Identification of Novel 1,3,5-Triphenylbenzene Derivative

International Journal of Molecular Sciences

Article Identiﬁcation of Novel 1,3,5-Triphenylbenzene Derivative Compounds as Inhibitors of Hen Lysozyme Amyloid Fibril Formation

Hassan Ramshini 1, Reza Tayebee 2 , Alessandra Bigi 3 , Francesco Bemporad 3 , Cristina Cecchi 3 and Fabrizio Chiti 3,* 1 Biology Department, Payam Noor University, Tehran 19395-4697, Iran; [email protected] 2 Department of Chemistry, School of Sciences, Hakim Sabzevari University, Sabzevar 96179-76487, Iran; [email protected] 3 Section of Biochemical Sciences, Department of Biomedical, Experimental and Clinical Sciences, University of Florence, I-50134 Florence, Italy; alessandra.bigi@unifi.it (A.B.); francesco.bemporad@unifi.it (F.B.); cristina.cecchi@unifi.it (C.C.) * Correspondence: fabrizio.chiti@unifi.it; Tel.: +39-055-275-1220

Received: 25 October 2019; Accepted: 2 November 2019; Published: 7 November 2019

Abstract: Deposition of soluble proteins as insoluble amyloid fibrils is associated with a number of pathological states. There is a growing interest in the identification of small molecules that can prevent proteins from undergoing amyloid fibril formation. In the present study, a series of small aromatic compounds with different substitutions of 1,3,5-triphenylbenzene have been synthesized and their possible effects on amyloid fibril formation by hen egg white lysozyme (HEWL), a model protein for amyloid formation, and of their resulting toxicity were examined. The inhibitory effect of the compounds against HEWL amyloid formation was analyzed using thioflavin T and Congo red binding assays, atomic force microscopy, Fourier-transform infrared spectroscopy, and cytotoxicity assays, such as the 3-(4,5-Dimethylthiazol)-2,5-Diphenyltetrazolium Bromide (MTT) reduction assay and caspase-3 activity measurements. We found that all compounds in our screen were efficient inhibitors of HEWL fibril formation and their associated toxicity. We showed that electron-withdrawing substituents such as –F and –NO2 potentiated the inhibitory potential of 1,3,5-triphenylbenzene, whereas electron-donating groups such as –OH, –OCH3, and –CH3 lowered it. These results may ultimately find applications in the development of potential inhibitors against amyloid fibril formation and its biologically adverse effects.

Keywords: protein misfolding; HEWL amyloid aggregation; drug discovery; small aromatic compounds; amyloid inhibitors; triphenylbenzene

1. Introduction The conversion of peptides or proteins from a soluble state into insoluble, β-sheet rich, fibrillar aggregates, generally called amyloid fibrils, is a key event in a large family of human pathologies, including neurodegenerative and non-neuropathic systemic amyloidosis [1,2]. Among these, Alzheimer’s disease (AD) is the most known and diffuse and it has an increasing incidence in the worldwide population. Similarly, Parkinson’s disease (PD), as well as Huntington’s, prion diseases, type II diabetes, and systemic amyloidosis share the common hallmark of amyloid fibrillar aggregates [1–3]. Although the proteins that comprise these deposits do not share any sequence or structural homology, amyloid fibrils formed by different proteins share similar structural and physicochemical properties and, possibly, a common pathogenic mechanism [4,5]. These diseases involve self-assembly of soluble proteins into large insoluble fibrils through nucleation-dependent

Int. J. Mol. Sci. 2019, 20, 5558; doi:10.3390/ijms20225558 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5558 2 of 18 assembly, often via the formation of oligomeric structures that possess toxic properties [1,6,7]. Therefore, preventing oligomer and fibril formation appears an attractive approach to tackling the progress of such diseases. Fibril formation is a polymerization process which can be simply described by a sigmoid curve with three phases, originally suggested to be a three-stage process consisting of nucleation, fibril elongation, and the final plateau [8], although the most recent progress has elucidated that each of the three phases involve a combination of the various microscopic steps [9,10]. Despite intensive efforts to treat amyloid-associated diseases, there is no fully effective and definitive treatment of such diseases [1,2,11,12]. Several strategies have been undertaken in the attempt to interfere with the formation and deposition of amyloid fibrils and their pathogenic role, including inhibition of the formation of the amyloidogenic form of proteins by stabilization of the native conformation, reduction in the production of the amyloidogenic sequence from a more complex protein, inhibition of protein self-assembly in oligomers and fibrils, and enhancement in the clearance of toxic aggregates [13–17]. The propensity of proteins and peptides to form amyloid fibrillar structures is correlated to their physicochemical properties such as net charge, secondary structure tendency, hydrophobicity, and aromatic interactions [18,19]. Regions of the sequence of proteins promoting amyloid fibril formation are often enriched with aromatic residues [20–22]. There are different hypotheses on the precise role of the aromatic residues in the amyloidogenic process. One of them suggests that aromatic amino acid residues promote peptide self-assembly for their hydrophobic nature, high β-sheet propensity and null charge, along with their planar geometry that are the most relevant factors in self-assembly [23–27]. According to this hypothesis, the aromatic nature of these residues does not contribute significantly to their amyloidogenicity. Other various studies suggest that aromatic amino acids contribute to peptide self-assembly through π-stacking interactions [22,28,29]. Other groups also believed that both hydrophobicity and aromaticity play a role in the self-assembly [30]. Regardless of the precise mechanism by which aromatic residues drive amyloid fibril formation, the parallel in-register alignment of β-strands in amyloid fibrils contributes to create a stacking of aromatic rings along the fibril axis. Thus, aromatic compounds may competitively interact with aromatic residues in amyloidogenic proteins, preventing their stacking, and block the self-assembly process [31]. It has been found that the effect of aromatic compounds on amyloid aggregation strongly depends on their structure and functional groups [32,33]. Multiple studies have reported that the number of phenyl rings and electron-withdrawing or -donating groups on the molecules, as well as their positions, are important characteristics that determine the effectiveness of the aromatic compounds in their inhibition of Aβ aggregation [34–36]. Following this concept, six aromatic molecules belonging to the category of 1,3,5-triphenylbenzene and having different electron-donor or -withdrawing substituents, have been synthesized and their possible effects on amyloid fibril formation by a sample protein have been tested. As a sample protein we have chosen hen egg white lysozyme (HEWL), as it is known to form amyloid fibrils under appropriate conditions [37–40] and because the properties of the resulting fibrils and the of the overall amyloid formation process have been investigated to a significant extent [41,42]. The effects of the compounds on amyloid fibril formation by HEWL and on the biological properties of the resulting aggregates have been investigated through the use of various techniques, including Congo red (CR) and thioflavin T (ThT) assays, Fourier-transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM), the MTT reduction assay, and caspase activity measurements. Our results showed the importance of the substituents on the efficiency of aromatic compounds, with compounds containing electron-withdrawing substituents having greater efficacy in HEWL amyloid inhibition, thus representing promising compounds for developing inhibitors of amyloid fibril formation. Int. J. Mol. Sci. 2019, 20, 5558 3 of 18

2. Results

2.1. Chemistry of the Compounds Scheme1 shows a general formula for the preparation of some para-substituted 1,3,5-triphenylbenzenesInt. J. Mol. Sci. 2019, 20, x FOR PEER (compounds REVIEW 1–6). These pharmaceutical important compounds are made3 of 19 by the condensation of aryl methyl ketones catalyzed by HPA/NCP under solvent-free conditions. condensation of aryl methyl ketones catalyzed by HPA/NCP under solvent-free conditions. Nanoclinoptilolite (NCP) refers to a natural zeolite composed of a microporous arrangement of silica and Nanoclinoptilolite (NCP) refers to a natural zeolite composed of a microporous arrangement of silica alumina and also HPA (H6P2W18O62) refers to the Wells–Dawson heteropolyacid. The heteropolyacid and alumina and also HPA (H6P2W18O62) refers to the Wells–Dawson heteropolyacid. The catalyst activates the carbonyl group toward the electrophilic substitution. Using this procedure, six heteropolyacid catalyst activates the carbonyl group toward the electrophilic substitution. Using this compounds belonging to the category of para-substituted 1,3,5-triarylbenzenes have been synthesized procedure, six compounds belonging to the category of para-substituted 1,3,5-triarylbenzenes have and puriﬁed as previously described [43,44]. These are 1,3,5-triphenylbenzene (Compound 1); been synthesized and purified as previously described [43,44]. These are 1,3,5-triphenylbenzene 1,3,5-tris(4-ﬂuorophenyl)benzene (Compound 2); 1,3,5-tris(4-nitrophenyl)benzene (Compound 3); (Compound 1); 1,3,5-tris(4-fluorophenyl)benzene (Compound 2); 1,3,5-tris(4-nitrophenyl)benzene 1,3,5-tris(4-methylphenyl)benzene (Compound 4); 1,3,5-tris(4-methoxyphenyl)benzene (Compound 5); (Compound 3); 1,3,5-tris(4-methylphenyl)benzene (Compound 4); 1,3,5-tris(4- and 1,3,5-tris(4-hydroxyphenyl)benzene (Compound 6). methoxyphenyl)benzene (Compound 5); and 1,3,5-tris(4-hydroxyphenyl)benzene (Compound 6). R

