technique allows to determine the kinetics of the amyloid fibril assembly process, as well as to screen for compounds that might inhibit this process, based on changes in the

intensity of ThT fluorescence13,14. The preparation of metabolite assemblies was performed as previously described36. To identify the metabolite concentrations that result in similar ThT

signals, and therefore presumably in similar levels of amyloid-like fibrils, different metabolite concentrations were examined prior to the addition of the inhibitors. Although resulting in

the formation of assemblies with similar morphologies, the required concentrations varied between the metabolites, as previously shown for different proteinaceous amyloids47. Next, these

concentrations were used for time-dependent monitoring of ThT fluorescence intensity in the presence of the same increasing concentrations of each inhibitor (Fig. 1b–g). Both EGCG and TA

were found to inhibit adenine (8 mg/ml, ~ 60 mM) fibrils in a dose-dependent manner, as demonstrated by the ThT fluorescence assay, presenting near complete inhibition of adenine aggregate

formation at their higher concentrations (Fig. 1b, c). Some adenine aggregates could be observed only at the lowest inhibitor concentration, where EGCG and TA treatment resulted in a

reduction of _ca_. 50% (Fig. 1b) and 70% (Fig. 1c) in ThT intensity, respectively, indicating a stronger inhibitory effect of TA. Similarly, in the case of phenylalanine (40 mg/ml, ~ 242 mM)

aggregates, both EGCG and TA inhibited the formation of the assemblies in a dose-dependent manner (Fig. 1d, e). EGCG presented a substantial inhibition at all concentrations (Fig. 1d), as

reflected by the great reduction in the ThT fluorescence intensity curves, whereas TA presented a complete inhibition only at its highest concentration, a significant reduction in

aggregation when applying the intermediate concentration and almost no effect using the lowest concentration (Fig. 1e). Finally, both inhibitors presented near complete inhibition of

tyrosine (2 mg/ml, ~ 10 mM) aggregates formation at their highest concentration and a significant reduction at the lower concentrations used, where nearly no formation of aggregates was

detected, as reflected from the ThT fluorescence intensity assay (Fig. 1f, g). It should be noted that ThT was previously indicated to promote amyloid formation48. Thus, the clear inhibitory

effect observed is even more notable. To examine whether the decrease in the ThT fluorescence signal was due to an interaction between the aromatic polyphenol compounds and the ThT dye,

rather than inhibition of structure formation, the inhibitory effect of both polyphenols at their highest concentration was also examined using a turbidity assay. Consistent with the ThT

fluorescence results, both EGCG and TA indeed hindered the formation of metabolite amyloids (Supplementary Fig. 1a–c). Moreover, the inhibitory effect was confirmed by dynamic light

scattering (DLS) measurements (Supplementary Fig. 2). The addition of both inhibitors to the adenine assemblies resulted in a dramatic decline of the autocorrelation function (ACF)

amplitude, indicating far fewer aggregates. In addition, there was a substantial reduction in the number of larger particles (longer correlation times) compared with the smaller particles

(shorter correlation times) (Supplementary Fig. 2a). In the case of the phenylalanine assemblies, the addition of TA resulted in a dramatic decline of the ACF amplitude and a reduction in

size, indicating the occurrence of far fewer aggregates. EGCG had a smaller effect compared with TA, resulting in a smaller amplitude and intermediate size shift (Supplementary Fig. 2b).

Tyrosine solution without the inhibitors was too turbid even at low concentrations, and thus could not be characterized by DLS. ULTRASTRUCTURAL ANALYSIS OF THE EFFECT OF POLYPHENOLS ON

METABOLITE AMYLOID FORMATION To gain insight into the morphological changes of the amyloid assemblies in the presence of the inhibitors, we employed transmission electron microscopy (TEM)

and extreme high-resolution scanning electron microscopy (XHR-SEM), using the same concentrations of both metabolites and inhibitors as described above. As a control, TEM and XHR-SEM images

of the polyphenolic inhibitors, at the highest concentrations used, were acquired (Supplementary Fig. 3). In the absence of the inhibitors, adenine and tyrosine presented the typical

fibrillar morphology of amyloid assemblies (Fig. 2a, o and Supplementary Fig. 4a, g), whereas in the presence of either EGCG or TA, inhibition of fibrils formation was observed

(Supplementary Fig. 4b, c, h, i) in a concentration-dependent manner (Fig. 2b–g, p–u). In the case of phenylalanine, lower metabolite concentrations resulted in the formation of a typical

fibrillar amyloid morphology (Fig. 1a), yet at the higher concentration of 40 mg/ml, the concentration used in the ThT fluorescence assay, a different morphology was observed (Fig. 2h and

Supplementary Fig. 4d). Both EGCG and TA hindered these structures (Supplementary Fig. 4e, f) in a concentration-dependent manner (Fig. 2i–n). Notably, the TA concentrations employed in both

ThT fluorescence assay and TEM imaging were at least an order of magnitude lower than those of EGCG, thus indicating a more potent inhibitory effect of TA. This is consistent with the

relative inhibitory effect of the two compounds toward the formation of amyloids by protein and peptide building blocks21. In addition, for all metabolites, the lack of the formation of

assemblies, as observed using TEM, correlates well with the results of the ThT fluorescence assay. INHIBITION OF FIBRILS CYTOTOXICITY BY POLYPHENOLS As mentioned above, the metabolite

assemblies display a dose-dependent cytotoxic effect on a neuronal cell model within six hours36. Therefore, we examined the correlation between the inhibition of metabolite assembly

formation by the polyphenol inhibitors and the resulting cytotoxicity. The adenine and phenylalanine concentrations used for this assay have been previously shown to reduce cell viability by

at least 50%36, whereas tyrosine concentration was lower than the one presenting this effect owing to its limited solubility. SH-SY5Y cells were incubated with metabolite amyloid fibrils

for 24 h, in the presence or absence of the inhibitors, a longer incubation period than was previously examined, thus resulting in a more significant reduction of cell viability. The

concentration of the inhibitors was lower by an order of magnitude than the one used in the in vitro experiments, as higher concentrations were toxic to the cells. In the case of adenine,

the inhibitors significantly increased cell viability in a dose-dependent manner (Fig. 3a, b), showing a threefold and twofold viability increase in the presence of the highest concentration

of EGCG (Fig. 3a) and TA (Fig. 3b), respectively. In the case of phenylalanine, cell viability was almost completely restored in the presence of both inhibitors at their highest

concentration, presenting a dose-dependent inhibition (Fig. 3c, d). Finally, in the case of tyrosine, for all the tested concentrations of both inhibitors, cell viability was significantly

increased to ~ 70% (Fig. 3e, f). To examine whether the improvement in cell viability was owing to the addition of the polyphenolic inhibitors, which can alter cell viability by themselves,

the effect of both inhibitors, when added to untreated cells or to cells following prior six hours treatment, with as the control or with the cytotoxic metabolite assemblies, was measured

(Supplementary Fig. 5a–e). In all cases, later addition of the inhibitors did not improve cell viability. This further indicates that the restoration of cell viability was a result of lack

of metabolites assembly formation owing to the addition of EGCG or TA. ACETYLSALICYLIC ACID AS A CONTROL In order to examine whether the inhibition presented by the polyphenol compounds was

a result of their interaction with the metabolite amyloid structures, it was crucial to examine the possible activity of a compound that is known to be ineffective as an amyloid formation

inhibitor. For this purpose, we used acetylsalicylic acid (ASA), a small-molecule applied to treat pain, fever, and inflammation, which has been previously used as a negative control for

amyloid inhibitors of the IAPP amyloid polypeptide, and did not affect the amyloid formation process16,49. When tested as an inhibitor of metabolite amyloid formation, ASA did not affect

aggregation, even at the highest concentration used for the other inhibitors, as was observed using ThT fluorescence assay, TEM imaging and the SH-SY5Y neuronal cell culture (Supplementary

Fig. 6–8). ANALYSIS OF THE MECHANISM UNDERLYING THE INHIBITION OF METABOLITE AMYLOID FORMATION In order to better understand the mechanism by which the polyphenol inhibitors affect the

metabolite fibril formation, EGCG and TA were added to the system at different time points of the metabolite fibrillation process (after 0, 0.5, 1, and 2 h), and the kinetics of metabolite

fibril formation was monitored using the ThT fluorescence assay. Both endpoint after overnight (Fig. 4a–c) and kinetics (Supplementary Fig. 9) data were recorded. Overall, EGCG inhibited

metabolite fibril formation even when added at later stages of fibrillation. TA inhibited the formation when added at an earlier time point, while showing a much lower impact when added at

the later stages of fibrillation. Next, we examined whether the addition of inhibitors at later time points of metabolite fibrillation had an effect on their resulting cytotoxicity (Fig. 