H6P2W18O62/nanoclinoptilolite + 3HO 3 solvent free, 100 ºC 2

R R R: R Compound 1: H; 1,3,5-Triphenylbenzene Compounds 1-6 Compound 2: 4-F; 1,3,5-Tris(4-fluorophenyl)benzene Compound 3: 4-NO2; 1,3,5-Tris(4-nitrophenyl)benzene Compound 4: 4-Me; 1,3,5-Tris(4-methylphenyl)benzene Compound 5: 4-OMe; 1,3,5-Tris(4-methoxyphenyl)benzene Compound 6: 4-OH; 1,3,5-Tris(4-hydroxyphenyl)benzene Scheme 1. General formulation for the cyclotrimerization of acetophenones. Scheme 1. General formulation for the cyclotrimerization of acetophenones. The names and chemical formulas of the six compounds are reported in Scheme1. Their NMR The names and chemical formulas of the six compounds are reported in Scheme 1. Their NMR spectra are reported in the supplementary information, again along with their chemical formulas spectra are reported in the supplementary information, again along with their chemical formulas (Figures S1–S6). (Figures S1–S6). 2.2. Effect of 1,3,5-Triarylbenzenes and Their Derivatives on Heat-Induced Fibrillation of HEWL Monitored with2.2. Effect ThT Fluorescence of 1,3,5-Triarylbenzenes and Their Derivatives on Heat-Induced Fibrillation of HEWL Monitored with ThT Fluorescence The inhibition potential of the six compounds was determined using the ThT fluorescence assay to measureThe inhibition the amount potential of amyloid of the six aggregates compounds after wa thes determined addition of using various the concentrationsThT fluorescence of assay each compoundto measure ranging the amount from of 0.03 amyloid to 2.6 µ M.aggregates Aggregation after of the HEWL addition was of induced various by concentrations incubating the proteinof each forcompound 48 h at aranging concentration from 0.03 of 2 to mg 2.6/mL µM. (140 AggregµM) ination 50 mM of glycineHEWL buwasff er,induced pH 2.5, by and incubating 57 ◦C, while the stirredprotein gently for 48 byh at magnetic a concentration bars (rpm of 250),2 mg/mL in the (140 absence µM) in or 50 presence mM glycine of 0.03–2.6 buffer,µM pH compounds 2.5, and 571 °C,–6. Allwhile compounds stirred gently clearly by showed magnetic a pattern bars of(rpm dose-dependent 250), in the inhibition, absence or as suggestedpresence byof the0.03–2.6 observed µM decreasecompounds of ThT 1–6. fluorescenceAll compounds intensity clearly (Figure showed1). a The pattern IC 50 ofvalues dose-dep of theendent compounds, inhibition, corresponding as suggested toby thethe concentrationobserved decrease at which of ThT the fluorescence inhibitory eintensityffect is half (Figure of the 1). maximum The IC50 values effect, of were the compounds, determined (Figurecorresponding2). The relativeto the concentration order of e ffi atcacy which of the the compounds inhibitory effect in terms is half of of IC the50 value maximum is compound effect, were2 > compounddetermined1 (Figure> compound 2). The3 ~relative compound order5 >of compoundefficacy of 4the> compound compounds6 and,in terms thus, of compound IC50 value2 isis thecompound best inhibitor 2 > compound and compound 1 > compound6 has minimum 3 ~ compound effect in 5 this > compound process. As 4 shown> compound in Figures 6 and,1 and thus,2, 0.32compoundµM of all2 is compounds the best inhibitor significantly and compound inhibited 6 the ha aggregations minimum effect of HEWL, in this thus process. this concentration As shown in wasFigures used 1 forand the 2, 0.32 subsequent µM of all experiments. compounds significantly inhibited the aggregation of HEWL, thus this concentration was used for the subsequent experiments. A control experiment was also designed to rule out the possibility that the low ThT fluorescence intensities measured in the presence of these compounds might result from the quenching of ThT fluorescence by the compounds themselves. These compounds were separately added at final concentrations of 2.6 µM to pre-formed HEWL aggregates and the ThT fluorescence assay was performed immediately before and after compound addition. The ThT fluorescence values were found to be similar in each case, before and after compound addition, indicating the lack of any significant quenching effect (Figure S7).

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 19

HEWL Agg + C1 0 μΜ HEWL Agg + C3 0 μΜ Int. J.800 Mol. Sci. 2019, 20, 5558 ThT alone 800 4ThT of alone 18 Int. J. Mol. Sci. 2019, 20, x FOR PEER2.6 REVIEW μΜ 4 2.6of μΜ 19 1.3 μΜ 1.3 μΜ 0.65 μΜ 0.65 μΜ 600 0.32 μΜ 600 0.32 μΜ 0.16 μΜ 0.16 μΜ HEWL Agg + C1 0 μΜ0.08 μΜ HEWL Agg + C3 0 μΜ 0.08 μΜ ThT0.06 alone μΜ 800 ThT alone 800 0.06 μΜ 400 2.60.03 μΜ μΜ 400 2.6 μΜ 1.3 μΜ 1.30.03 μΜ μΜ 0.65 μΜ 0.65 μΜ 600 0.32 μΜ 600 0.32 μΜ 0.16 μΜ 200 200 0.16 μΜ 0.08 μΜ ThT florescence (a.u.) ThT florescence (a.u.) 0.08 μΜ 0.06 μΜ 0.06 μΜ 400 0.03 μΜ 400 0 0 0.03 μΜ 460 480 500 520 540 460 480 500 520 540 Wavelength (nm) Wavelength (nm) 200 200 ThT florescence (a.u.) ThT florescence (a.u.)

HEWL Agg + C6 0 μΜ 0 8000 ThT alone 460 480 500 520 540 460 480 500 520 5402.6 μΜ HEWL Agg + C5 0 μΜ HEWL AggWavelength + C4 (nm) 0 μΜ Wavelength (nm) 1.3 μΜ ThT alone 800 ThT alone 800 0.65 μΜ 2.6 μΜ 2.6 μΜ 600 0.32 μΜ 1.3 μΜ 1.3 μΜ 0 μΜ0.16 μΜ 0.65 μΜ HEWL Agg + C6 0.65 μΜ 0.08 μΜ 600 600 0.32 μΜ 800 ThT alone 0.32 μΜ 2.60.06 μΜ μΜ 0.16 μΜ HEWL Agg + C5 0 μΜ0.16 μΜ 400 HEWL Agg + C4 0 μΜ 1.30.03 μΜ μΜ 0.08 μΜ ThT0.08 alone μΜ 800 ThT alone 800 0.65 μΜ 0.06 μΜ 2.60.06 μΜ μΜ 400 2.6 μΜ 400 600 0.32 μΜ 0.03 μΜ 1.30.03 μΜ μΜ 1.3 μΜ 200 0.16 μΜ 0.65 μΜ 0.65 μΜ

ThT florescence (a.u.) 0.08 μΜ 600 600 0.32 μΜ 0.32 μΜ 0.06 μΜ 200 0.16 μΜ 200 0.16 μΜ 400

ThT florescence (a.u.) florescence ThT 0.03 μΜ 0.08 μΜ ThT florescence (a.u.) 0.08 μΜ 0 400 0.06 μΜ 400 0.06 μΜ 460 480 500 520 540 0 0.03 μΜ 0.03 μΜ 460 480 500 520 540 0 200 Wavelength (nm) Wavelength (nm) 460 480 500 520 540 ThT florescence (a.u.) 200 200 Wavelength (nm) ThT florescence (a.u.) florescence ThT