4d). When EGCG was added after 2 hours, cell viability was significantly restored. However, the addition of TA did not restore cell viability, which decreased to similar levels as the

control without the inhibitor. These results are in agreement with the ThT fluorescence assay (Fig. 4a–c), suggesting that TA mostly inhibits fibrilization at the early stages of nucleation,

whereas EGCG acts both at the early and later stages of fibril formation. Taken together, the combination of results obtained via both the in vitro and neuronal cell model systems provides

important insights into the differential mode of action of each inhibitor. MOLECULAR DYNAMICS MECHANISTIC STUDIES To gain further insight into the distinct inhibition mechanisms of the two

inhibitors, we performed molecular dynamics (MD) simulations of the potential inhibition activity of EGCG, TA, and ASA during the formation of metabolites fibrillar assemblies. In

particular, we examined the interactions between inhibitor molecules and both free solvated metabolite molecules and small metabolite fibril nuclei. Figure 5a–d presents the MD-simulation of

phenylalanine coupled to TA and EGCG (see Supplementary Fig. 10–13 for additional data). The interaction energy was determined by selecting an inhibitor and calculating the average binding

energy per metabolite within 4.5 Å of that inhibitor molecule. We partitioned the trajectory into two sections and averaged the binding energy of each section. The midpoint between the

averages was chosen as the final value. Error bars were determined from the deviation of the averages of each partition and the midpoint. As revealed by the simulations, the average binding

energies of the metabolites to both TA and EGCG are similar, whereas the binding energies to ASA are 2–3 times lower. Moreover, there is a small variation of these energies between the

studied metabolites, with the binding energy of adenine found to be ~ 50% of those of phenylalanine and tyrosine (Fig. 5e). Next, we calculated the number of free metabolites coupled to

inhibitors, normalized by the concentrations of metabolites and inhibitors (Fig. 5f). We determined averages and uncertainties using the same procedure we used in determining the interaction

energy. This analysis shows that a larger number of free metabolites can adsorb to TA as compared with EGCG or ASA, despite similar binding energies of these inhibitors to individual

metabolites (Fig. 5e). This difference results from the significantly larger surface area of TA, as compared with EGCG or ASA. These observations correlate well with the experimental results

showing that TA efficiently binds free metabolites, whereas EGCG reaches the same efficiency only at about an order of magnitude higher concentration. In addition, the number of inhibitors

bound to the metabolites in their crystalline state and normalized to the concentrations of inhibitors and the surface areas of the metabolite crystals was calculated (Fig. 5g). The data

reveal that at the same concentrations, TA and EGCG have a similar propensity for binding to the ordered lattice, whereas ASA has a very low binding propensity. This explains the

experimental observations showing that EGCG can block the growth of fibrils even when added at later stages, as it binds to the crystal at about an order of magnitude higher concentration.

Given the relative dimensions of TA and EGCG, EGCG can cover the crystals to a larger extent at the higher concentrations used. Moreover, there is a twofold decrease of inhibitors bound to

the tyrosine crystal, probably owing to the fact that the preferable binding sites are not fully exposed in this form as its facets are jagged, unlike adenine and phenylalanine surfaces,

preventing the formation of π–π stacking interactions (Fig. 5g). DISCUSSION One of the interesting observations regarding the formation of amyloid fibrils is their generic inhibition by

various polyphenol molecules. Different polyphenols can serve as universal inhibitors of the process of amyloid fibril formation regardless of the amino-acid composition of the protein and

polypeptide assemblies. Nevertheless, the mechanism of inhibition and the specificity and dynamics of inhibitor interaction are not fully understood, giving rise to a genuine need for

minimalistic models on which experimental and computational approaches can be applied. Here, we reveal that two aromatic polyphenolic compounds, EGCG and TA, which have been previously shown

to generically inhibit the formation of protein and peptide-based amyloid structures, can also inhibit the formation of metabolite amyloid fibrils, even when applied at a very low molar

ratio. The examined concentrations, resulting in the same end-effects, varied between the metabolites, as previously shown for different protein and polypeptide amyloid aggregates47. We

demonstrated the activity of these inhibitors both in vitro, using ThT fluorescence assay and TEM analysis, and in vivo, using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide

(MTT) cell viability assay preformed on a neuronal cell model. The addition of the polyphenols at different time points provided a key mechanistic information about the process of

inhibition by EGCG and TA and its correlation to reduced cytotoxicity. We demonstrated that in spite of the generic inhibition, the compounds function via two different mechanisms. Although

EGCG affects both early and later stages of fibrillation, TA is only effective in the early state. To gain further insights on these different inhibition mechanisms, we preformed MD

simulations. The simple chemical composition of the metabolites and the information about their molecular packing allowed to decipher the mechanism of amyloid formation, making them a

potential model for understanding and controlling protein amyloid formation. The simulation provided molecular details about the nature of the binding of the two inhibitors to the studied

metabolites in both monomeric and crystalline forms. It appears that each metabolite binds to both inhibitors with similar energy. At the molarity experimentally used here, the two

inhibitors also bind a comparable number of free metabolites. However, at this molarity, the number of EGCG molecules binding to the crystalline form is several fold higher than the TA

molecules, providing a theoretical framework to understand the distinct effect of the inhibitors when added at different stages of amyloid self-assembly. The metabolites studied in the scope

of this work are known to accumulate in different inborn error of metabolism disorders and, as we have previously shown, can self-assemble and form supramolecular β-sheet-like amyloid

structures. In protein and peptide-based amyloids aggregation, the core β-sheet structure seems to be primarily stabilized by hydrogen bonds50,51. Based on their structural resemblance and

previous findings regarding phenylalanine and tyrosine assemblies’ formation41, we can speculate that the amyloid metabolite supramolecular structures are also stabilized via hydrogen bonds,

which may support the findings derived from the simulations presented here regarding the nature of inhibitor-metabolite interactions. This was indeed the case for phenylalanine, for which

the newly determined crystal structure of the zwitterionic state, that promotes fibrils formation, shows a clear hydrogen-bonding network as observed in peptides and proteins52. Moreover,

the overlay of computer-generated putative poly-phenylalanine β-strands on the crystal structures resulted in remarkable superposition36. The use of known protein and peptide amyloid

inhibitors in order to affect metabolite amyloid structure formation further supports the notion of an extended family of amyloid structures that includes protein, peptides, and metabolites.

Finally, the less-explored concept of error of metabolism disorders as amyloid diseases may thus lead to the development of new therapeutic strategies, in addition to the current dietary

restrictions. METHODS MATERIALS Metabolites (phenylalanine and tyrosine purity ≥ 98%; adenine purity ≥ 99%), thiazolyl blue tetrazolium bromide 98% and thioflavine T were purchased from

Sigma-Aldrich (Rehovot, Israel). Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F12 (Ham’s) (1:1) was purchased from Biological Industries (BI) (Kibbutz Beit Haemek, Israel).

Dimethylformamide (DMF) was purchased from Bio-lab (Jerusalem, Israel). Sodium _n_-dodecyl sulfate (SDS) 99% was purchased from Tzamal D-Chem (Petah Tikva, Israel). THT KINETICS FLUORESCENCE

ASSAY Adenine, phenylalanine, and tyrosine were dissolved at 90 °C in PBS to a final concentration of 8, 40, and 2 mg/ml, respectively, and monomeric solutions of the metabolites were

obtained. Next, these solutions were mixed with the inhibitors, EGCG or TA, at the stated concentrations in a black 96-well, clear, and flat bottom microplate (Greiner). As a control,

metabolites were diluted with PBS alone to the same final concentrations. For the time points experiments, the inhibitors, EGCG (1 mM) and TA (0.1 mM), or PBS as a control, were added at

different time points (0, 0.5, 1, and 2 h). ThT in PBS was added to a final concentration of 40 µM (for phenylalanine) or 20 µM (for adenine and tyrosine). Following excitation at 450 nm,

ThT emission data at 480 nm were measured over time and recorded using a TECAN Infinite 200 PRO plate reader. Data processing was preformed using OriginLab software. TURBIDITY MEASUREMENTS

Adenine, phenylalanine, and tyrosine were dissolved at 90 °C in PBS to a final concentration of 8, 40, and 2 mg/ml, respectively, and monomeric solutions of the metabolites were obtained.