ThT florescence (a.u.) 0 460 480 500 520 540 0 0 Wavelength (nm) 460 480 500 520 540 460 480 500 520 540 Figure Wavelength1. Dose-dependent (nm) inhibition of 0.03–2.6Wavelength µM (nm)compounds 1–6 on hen egg white lysozyme (HEWL) amyloid fibril formation at 2 mg/mL (140 µM), in 50 mM glycine buffer, pH 2.5, and 57 °C, Figureunder stirring 1. Dose-dependent (250 rpm), for inhibition 48 h, monitored of 0.03–2.6 by chanµMges compounds in thioflavin1–6 Ton (ThT) hen florescence egg white lysozymeintensity. (HEWL) amyloid fibril formation at 2 mg/mL (140 µM), in 50 mM glycine buffer, pH 2.5, and 57 ◦C, FigureThe spectra 1. Dose-dependent refer to ThT alone inhibition (gray), ofHEWL 0.03–2.6 pre-incubated µM compounds with 0 1–6(black), on hen0.03 egg(violet), white 0.06 lysozyme (indigo), under stirring (250 rpm), for 48 h, monitored by changes in thioflavin T (ThT) florescence intensity. (HEWL)0.08 (dark amyloid blue), 0.16fibril (blue), formation 0.32 (green),at 2 mg/mL 0.65 (140(yellow), µM), 1.3in 50(orange), mM glycine and 2.6 buffer, (red) pHµM 2.5, of compounds and 57 °C, The spectra refer to ThT alone (gray), HEWL pre-incubated with 0 (black), 0.03 (violet), 0.06 (indigo), under1–6. stirring (250 rpm), for 48 h, monitored by changes in thioflavin T (ThT) florescence intensity. 0.08 (dark blue), 0.16 (blue), 0.32 (green), 0.65 (yellow), 1.3 (orange), and 2.6 (red) µM of compounds 1–6. The spectra refer to ThT alone (gray), HEWL pre-incubated with 0 (black), 0.03 (violet), 0.06 (indigo), 0.08 (dark blue), 0.16 (blue), 0.32 (green), 0.65 (yellow), 1.3 (orange), and 2.6 (red) µM of compounds 1–6.

Figure 2. Concentration-dependent prevention of HEWL amyloid fibril formation by compounds 1–6 Figure 2. Concentration-dependent prevention of HEWL amyloid fibril formation by compounds 1– incubated as described in Figure1 at di fferent compound concentrations, ranging from 0.03 to 2.6 µM. 6 incubated as described in Figure 1 at different compound concentrations, ranging from 0.03 to 2.6 The traces refer to compound 1 (red), compound 2 (orange), compound 3 (yellow), compound 4 (green), µM. The traces refer to compound 1 (red), compound 2 (orange), compound 3 (yellow), compound 4 compound 5 (blue), and compound 6 (indigo). The dotted line indicates a level of 50%. Figure(green), 2. compoundConcentration-dependent 5 (blue), and compound prevention 6 (i ofndigo). HEWL The amyloid dotted fibril line indicatesformation a bylevel compounds of 50%. 1– 6A incubated control experimentas described wasin Figure also designed1 at different to rulecompound out the concentrations, possibility that ranging the low from ThT 0.03 fluorescence to 2.6 intensitiesµM. The measured traces refer in to the compound presence 1 (red), of these compou compoundsnd 2 (orange), might compound result from 3 (yellow), the quenching compound of4 ThT fluorescence(green), compound by the compounds 5 (blue), and compound themselves. 6 (indigo). These The compounds dotted line wereindicates separately a level of 50%. added at final

concentrations of 2.6 µM to pre-formed HEWL aggregates and the ThT ﬂuorescence assay was

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 19

2.3. Effect of 1,3,5-Triarylbenzenes and Their Derivatives on Heat-Induced Fibrillation of HEWL Monitored with CR Assay Aliquots of the same samples used for ThT experiments were also used to carry out the CR assays. The CR absorbance measured in the absence of HEWL and any of the compounds was found to be low, with a peak at 490 nm (Figure 3A). The CR absorbance measured in the presence of HEWL

Int.pre-incubated J. Mol. Sci. 2019 for, 20 ,48 5558 h under aggregating conditions in the absence of any compound exhibited5 of 18a weak increase accompanied by a red shift of the peak to 520–540 nm (Figure 3A). The higher CR absorbance obtained in the presence of HEWL is likely to originate from the presence of protein performedaggregates immediatelythat scatter light, before whereas and after the compound red shift addition.of the peak The from ThT 490 fluorescence to 520–540 values nm indicates were found the topresence be similar of β in-sheet each containing case, before aggregates. and after compound The CR abso addition,rbance indicatingmeasured thein the lack presence of any significant of HEWL quenchingpre-incubated effect for (Figure 48 h under S7). aggregating conditions with 0.32 µM compounds 1, 2, 3, 4, and 5 were found to be lower than that measured in the absence of the compounds (Figure 3A). This indicates 2.3. Effect of 1,3,5-Triarylbenzenes and Their Derivatives on Heat-Induced Fibrillation of HEWL Monitored that these compounds were able to inhibit HEWL amyloid fibril formation. The CR absorbance with CR Assay measured in the presence of HEWL and 0.32 µM compound 6 was found to be markedly higher, with a higherAliquots red shift, of the indicating same samples that this used compound for ThT experiments was not effective were alsoin inhibiting used to carry HEWL out amyloid the CR assays.formation. The These CR absorbance results were measured confirmed in theby analyzin absence ofg the HEWL difference and any spectra, of the obtained compounds by subtracting was found toin beeach low, case with the a spectrum peak at 490 of nmCR (Figurealone from3A). Thethos CRe obtained absorbance with measured HEWL aggregates in the presence and CR of HEWL(Figure pre-incubated3B). The absorbance for 48 hpeak under at aggregating540 nm, obtained conditions without in the compounds absence of any1–6 compoundand readily exhibited assigned a to weak CR increasebound to accompanied the aggregates, by awas red found shift of to the be peaksuppress to 520–540ed in the nm presence (Figure3 ofA). the The various higher compounds CR absorbance 1–5, obtainedwith only in a themodest presence decrease of HEWL in the ispresence likely to of originate compound from 6. the presence of protein aggregates that scatterIn light,this study whereas, we the used red dimethyl shift of the sulfoxide peak from (DMSO) 490 to 520–540for dissolving nm indicates our compounds, the presence and, of β thus,-sheet a containingcontrol experiment aggregates. was The carried CR absorbance out to determine measured wh inether the presenceDMSO itself of HEWL had an pre-incubated effect on the forHEWL 48 h underaggregation. aggregating However, conditions 2% (v/v with) DMSO 0.32 µusedM compounds here has a minimal1, 2, 3, 4, effect and 5 onwere the found aggregation to be lower of HEWL than that(Figure measured S8). Therefore, in the absence all compounds of the compounds were dissolved (Figure 3inA). DMSO This indicatesprior to use. that these compounds were able toThe inhibit results HEWL observed amyloid by the fibril ThT formation. and CR assay The CR depicted absorbance in Figures measured 1–3 could in the be presence due to an of HEWLability andof the 0.32 sixµ Mcompounds compound to6 wasinhibit found HEWL to be aggregation. markedly higher, Alternatively, with a higher they red might shift, just indicating inhibit amyloid that this compoundfibril formation was notwithout effective preventing in inhibiting the HEWLformatio amyloidn of protein formation. aggregates These results with werea non-amyloid confirmed bystructure. analyzing To assess the di ffeithererence possibility, spectra, obtained we centri byfuged subtracting the various in each HEWL case samples the spectrum and measured of CR alone the fromprotein those concentration obtained with of the HEWL resuspended aggregates pellets and CR, showing (Figure 3B).that The pellets absorbance obtained peak from at 540samples nm, obtainedpreincubated without with compounds compounds1 –1–66 and had readily a higher assigned protein to CRcontent bound than to the those aggregates, incubated was without found tocompounds. be suppressed This in indicated the presence that of the the aromatic various compoundsmolecules did1–5 ,not with inhibit only a modestaggregation decrease of HEWL in the presencegenerally, of but compound more specifically6. its process of amyloid fibril formation.

0.6 CR alone HEWL Agg 0.5 HEWL Agg + C1 HEWL Agg + C2 HEWL Agg + C3 HEWL Agg + C4 0.4 HEWL Agg + C5 HEWL Agg + C6

0.3

0.2

0.1 Congo red Absorbance

0 400 450 500 550 600 650 Wavelength (nm)

(A) (B)

Figure 3. Effects of compounds 1–6, at a concentration of 0.32 µM, on HEWL aggregation at 2 mg/mL, Figure 3. Effects of compounds 1–6, at a concentration of 0.32 µM, on HEWL aggregation at 2 mg/mL, 50 mM glycine buffer, pH 2.5, 57 ◦C, under stirring (250 rpm), monitored by changes in CR absorbance after50 mM 48 glycine h. (A) The buffer, spectra pH 2.5, refer 57 to °C, CR under alone stirring (gray), HEWL(250 rpm), pre-incubated monitored underby changes aggregating in CR absorbance conditions withoutafter 48 compoundsh. (A) The (black),spectra withrefer compoundto CR alone1 (red), (gray) compound, HEWL pre-incubated2 (orange), compound under aggregating3 (yellow), compound 4 (green), compound 5 (blue), and compound 6 (indigo). (B) Difference spectra obtained by subtracting the spectrum of CR alone from the spectra of HEWL aggregates with CR reported in (A). Color code as in (A).

In this study, we used dimethyl sulfoxide (DMSO) for dissolving our compounds, and, thus, a control experiment was carried out to determine whether DMSO itself had an effect on the HEWL Int. J. Mol. Sci. 2019, 20, 5558 6 of 18 aggregation. However, 2% (v/v) DMSO used here has a minimal effect on the aggregation of HEWL (Figure S8). Therefore, all compounds were dissolved in DMSO prior to use. The results observed by the ThT and CR assay depicted in Figures1–3 could be due to an ability of the six compounds to inhibit HEWL aggregation. Alternatively, they might just inhibit amyloid fibril formation without preventing the formation of protein aggregates with a non-amyloid structure. ToInt. assess J. Mol.either Sci. 2019 possibility,, 20, x FOR PEER we REVIEW centrifuged the various HEWL samples and measured the6 protein of 19 concentration of the resuspended pellets, showing that pellets obtained from samples preincubated with compoundsconditions1–6 had without a higher compounds protein (black), content with than compound those incubated 1 (red), compound without 2 compounds.(orange), compound This indicated 3 (yellow), compound 4 (green), compound 5 (blue), and compound 6 (indigo). (B) Difference spectra that the aromatic molecules did not inhibit aggregation of HEWL generally, but more specifically its obtained by subtracting the spectrum of CR alone from the spectra of HEWL aggregates with CR process of amyloid fibril formation. reported in (A). Color code as in (A). 2.4. Effect of 1,3,5-Triarylbenzenes and Their Derivatives on Heat-Induced Fibrillation of HEWL Monitored with2.4. FTIR Effect Spectroscopy of 1,3,5-Triarylbenzenes and Their Derivatives on Heat-Induced Fibrillation of HEWL Monitored with FTIR Spectroscopy New samples of HEWL aggregates with or without compounds 1–6 at a concentration of 0.32 µM New samples of HEWL aggregates with or without compounds 1–6 at a concentration of 0.32 were prepared, under the same conditions as those used for ThT and CR experiments, centrifuged µM were prepared, under the same conditions as those used for ThT and CR experiments, after 48 h to collect the aggregates, resuspended at high concentration in D2O and analyzed with FTIR centrifuged after 48 h to collect the aggregates, resuspended at high concentration in D2O and spectroscopy,analyzed with to determineFTIR spectroscopy, the effects to ofdetermine the six compounds the effects of on the the six secondary compounds structure on the ofsecondary the HEWL aggregates. The HEWL aggregates without compounds showed a clear maximum at 1624 1 cm 1 structure of the HEWL aggregates. The HEWL aggregates without compounds showed a± clear − inmaximum the amide at I region1624 ± of1 cm the−1 FTIRin the spectrum,amide I region which of the can FTIR readily spectrum, be assigned which to can intermolecular readily be assignedβ-sheet 1 structureto intermolecular (Figure4). β-sheet Another structure peak is(Figure present 4). Another at ca. 1650 peak cm is present− attributable at ca. 1650 to cm either−1 attributable unordered to or α-helicaleither unordered secondary or structure. α-helical secondary All the HEWL structure. aggregates All the HE formedWL aggregates in the presence formed of in the the compounds presence 1 1 showedof the acompounds lower absorbance showed a peak lower at absorbance 1623–1625 peak cm− atand 1623–1625 a higher cm absorption−1 and a higher at 1648–1652 absorption cm at − , indicating1648–1652 loss cm of−1, βindicating-sheet structure loss of β and-sheet gain structure of other and types gain ofof secondaryother types structureof secondary in the structure aggregates in (Figurethe aggregates4). In particular, (Figure 4). compound In particular,2 led compound to the most 2 led remarkable to the most change, remarkable followed change, by followed compound by 1, compoundcompound4, 1, compound compound3 ,4, compound compound5 3,, andcompound compound 5, and6, acompound ranking similar 6, a ranking to that similar observed to that with ThTobserved fluorescence. with ThT fluorescence.

FigureFigure 4. 4.Inhibitory Inhibitory e ffeffectsects of of the the compounds compounds 11–6–6 on the formation of of ββ-sheet-sheet secondary secondary structure structure of of HEWLHEWL monitored monitored by by FTIR. FTIR. HEWL HEWL amyloidamyloid fibrilfibril fo formationrmation was was pre-incubated pre-incubated at at2 mg/mL 2 mg/mL (140 (140 µM),µM), inin 50 50 mM mM glycine glycine bubuffer,ffer, pHpH 2.5, and and 57 57 °C,◦C, under under stirring stirring (250 (250 rpm), rpm), for 48 for h in 48 the hin presence the presence of 0.32 of 0.32µMµ Mcompounds compounds 1–6,1 –centrifuged6, centrifuged at 13,000 at 13,000g for 10g minfor 10and min resuspended and resuspended in D2O to in a DfinalO toHEWL a final × 2 HEWLconcentration concentration of 27.8 of 27.8mg/mL. mg/mL. Blank-subtracted, Blank-subtracted, baseline-corrected, baseline-corrected, and and normalized normalized FTIR FTIR spectra spectra (amide(amide I region) I region) of HEWLof HEWL aggregates aggregates without without (black) (black) or with or compoundwith compound1 (red), 1 compound(red), compound2 (orange), 2 compound(orange), 3compound(yellow), 3 compound (yellow), compound4 (green), compound4 (green), compound5 (blue), and5 (blue), compound and compound6 (indigo). 6 (indigo).

2.5. Effect of the Compounds on the Kinetic of HEWL Aggregation The time course of HEWL amyloid fibril formation in the absence of the compounds monitored by measuring the ThT fluorescence emission intensity over a period of 48 h was nucleation dependent, with a typical sigmoidal profile, including lag, exponential, and equilibrium phases [8]. This kinetic trace was meant to be qualitative rather than quantitative and, for this reason, data were

Int. J. Mol. Sci. 2019, 20, 5558 7 of 18

2.5. Effect of the Compounds on the Kinetic of HEWL Aggregation The time course of HEWL amyloid fibril formation in the absence of the compounds monitored by measuring the ThT fluorescence emission intensity over a period of 48 h was nucleation dependent, with a typical sigmoidal profile, including lag, exponential, and equilibrium phases [8]. This kinetic traceInt. wasJ. Mol. meant Sci. 2019 to, 20 be, x qualitativeFOR PEER REVIEW rather than quantitative and, for this reason, data were acquired7 of only19 every 6 h until 48 h. Inhibition of amyloid aggregation by the various inhibitors is generally quantified acquired only every 6 h until 48 h. Inhibition of amyloid aggregation by the various inhibitors is bygenerally assessing quantified changes of by the assessing lag time, changes exponential of the phase,lag time, and exponential aggregation phase, extent and at aggregation equilibrium extent plateau. Theat lagequilibrium times measured plateau. in The the lag presence times measured of compounds in the1 –presen5, thusce except of compounds compound 1–5,6, wasthus relativelyexcept longcompound (Figure5 ).6, Inwas addition, relatively in long presence (Figure of all5). In compounds, addition, in again presence except of all compound compounds,6, the again exponential except phasecompound changed 6, butthe exponential with different phase magnitudes changed but (Figure with5 different). magnitudes (Figure 5).

800

HEWL Agg 700 HEWL Agg + C1 HEWL Agg + C2 HEWL Agg + C3 600 HEWL Agg + C4 HEWL Agg + C5 HEWL Agg + C6 500

400

300

200

100 ThT fluorescence intensity (a.u.) intensity fluorescence ThT 0 0 1020304050 Time (h)

FigureFigure 5. 5.Kinetics Kinetics of of HEWL HEWL aggregation aggregation at 2 mg/mL mg/mL (140 (140 µMµM),), in in 50 50 mM mM glycine glycine buffer, buﬀer, pH pH 2.5, 2.5, 57 57°C,◦ C, underunder stirring stirring (250 (250 rpm), rpm), in in the the presence presence ofof 0.320.32 µµMM of compounds 1–61–6 followedfollowed by by monitoring monitoring ThT ThT ﬂuorescencefluorescence intensity. intensity.The The kinetickinetic tracestraces referrefer to HEWL without without compounds compounds (black), (black), HEWL HEWL with with compoundcompound1 (red),1 (red), HEWL HEWL with with compound compound 22 (orange), HEWL HEWL with with compound compound 3 3(yellow),(yellow), HEWL HEWL with with compoundcompound4 (green),4 (green), HEWL HEWL with with compound compound 55 (blue),(blue), and HEWL HEWL with with compound compound 6 6(indigo).(indigo).