Next, these solutions were mixed with the inhibitors, EGCG (0.1 mM) or TA (0.01 mM), in a 96-well microplate (Corning). As a control, metabolites were diluted with PBS alone to the same

final concentrations. Time-dependent changes in solution turbidity were determined overnight by optical density measurements (600 nm) using a Biotek Synergy HT microplate reader at 25 °C.

DLS For DLS experiments, adenine (2 mg/ml) and phenylalanine (4 mg/ml) were dissolved at 90 °C in PBS and mixed with the inhibitors, either EGCG (0.1 mM) or TA (0.01 mM), or with PBS as a

control. The PBS and inhibitors solutions were both filtered using a 0.02 µm syringe filter prior to the addition of metabolites. After an overnight incubation, 200 µl of the solutions were

loaded in quadruplicates into a black 96-well, clear and flat bottom microplate (Greiner). The autocorrelation amplitude of the solutions was measured applying the DynaPro II Plate Reader

system (Wyatt Technology). For adenine samples, 5 × 5 s per well was acquired, and for phenylalanine, 4 × 5 acquisitions × 5 s per well was acquired. The results represent an average of each

solution quadruplicate. TRANSMISSION ELECTRON MICROSCOPY Adenine, phenylalanine, and tyrosine were dissolved at 90 °C in PBS to a final concentration of 8, 40, and 2 mg/ml, respectively, to

obtain monomeric solutions of the metabolites. Next, these solutions were mixed with the inhibitors, EGCG or TA, at the stated concentrations. As controls, metabolites were diluted with PBS

alone to the same final concentrations and the inhibitors alone without the metabolites were examined as well. In Fig. 1a, phenylalanine was dissolved at 90 °C in PBS to a final

concentration of 4 mg/ml. Samples of the metabolites, in the presence or absence of inhibitors, or samples of inhibitors without the metabolites, were incubated overnight at room

temperature. Subsequently, 10 µl samples were placed on 400-mesh copper grids. After 1 min, excess fluids were removed. Samples were viewed using a JEOL 1200EX electron microscope operating

at 80 kV. EXTREME HIGH-RESOLUTION SCANNING ELECTRON MICROSCOPY Adenine, phenylalanine, and tyrosine were dissolved at 90 °C in PBS to a final concentration of 8, 40, and 2 mg/ml,

respectively, and monomeric solutions of the metabolites were obtained. Next, these solutions were mixed with the inhibitors, EGCG (1 mM) or TA (0.1 mM). As controls, metabolites were

diluted with PBS alone to the same final concentrations and the inhibitors alone without the metabolites were examined as well. Samples of the metabolites, in the presence or absence of

inhibitors, or samples of inhibitors without the metabolites, were incubated overnight at room temperature. Subsequently, 10 µl samples of these solutions were placed on a glass slide and

left to dry at room temperature. Samples were then coated with 3 nm chrome and viewed using Verios XHR 460 L SEM operating at 3 kV. Micrographs displayed are representative of three

independent experiments conducted. CELL CYTOTOXICITY EXPERIMENTS SH-SY5Y cells (2 × 105 cells/ml) were cultured in 96-well tissue microplates (100 µl per well) and allowed to adhere

overnight at 37 °C. Each plate was divided and only half of it was plated with cells, with the other half later serving as a control for the solutions alone. The treatment solutions were

prepared as follows: adenine, phenylalanine, and tyrosine were dissolved at 90 °C in DMEM/Nutrient Mixture F12 (Ham’s) (1:1) (Biological Industries) without fetal bovine serum (FBS), to a

final concentration of 2, 4, and 2 mg/ml, respectively, to obtain monomeric solutions of the metabolites. Next, these solutions were mixed with the inhibitors, EGCG or TA, at the stated

concentrations. As a control, metabolites were diluted with DMEM/Nutrient Mixture F12 without FBS to the same final concentrations. The inhibitors without the metabolites were examined as

well. For the inhibition mechanism experiments, EGCG (100 µM), TA (10 µM), or DMEM/Nutrient Mixture F12 (Ham’s) without FBS as a control, were added to the metabolites samples after 2 hours.