2.6.2.6. Eff Effectect of of the the Compounds Compounds on on HEWL HEWL AggregationAggregation Morphology InIn order order to to define define further further the the nature nature ofof thethe aggregated species, species, an an analysis analysis of of their their morphologies morphologies waswas carried carried out out using using AFM. AFM. HEWL HEWL was was incubated incubated under under the the same same aggregation aggregation conditions conditions used used for for the ThT,the CR, ThT, and CR, FTIR andexperiments, FTIR experiments, i.e., for i.e., 48 for h at 48 a h concentration at a concentration of 2 mg of /2mL mg/mL (140 µ (140M) inµM) 50 in mM 50 glycinemM buglycineffer, pH buffer, 2.5, and pH 57 2.5,◦C, and under 57 stirring°C, under (250 stirring rpm), (250 in the rpm), absence in the or absence presence or ofpresence 0.32 µM of of 0.32 compounds µM of 1–6compoundsand the resulting 1–6 and samples the resulting were samples examined were by AFMexamined (Figure by 6AFM). HEWL (Figure aggregates 6). HEWL in aggregates the absence in of compoundsthe absence showed of compounds typical long,showed unbranched typical long, fibrils. unbranched On the fibrils. other hand, On the when other HEWL hand, when was incubated HEWL inwas presence incubated of compounds in presence1– of4, verycompounds few fibrils 1–4, werevery observedfew fibrils after were 48 observed h. Rather, after oligomeric 48 h. Rather, and/ or amorphousoligomeric species and/or wereamorphous predominant. species HEWLwere predominant. pre-incubated HEWL in presence pre-incubated of compound in presence5 showed of a fewcompound short fibrillar 5 showed assemblies a few alongshort withfibrillar oligomeric assemblies species along and, with in oligomeric the presence species of compound and, in the6 , a mixturepresence of unbranchedof compound filamentous 6, a mixture assemblies of unbranched appeared. filamentous These resultsassemblies again appeared. showed thatThese compound results again showed that compound 6 cannot inhibit the formation of HEWL aggregates in agreement with 6 cannot inhibit the formation of HEWL aggregates in agreement with the ThT and CR results. the ThT and CR results. 2.7. Effect of the Compounds on HEWL Amyloid Induced Cytotoxicity We then assessed the cytotoxicity of HEWL aggregates, formed in the absence or presence of compounds 1–6, according to the aggregation protocol described for the ThT experiments. HEWL aggregates were collected by centrifugation, dried under N2, dissolved in cell culture medium, and

Int. J. Mol. Sci. 2019, 20, 5558 8 of 18 added to SH-SY5Y human neuroblastoma cells at a concentration of 2 µM (monomer equivalents). The ability of the resulting aggregates to cause cellular dysfunction was assessed by performing the MTT reduction inhibition assay, a widely used indicator of mitochondrial dysfunction [45]. The ability of SH-SY5Y cells to reduce MTT was significantly decreased to 73.9 2.1% (relative to untreated cells) ± after a 24 h exposure to HEWL aggregates, whereas it was unaffected by native HEWL treatment (Figure7A). The ability of cells to reduce MTT was significantly increased when HEWL aggregates were formed in the presence of compound 1 (to 92.7 3.2%), 2 (to 96.2 3.2%) and 5 (to 93.9 2.9%). ± ± ± By contrast, incubation with compounds 3, 4, and 6 resulted in a partial rescue of the cell viability. Very similar results were obtained by measuring the activity of caspase-3, a well-recognized apoptotic marker [46]. A significant activation of caspase-3 (320 12%, as compared to untreated cells) was ± observed following treatment for 24 h of SH-SY5Y cells with HEWL aggregates at 2 µM (Figure7B). Moreover, the presence of compounds 1, 2, 4, and 5 during HEWL aggregation significantly prevented the apoptotic response induced by aggregates (Figure7B). Furthermore, in this case, compounds 3 and 6 generatedInt. J. Mol. Sci. only 2019, a 20 slight, x FOR protective PEER REVIEW eff ect on apoptotic pathway activation. 8 of 19

HEWL Agg without compounds

HEWL Agg + C1 HEWL Agg + C2 HEWL Agg + C3

HEWL Agg + C4 HEWL Agg + C5 HEWL Agg + C6

FigureFigure 6. 6.Atomic Atomic force force microscopy microscopy (AFM)(AFM) imagesimages of HEWL HEWL aggregates aggregates formed formed in in absence absence or orpresence presence ofof compounds compounds1– 1–6.6. HEWL HEWL was was pre-incubated pre-incubated under conditions promoting promoting amyloid amyloid fibril ﬁbril formation formation

thatthat is 48is 48 h ath at a concentrationa concentration of of 140 140µ µM,M, inin 5050 mMmM glycine buffer, buﬀer, pH pH 2.5, 2.5, 57 57 °C,◦C, under under stirring stirring (250 (250 rpm),rpm), in in the the absence absence or or presence presence of of 0.32 0.32µ µMM compoundscompounds 11–6.–6.

2.7. Effect of the Compounds on HEWL Amyloid Induced Cytotoxicity We then assessed the cytotoxicity of HEWL aggregates, formed in the absence or presence of compounds 1–6, according to the aggregation protocol described for the ThT experiments. HEWL aggregates were collected by centrifugation, dried under N2, dissolved in cell culture medium, and added to SH-SY5Y human neuroblastoma cells at a concentration of 2 µM (monomer equivalents). The ability of the resulting aggregates to cause cellular dysfunction was assessed by performing the MTT reduction inhibition assay, a widely used indicator of mitochondrial dysfunction [45]. The ability of SH-SY5Y cells to reduce MTT was significantly decreased to 73.9 ± 2.1% (relative to untreated cells) after a 24 h exposure to HEWL aggregates, whereas it was unaffected by native HEWL treatment (Figure 7A). The ability of cells to reduce MTT was significantly increased when HEWL aggregates were formed in the presence of compound 1 (to 92.7 ± 3.2%), 2 (to 96.2 ± 3.2%) and 5 (to 93.9 ± 2.9%). By contrast, incubation with compounds 3, 4, and 6 resulted in a partial rescue of the cell viability. Very similar results were obtained by measuring the activity of caspase-3, a well-

Int. J. Mol. Sci. 2019, 20, 5558 9 of 18 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 10 of 19

. Figure 7. Impairment of cell viability after exposure to HEWL aggregates. (A) MTT reduction in SH-SY5YFigure 7. cells Impairment treated for of 24cell h viability with 2 µ afterM (monomer exposure equivalents)to HEWL aggregates. HEWL aggregates (A) MTT reduction formed in in the SH- absenceSY5Y cells or presence treated for of the24 h indicated with 2 µM compounds. (monomer eq (Buivalents)) Caspase-3 HEWL activity aggregates in SH-SY5Y formed cells in treated the absence for 24or h presence with 2 µ Mof HEWLthe indicated aggregates compounds. formed (B in) theCaspase-3 absence activity or presence in SH-SY5 of theY cells indicated treated compounds. for 24 h with The2 µM green HEWL ﬂuorescence aggregates arises formed from intracellularin the absence caspase-3 or presence activity. of the The indicate resultsd ofcompounds. a semi-quantitative The green analysisfluorescence of the arises caspase-3-derived from intracellula ﬂuorescencer caspase-3 data activity. are expressed The results as theof a percentagesemi-quantitative of the valueanalysis for of untreatedthe caspase-3-derived cells. In both panels, fluorescence experimental data are errors expresse are standardd as the errorpercentage of mean of (S.E.M).the value Samples for untreated were analyzedcells. In byboth one-way panels, ANOVA experimental followed errors by Bonferroni’sare standard multiple error of comparisonmean (S.E.M). test Samples relative were to untreated analyzed cellsby (*one-wayp < 0.05, ANOVA ** p < 0.01, followed *** p < by0.001), Bonferroni’s or to cells multiple treated comparison with HEWL test aggregates relative to in untreated the absence cells of (* compoundsp < 0.05, ** (§ p << 0.05,0.01, §§***p p< 0.01,< 0.001), §§§ por< to0.001). cells treated with HEWL aggregates in the absence of compounds (§ p < 0.05, §§ p < 0.01, §§§ p < 0.001).