The samples were incubated overnight at room temperature. Then, cells were washed and treated with the solutions (100 µl per well), followed by overnight incubation at 37 °C. In order to

explore the effect of a later addition of the inhibitors on cells viability, EGCG (100 µM), and TA (10 µM), were added after 6 h to untreated cells or to cells that were pre-treated with

metabolite fibrils, followed by overnight incubation at 37 °C. Cell viability was evaluated using the MTT assay. In brief, 10 µl of 5 mg/ml MTT dissolved in PBS were added to each well.

After a 4 h incubation at 37 °C, 100 µl extraction buffer (20% SDS dissolved in a mixture of 50% DMF and 50% DDW (pH 4.7)) were added to each well, and the plates were incubated again at 37 

°C for 30 min. Finally, color intensity was measured using an ELISA reader at 570 nm. The data are presented as mean ± SEM. STATISTICAL ANALYSIS Two-tailed Student’s _t_-test was performed

when two groups were compared. FORCE FIELD FITTING The force field parameters for inhibitor molecules were calculated using Gaussian0953. Owing to the large size of TA, this molecule was

divided to two sections in the calculations: (1) an inner core, which is a beta-d glucose derivative, with each hydroxyl group being methylated; (2) ligands, with the ester group that is

bonded to the core methylated. In the calculations of the EGCG core, the two main fused rings and the ester group were considered; the aromatic groups of EGCG were replaced with methyl

groups. Force field parameters for aromatic groups used the results from TA calculations or were approximated with methyl groups. Geometries and force constants of inhibitors were determined

using MP2/6-31 g(d)//MP2/6-31 g(d) level for the EGCG core and TA core. Owing to the extent of ligand calculations, MP2/6-31 g(d)//MP2/6-31 g methods were used instead. Force field fitting

was performed using VMD Force Field Toolkit Plugin54. To determine dihedral parameters, MP2 level of calculations were also used to derive a quantum target, with the angle scanned six steps

in both positive and negative directions at a step size of 15˚. Charges were determined using the ChelpG algorithim55. SOLVATED METABOLITE SIMULATIONS Systems formed by 288 ADN, PHE, and TYR

freely solvated metabolite molecules were simulated in the presence of a small nucleation crystal of 125, 108, and 100 metabolite molecules, respectively. Each system consisted of 7 TA, 10

EGCG, or 15 ASA molecules. Systems with TA or EGCG were simulated for 20 ns, and those with ASA for 30 ns, using NAMD256 and the CHARMM force field. The systems were simulated in (NaCl) = 

0.15 M aqueous solution, in order to imitate physiological conditions. The Langevin dynamics with a damping coefficient of 1 ps−1 and a time step of 2 fs was used to describe systems in a

NPT ensemble at a pressure of 1 atm and a temperature of 310 K. During minimization, pre-equilibration, and equilibration, Particle Mesh Ewald57 was used with a grid spacing of 1.0. The

non-bonded interactions used the SHAKE switching algorithm with a switch on/off distance of 10/12 Å. Non-bonded pairs lists were 13.5 Å, with the list updated every 20 steps; 1–4 non-bonded

interactions were not scaled. There were 50,000 steps of minimization followed by 2 ns of equilibration, after which the simulations were performed. The same approach was used in the

simulations of inhibitors binding to metabolite crystals. CRYSTALLINE METABOLITE SIMULATIONS Simulated bulk-like crystals of ADN, PHE, and TYR had 564, 768, and 800 molecules, respectively,

which were cut from their bulk crystal structures52,58,59. The solutions of simulated bulk crystals contained 25 TA, 50 EGCG, or 50 ASA molecules, and no free metabolite molecules. No

specific fibril structures were considered. SOLVATED METABOLITE INTERACTION ENERGIES Average interaction energies between inhibitors and free metabolites or crystals were calculated from the

simulated systems. The CHARMM force field60,61,62,63,64 and NAMDenergy plugin in VMD65 were used to determine the strength of total interaction energy for each inhibitor by calculating vdW