Int. J. Mol. Sci. 2019, 20, 5558 10 of 18

3. Discussion Design and synthesis of novel small-compound inhibitors of the process of amyloid fibril formation is one of the leading therapeutic approaches for the treatment of amyloid diseases [47–50]. The studies on different amyloidogenic proteins showed that small aromatic compounds are able to inhibit fibril formation, possibly due to their ability to interfere with the β-sheet architecture of the fibrils and/or aromatic stacking in fibril stabilization [22,30,51,52]. In fact, the exact contribution of aromaticity to amyloid formation is controversial and remains under debate [53]. Various findings demonstrate that the functional groups appended to the aromatic rings [54,55], geometry and steric profile of the functional groups on the aromatic rings [53], and also number of rings [36] can dramatically affect amyloid formation. We have recently reported that bis(indolyl)phenylmethane compounds inhibited the formation of amyloid fibrils and associated toxicity in a concentration-dependent manner [33]. We also showed that all tested compounds were able to inhibit HEWL amyloid fibril formation but with different efficiencies, which showed the importance of slight variations of the functional groups within the same scaffold as a small molecule [33]. In this work, a series of novel 1,3,5-triarylbenzenes derivatives having four aromatic rings and different electron-donor or -acceptor substitutions were synthesized and evaluated for their potential to prevent HEWL amyloid aggregation. These compounds, similar to bis(indolyl)phenylmethanes studied previously, were shown to inhibit HEWL amyloid fibril formation under conditions in which the protein is initially partially unfolded, but with different magnitude. In particular, considering the various techniques used here, including the ThT and CR assays, AFM, and FTIR, compounds 1–3 were found to be clearly more effective as inhibitors in comparison to compounds 4–6, particularly the latter that did not show any detectable effect (Figures1–5). These findings showed that electron-donating substituents have less efficacy in preventing HEWL amyloid formation, whereas electron-withdrawing groups have much more influence in amyloid inhibition. The IC50 values of the compounds were between 0.05 and 0.98 µM, which are two orders of magnitude lower than the IC50 value measured by a representative bis(indolyl)phenylmethane, i.e., 12.3 1 µM[33]. ± The amorphous and unstructured HEWL aggregates formed in the presence of all compounds were less toxic than the HEWL fibrils formed in their absence, as shown by MTT cell viability and caspase activity assays (Figure7). Moreover, our results showed that HEWL aggregates formed in the presence of 1,3,5-triarylbenzenes derivatives, similar to results obtained with bis(indolyl)phenylmethane [33] and curcumin [56] derivatives, were non-toxic or retain a weak toxicity, allowing all these compounds to be classified as class I molecules, based on the classification proposed by Ladiwala et al., i.e., molecules that remodel soluble oligomers into species (often large off-pathway aggregates) that are non-toxic [57]. It has been proposed that π–π stacking of aromatic rings can be enhanced when two rings are electron poor and rich, respectively [36]. Our work and results presented by other scientists [36] are in agreement with this theory, as the addition of a strong electron-withdrawing group, such as –NO2, –F, or –CN [36] on the aromatic inhibitor increases the inhibitory effect, possibly due to the formation of a strong electron donor–electron acceptor complex between the inhibitory compounds and the aromatic residues of HEWL. In particular, the –F group was found to be the most effective, probably due to its high electronegativity, followed by the –NO2 group. On the other hand, when an electron-donating group, such as a –OH, –CH3, or –OCH3, was added, amyloid inhibition is decreased due to a repulsion between the electron-rich aromatic ring of the inhibitor and the electron-rich aromatic residues of HEWL, which does not allow the establishment of a stable complex between the inhibitor and the protein undergoing aggregation. In this context, the –OH group is the least effective in inhibiting HEWL aggregation. Inhibitory effects of bis(indolyl)phenylmethanes may be discussed from different points of view, including different factors, such as the importance of inter- and intra-molecular hydrogen bonds, electronic effects and steric hindrances, the intrinsic steric congestion originating from the structural framework of bis-indolylarylmethanes, all affected by the substituents. In the case of Int. J. Mol. Sci. 2019, 20, 5558 11 of 18

1,3,5-triarylbenzenes, a fully planar formula presents three aryl substituents in the same plane as the central benzene ring. In this case, only the aryl substituents may exert a little steric congestion around the aryl rings. Therefore, the net structure should have a planar identity and we can assume an effective diffusion of these types of compounds into amyloid fibrils. This can explain both the most effective inhibitory potential of 1,3,5-triarylbenzenes relative to bis-indolylarylmethanes and the critical role played by the electron releasing/donating substituents on the aryl rings in determining such an inhibition.

4. Materials and Methods

4.1. Materials Hen egg white lysozyme (HEWL), thioflavin T (ThT), and Congo red (CR) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other reagents were purchased from Merck (Darmstadt, Germany), unless stated otherwise. All synthesized molecules were compared, in terms of their spectral and Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 19 physical data, with those reported previously [58]. 4.2. General Chemistry 4.2. General Chemistry All reagents and starting materials were commercially available and were used as received. All reagents and starting materials were commercially available and were used as received. Natural clinoptilolite-rich tuffs were obtained from the Sabzevar region in the north-east of Iran. Natural clinoptilolite-rich tuffs were obtained from the Sabzevar region in the north-east of Iran. Fourier-transform infrared (FT-IR) spectra were recorded on a 8700 Shimadzu Fourier-transform Fourier-transform infrared (FT-IR) spectra were recorded on a 8700 Shimadzu Fourier-transform −1 1 spectrophotometer (Shimadzu, Tokyo, Japan) in the region of 400–4000 cm1 using KBr pellets.1 H spectrophotometer (Shimadzu, Tokyo, Japan) in the region of 400–4000 cm− using KBr pellets. H and and 13C NMR spectra were recorded on a Bruker AVANCE 300 MHz instrument (Bruker, MA, USA) 13C NMR spectra were recorded on a Bruker AVANCE 300 MHz instrument (Bruker, MA, USA) using using TMS as internal reference. All products were identified by comparison of their spectral and TMS as internal reference. All products were identified by comparison of their spectral and physical data physical data with those previously reported. Wells–Dawson diphosphooctadecatungstic acid with those previously reported. Wells–Dawson diphosphooctadecatungstic acid H6P2W18O62 24H2O H6P2W18O62·24H2O was prepared according to the literature method [59]. · was prepared according to the literature method [59].

4.2.1.General General Procedure Procedure for the for Conversion the Conversion of Acetophenone of Acetophenone into 1,3,5-Triphenylbenzene into 1,3,5-Triphenylbenzene In a typical typical reaction, reaction, acetophenone acetophenone (1 (1 mmoL) mmoL) and and the the surface-modified surface-modified HPA/NCP HPA/NCP (15 mg) were added to a small test tube and the reaction mixture was stirred for 2.5 h at 100 °C. After completion added to a small test tube and the reaction mixture was stirred for 2.5 h at 100 ◦C. After completion of the reaction, reaction, as as indicated indicated by by thin thin layer layer chromato chromatographygraphy (TLC), (TLC), the the reaction reaction mass mass was was cooled cooled to 25 to °C; then, hot ethanol was added to the reaction mixture and the mixture was stirred for 5 min. The 25 ◦C; then, hot ethanol was added to the reaction mixture and the mixture was stirred for 5 min. insolubleThe insoluble catalyst catalyst was wasisolated isolated via viasimple simple filtration. filtration. The The filtrate filtrate containing containing water water as as the the only byproduct ofof the the cyclotrimerization cyclotrimerization was was concentrated concentrated under under reduced reduced pressure, pressure, and finally andthe finally obtained the obtained crude product was purified through re-crystallization in EtOH:H2O (3:1) as confirmed by crude product was purified through re-crystallization in EtOH:H2O (3:1) as confirmed by an intense ansingle intense spot insingle TLC. spot The in pure TLC. products The pure were products specified were based specified on the spectral based dataon the and spectral determination data and of determinationtheir melting points. of their melting points.

4.2.1.1.1,3,5-Triphenylbenzene 1,3,5-Triphenylbenzene (Compound (Compound1) 1)

−11 Light yellow solid; m.m. p. 171–173 ◦°C.C. IRIR (KBr):(KBr): vvmaxmax == 3053,3053, 1651, 1651, 1494, 1494, 1431, 1431, 1125, 758, 700 cm− .. 1 HH NMR NMR (DMSO-d (DMSO-d6,, 500 500 MHz): MHz): δδ 7.887.88 (s, (s, 3H), 3H), 7.87–7.86 7.87–7.86 (m, 6H), 7. 7.52–7.4952–7.49 (t, J = 7.57.5 Hz, Hz, 6H), 6H), 7.43–7.40 7.43–7.40 1313 (t, J = 7.57.5 Hz, Hz, 3H). 3H). CC NMR NMR (DMSO-d (DMSO-d6,6 ,125 125 MHz): MHz): δ δ141.6,141.6, 140.1, 140.1, 128.9, 128.9, 127.7, 127.7, 127.2, 127.2, 124.4. 124.4.