and electrostatic interaction energies. Interaction energy between each inhibitor and any metabolite molecule, which had at least one atom within 3.5 Å of any atom of the selected inhibitor,

was considered for the calculation. For each snapshot, the total energy and number of metabolites interacting with the inhibitor was recorded. Interaction energy per metabolite and

inhibitor was determined at each snapshot. When averaging the interaction energy, only cases with at least one metabolite interacting with the inhibitor were included. The average number of

free metabolites was computed for the entire trajectory. The average number of interacting metabolites was normalized with respect to the concentrations of both inhibitors and solvated

metabolites (Supplementary Table 1). CRYSTALLINE METABOLITES INTERACTION ENERGIES Similar to solvated metabolites, we chose one inhibitor for the crystal cases and evaluated its interaction

energies with metabolites on the crystal. Metabolites on crystal, which were within 3.5 Å of the inhibitor, were considered. The average interaction energy was computed using the same

procedure as for the solvated metabolites. The number of inhibitors interacting with the crystal was determined by calculating any inhibitor with at least one atom within 3.5 Å of the

crystal. This number was averaged throughout the trajectory. Then, it was normalized with respect to the concentration of inhibitors and surface area of the crystal, obtained at the end of

the trajectory (Table S1), because some metabolite molecules dissolved from the crystals during the simulation. DATA AVAILABILITY The authors declare that all of the data supporting the

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ACKNOWLEDGEMENTS This work was supported by the Israel Science Foundation (grant no. 802/15; E.G.), The FEBS Long-Term Fellowship (A.L.), The Strauss Institute (fellowship; S.S.-N.), The

Adelis Forever Foundation (E.G.), and the NSF Division of Materials Research (grant no. 1506886; P.K.). We thank D. Some of Wyatt Technology Corp. and A. Tsadok of Danyel Biotech for

performing the dynamic light scattering analysis and the members of the Gazit, Adler-Abramovich, Vuković, and Král groups for helpful discussions. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS

* Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel Shira Shaham-Niv, Dor Zaguri, Aviad Levin 

& Ehud Gazit * Department of Chemistry, University of Illinois at Chicago, Chicago, 60607, IL, USA Pavel Rehak & Petr Král * Department of Chemistry, University of Cambridge,

Cambridge CB2 1EW, UK Aviad Levin * Department of Oral Biology, The Goldschleger School of Dental Medicine, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 6997801, Israel Lihi

Adler-Abramovich * Department of Chemistry and Biochemistry, University of Texas at El Paso, El Paso, 79968, TX, USA Lela Vuković * Department of Physics, University of Illinois at Chicago,

Chicago, 60607, IL, USA Petr Král * Department of Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, 60612, IL, USA Petr Král * Department of Materials Science and

Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv, 6997801, Israel Ehud Gazit * BLAVATNIK CENTER for Drug Discovery, Tel Aviv University, Tel Aviv,

6997801, Israel Ehud Gazit Authors * Shira Shaham-Niv View author publications You can also search for this author inPubMed Google Scholar * Pavel Rehak View author publications You can

also search for this author inPubMed Google Scholar * Dor Zaguri View author publications You can also search for this author inPubMed Google Scholar * Aviad Levin View author publications

You can also search for this author inPubMed Google Scholar * Lihi Adler-Abramovich View author publications You can also search for this author inPubMed Google Scholar * Lela Vuković View

author publications You can also search for this author inPubMed Google Scholar * Petr Král View author publications You can also search for this author inPubMed Google Scholar * Ehud Gazit

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.S.-N., L.A.-A., A. L., and E.G. conceived and designed the experiments. S.S.-N. and D.Z

planned and performed the experiments. P.R., L.V., and P. K. planned and performed the MD simulations. S.S.-N., L.A.-A., P.R, L. V., P.K, and E.G. wrote the paper. All authors discussed the

results, provided intellectual input and critical feedback, and commented on the manuscript. CORRESPONDING AUTHOR Correspondence to Ehud Gazit. ETHICS DECLARATIONS COMPETING INTERESTS The

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metabolite amyloid formation by generic fibrillation-modifying polyphenols. _Commun Chem_ 1, 25 (2018). https://doi.org/10.1038/s42004-018-0025-z Download citation * Received: 08 October

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