4.2.1.2. 1,3,5-Tris(4-Fluorophenyl)Benzene (Compound 2)

White solid; m. p. 238–240 °C. IR (KBr): vmax = 3059, 1604, 1508, 1449, 1224, 1159, 825, 771 cm−1. 1H NMR (CDCl3, 500 MHz): δ 7.67 (s, 3H), 7.61 (d, J = 8.5 Hz, 6H), 7.59 (d, J = 8.5 Hz, 6H). 13C NMR (CDCl3, 125 MHz): 161.7, 137.6, 132.4, 129.0 (d, J = 7.5 Hz), 125.3, 115.9 (d, J = 22.5 Hz). F

4.2.1.3. 1,3,5-Tris(4-Nitrophenyl)Benzene (Compound 3)

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 19

4.2. General Chemistry All reagents and starting materials were commercially available and were used as received. Natural clinoptilolite-rich tuffs were obtained from the Sabzevar region in the north-east of Iran. Fourier-transform infrared (FT-IR) spectra were recorded on a 8700 Shimadzu Fourier-transform spectrophotometer (Shimadzu, Tokyo, Japan) in the region of 400–4000 cm−1 using KBr pellets. 1H and 13C NMR spectra were recorded on a Bruker AVANCE 300 MHz instrument (Bruker, MA, USA) using TMS as internal reference. All products were identified by comparison of their spectral and physical data with those previously reported. Wells–Dawson diphosphooctadecatungstic acid H6P2W18O62·24H2O was prepared according to the literature method [59].

4.2.1. General Procedure for the Conversion of Acetophenone into 1,3,5-Triphenylbenzene In a typical reaction, acetophenone (1 mmoL) and the surface-modified HPA/NCP (15 mg) were added to a small test tube and the reaction mixture was stirred for 2.5 h at 100 °C. After completion of the reaction, as indicated by thin layer chromatography (TLC), the reaction mass was cooled to 25 °C; then, hot ethanol was added to the reaction mixture and the mixture was stirred for 5 min. The insoluble catalyst was isolated via simple filtration. The filtrate containing water as the only byproduct of the cyclotrimerization was concentrated under reduced pressure, and finally the obtained crude product was purified through re-crystallization in EtOH:H2O (3:1) as confirmed by an intense single spot in TLC. The pure products were specified based on the spectral data and determination of their melting points.

4.2.1.1. 1,3,5-Triphenylbenzene (Compound 1)

Light yellow solid; m. p. 171–173 °C. IR (KBr): vmax = 3053, 1651, 1494, 1431, 1125, 758, 700 cm−1. 1H NMR (DMSO-d6, 500 MHz): δ 7.88 (s, 3H), 7.87–7.86 (m, 6H), 7.52–7.49 (t, J = 7.5 Hz, 6H), 7.43–7.40 (t, J = 7.5 Hz, 3H).13C NMR (DMSO-d6, 125 MHz): δ 141.6, 140.1, 128.9, 127.7, 127.2, 124.4.

Int. J. Mol. Sci. 2019, 20, 5558 12 of 18

1,3,5-Tris(4-Fluorophenyl)Benzene (Compound 2) 4.2.1.2. 1,3,5-Tris(4-Fluorophenyl)Benzene (Compound 2) 1 White solid; m. p. 238–240 ◦C. IR (KBr): vmax = 3059, 1604, 1508, 1449, 1224, 1159, 825, 771 cm− . White solid; m. p. 238–240 °C. IR (KBr): vmax = 3059, 1604, 1508, 1449, 1224, 1159, 825, 771 cm−1. 1 13 H NMR (CDCl3, 500 MHz): δ 7.67 (s, 3H), 7.61 (d, J = 8.5 Hz, 6H), 7.59 (d, J = 8.5 Hz, 6H). C NMR 1H NMR (CDCl3, 500 MHz): δ 7.67 (s, 3H), 7.61 (d, J = 8.5 Hz, 6H), 7.59 (d, J = 8.5 Hz, 6H). 13C NMR (CDCl3, 125 MHz): 161.7, 137.6, 132.4, 129.0 (d, J = 7.5 Hz), 125.3, 115.9 (d, J = 22.5 Hz). (CDCl3, 125 MHz): 161.7, 137.6, 132.4, 129.0 (d, J = 7.5 Hz), 125.3, 115.9 (d, J = 22.5 Hz). F

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 13 of 19 Int.1,3,5-Tris(4-Nitrophenyl)Benzene4.2.1.3. J. Mol. 1,3,5-Tris(4-Nitrophenyl)Benzene Sci. 2019, 20, x FOR PEER REVIEW (Compound (Compound3) 3) 13 of 19 1 1 White solid; m. p. 151–152 C. IR (KBr): v = 3043, 1595, 1489, 1379, 1085, 1007, 849 cm −1. 1H White solid; m. p. 151–152 ◦°C. IR (KBr): vmaxmax = 3043, 1595, 1489, 1379, 1085, 1007, 849 cm− . H White solid; m. p. 151–152 °C. IR (KBr): vmax = 3043, 1595, 1489, 1379,13 1085, 1007, 849 cm−1. 1H NMR (CDCl ): δ = 7.56 (d, J 8.1 Hz, 6H), 7.62 (d, J 8.2 Hz, 6H), 7.70 (s, 3H). 13 C NMR (125 MHz, CDCl ): NMR (CDCl33): δ = 7.56 (d, J 8.1 Hz, 6H), 7.62 (d, J 8.2 Hz, 6H), 7.70 (s, 3H). C NMR (125 MHz, CDCl3): NMR (CDCl3): δ = 7.56 (d, J 8.1 Hz, 6H), 7.62 (d, J 8.2 Hz, 6H), 7.70 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 122.3, 125.0, 128.9, 132.1, 139.7, 141.6. δ 122.3, 125.0, 128.9, 132.1, 139.7, 141.6.

NO2 NO2

O2N NO2 O2N NO2

4.2.1.4.1,3,5-Tris(4-Methylphenyl)Benzene 1,3,5-Tris(4-Methylphenyl)Benzene (Compound (Compound4) 4) 4.2.1.4. 1,3,5-Tris(4-Methylphenyl)Benzene (Compound 4) 1 1 White solid; m. p. 178–179 ◦C. IR (KBr): vmax = 3017, 2922, 1612, 1511, 1491, 1109, 812 cm−−1. 1H White solid; m. p. 178–179 °C. IR (KBr): vmax = 3017, 2922, 1612, 1511, 1491, 1109, 812 cm−1. 1H White solid; m. p. 178–179δ °C. IR (KBr): vmax = 3017, 2922, 1612, 1511, 1491, 1109, 812 cm .13 H NMR (CDCl33, 500 MHz): δ 7.74 (s, 3H), 7.62 (d, JJ == 8.0 Hz, 6H), 7.30 (d, JJ == 7.5 Hz, 6H), 2.44 (s,(s, 9H).9H). 13C NMR (CDCl3, 500 MHz): δ 7.74 (s, 3H), 7.62 (d, J = 8.0 Hz, 6H), 7.30 (d, J = 7.5 Hz, 6H), 2.44 (s, 9H). 13C NMR (CDCl 3,, 125 125 MHz): MHz): 142.2, 142.2, 138.3, 138.3, 137.4, 137.4, 129.4, 129.4, 127.3, 127.3, 124.7, 124.7, 21.1. 21.1. NMR (CDCl3, 125 MHz): 142.2, 138.3, 137.4, 129.4, 127.3, 124.7, 21.1. CH3 CH3

H3CCH3 H3CCH3 4.2.1.5. 1,3,5-Tris(4-Methoxyphenyl)Benzene (Compound 5) 4.2.1.5.1,3,5-Tris(4-Methoxyphenyl)Benzene 1,3,5-Tris(4-Methoxyphenyl)Benzene (Compound (Compound5) 5) White solid; m. p. 142–143 °C. IR(KBr): vmax = 2953, 2918, 2848, 1598, 1460, 1378, 1263, 1097, 802 White solid; m. m. p. p. 142–143 142–143 °C.◦C. IR(KBr): IR(KBr): vmax vmax = 2953,= 2953, 2918, 2918, 2848, 2848, 1598, 1598, 1460, 1460, 1378, 1378, 1263, 1263, 1097, 1097, 802 cm−1. 1H 1NMR1 (CDCl3, 500 MHz): δ 7.59 (d, J = 9.0 Hz, 6H), 7.37 (s, 3H), 6.99 (d, J = 8.5 Hz, 6H), 3.86 cm802−1 cm. 1H− NMR. H NMR (CDCl (CDCl3, 5003 MHz):, 500 MHz): δ 7.59δ (d,7.59 J = (d, 9.0 J =Hz,9.0 6H), Hz, 7.37 6H), (s, 7.37 3H), (s, 6.99 3H), (d, 6.99 J = (d, 8.5 J =Hz,8.5 6H), Hz, 6H),3.86 (s, 9H). 13C NMR13 (CDCl3, 125 MHz): 159.7, 141.7, 134.0, 129.3, 122.7, 114.2, 55.9. (s,3.86 9H). (s, 9H).13C NMRC NMR (CDCl (CDCl3, 1253 MHz):, 125 MHz): 159.7, 159.7, 141.7, 141.7, 134.0, 134.0, 129.3, 129.3, 122.7, 122.7, 114.2, 114.2, 55.9. 55.9. OCH3 OCH3

H3CO OCH3 H3CO OCH3

4.2.1.6. 1,3,5-Tris(4-Hydroxyphenyl)Benzene (Compound 6) 4.2.1.6. 1,3,5-Tris(4-Hydroxyphenyl)Benzene (Compound 6) White solid; m. p. 237–239 °C. IR (KBr): vmax = 2953, 2918, 2848, 1606, 1460, 1378, 1263, 1097, 802 White solid; m. p. 237–239 °C. IR (KBr): vmax = 2953, 2918, 2848, 1606, 1460, 1378, 1263, 1097, 802 cm−1. 1H NMR (CDCl3): δ = 3.81 (s, 9H), 6.94–7.41 (m, 12H), 7.79 (s, 3H). 13C NMR (CDCl3, 125 MHz): cm−1. 1H NMR (CDCl3): δ = 3.81 (s, 9H), 6.94–7.41 (m, 12H), 7.79 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 55.4, 114.3, 123.8, 128.3, 133.9, 141.8, 159.3. δ 55.4, 114.3, 123.8, 128.3, 133.9, 141.8, 159.3.

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 13 of 19

White solid; m. p. 151–152 °C. IR (KBr): vmax = 3043, 1595, 1489, 1379, 1085, 1007, 849 cm−1. 1H NMR (CDCl3): δ = 7.56 (d, J 8.1 Hz, 6H), 7.62 (d, J 8.2 Hz, 6H), 7.70 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 122.3, 125.0, 128.9, 132.1, 139.7, 141.6.

NO2

O2N NO2

4.2.1.4. 1,3,5-Tris(4-Methylphenyl)Benzene (Compound 4)

White solid; m. p. 178–179 °C. IR (KBr): vmax = 3017, 2922, 1612, 1511, 1491, 1109, 812 cm−1. 1H NMR (CDCl3, 500 MHz): δ 7.74 (s, 3H), 7.62 (d, J = 8.0 Hz, 6H), 7.30 (d, J = 7.5 Hz, 6H), 2.44 (s, 9H). 13C NMR (CDCl3, 125 MHz): 142.2, 138.3, 137.4, 129.4, 127.3, 124.7, 21.1.

CH3

H3CCH3

4.2.1.5. 1,3,5-Tris(4-Methoxyphenyl)Benzene (Compound 5)

White solid; m. p. 142–143 °C. IR(KBr): vmax = 2953, 2918, 2848, 1598, 1460, 1378, 1263, 1097, 802 Int. J. Mol. Sci. 2019, 20, 5558 13 of 18 cm−1. 1H NMR (CDCl3, 500 MHz): δ 7.59 (d, J = 9.0 Hz, 6H), 7.37 (s, 3H), 6.99 (d, J = 8.5 Hz, 6H), 3.86 (s, 9H). 13C NMR (CDCl3, 125 MHz): 159.7, 141.7, 134.0, 129.3, 122.7, 114.2, 55.9.

OCH3

H3CO OCH3

4.2.1.6.1,3,5-Tris(4-Hydroxyphenyl)Benzene 1,3,5-Tris(4-Hydroxyphenyl)Benzene (Compound (Compound6) 6)

White solid; m. m. p. p. 237–239 237–239 °C.◦C. IR IR (KBr): (KBr): vmax vmax = 2953,= 2953, 2918, 2918, 2848, 2848, 1606, 1606, 1460, 1460, 1378, 1378, 1263, 1263, 1097, 1097, 802 −1 1 1 1 13 13 cm802 cm. H− NMR. H (CDCl NMR3 (CDCl): δ = 3.813): δ(s,= 9H),3.81 6.94–7.41 (s, 9H), 6.94–7.41 (m, 12H), (m, 7.79 12H), (s, 3H). 7.79 C (s, NMR 3H). (CDClC NMR3, 125 (CDCl MHz):3, 125δInt. 55.4, J. MHz): Mol. 114.3, Sci.δ 2019 55.4,123.8,, 20 114.3,, x128.3, FOR 123.8,PEER 133.9, REVIEW 128.3, 141.8, 133.9, 159.3. 141.8, 159.3. 14 of 19

HO OH

4.3. Protein Protein Aggregation 1 1 HEWL concentration waswas determineddetermined by by optical optical absorption absorption at at 280 280 nm nm (ε (ε280= =2.65 2.65 L L·gg −1·cmcm −1))[ [60].60]. 280 · − · − HEWL was dissolved to to a a concentration of of 2 2 mg/m mg/mLL (140 (140 µM)µM) in in 50 50 mM mM glycine glycine buffer, buffer, pH pH 2.5, 2.5, centrifuged for 10 min at 13,000 rpm, then incubate incubatedd at 57 ◦°CC for the specifiedspecified time lengths in the absence oror presencepresence of of the the indicated indicated concentrations concentrations of of compounds compounds1–6 1–6,, under under gentle gentle stirring stirring at 250 at rpm250 rpmby a Teflonby a Teflon magnetic magnetic bar [ 33bar]. [33].

4.4. ThT Fluorescence Assay A mother solution of 2.5 mM ThT was prepared in 10 mM sodium phosphate buffer, buffer, 150 mM NaCl, pH 7.0. It It was was then then passed through a 0.45 µmµm filter filter paper and stored at 4 ◦°CC until it was placed at room temperature immediately before use. At At regular regular time time intervals, intervals, a a 10 10 µLµL aliquot of protein solution was mixed with 990 µL of ThT solution in a 10 10 mm quartz cuvette and the fluorescence solution was mixed with 990 µL of ThT solution in a 10 × 10 mm quartz cuvette and the fluorescence emission spectra were recorded at room temper temperatureature using a Cary Eclipse VARIAN fluorescencefluorescence spectrophotometer (Varian, Mulgrave,Mulgrave, Australia)Australia) andand anan excitationexcitation wavelengthwavelength ofof 440440 nmnm [[61].61].

4.5. CR Assay Since compounds 1–61–6 have optical absorption sp spectraectra partially overlapped to that of CR, all protein samples were centrifuged at 10,000 rpm an andd the the pellets pellets resuspended resuspended in in 50 50 mM mM glycine glycine buffer, buffer, pH 2.5,2.5, at at room room temperature. temperature. A motherA mother solution solution of 20 of mM 20 CR mM was CR prepared was prepared in 5 mM sodiumin 5 mM phosphate sodium phosphatebuffer, 150 mMbuffer, NaCl, 150 pHmM 7.4, NaCl, it was pH then 7.4, filteredit was then using filtered a center-glass using a N4center-glass filter and N4 stored filter at and 4 ◦C, stored until atit was4 °C, placed until atit was room placed temperature at room immediately temperature before immediately use. At before different use. time At pointsdifferent during time protein points µ µ duringaggregation, protein a 60aggregation,L aliquot a of 60 resuspended µL aliquot of protein resuspended solution protein was mixed solution with was 440 mixedL of CRwith solution. 440 µL ofOptical CR solution. absorption Optical spectra absorption were acquired spectra were after acquired 2–3 min equilibrationafter 2–3 min atequilibration 25 ◦C, using at a25 Cecil °C, using 7200 aUV–visible Cecil 7200 spectrophotometer UV–visible spectrophotometer (Aquarius, Cambridge, (Aquarius, England)Cambridge, and England) a 1 cm path-length and a 1 cm cell.path-length cell.

4.6. FTIR Spectroscopy Samples were incubated for 48 h to promote aggregation as described above in the presence of 0.32 µM compounds 1–6, centrifuged at 13,000× g for 10 min and resuspended in D2O to a final volume of 72.8 µL, to reach a final concentration of 27.8 mg/mL. For each sample, 40 µL was deposited onto an Omni-Cell transmission cell with a 25 µm spacer (Specac, Orpington, UK), and Fourier- transform infrared (FTIR) spectra were recorded using a FTS-40 FTIR spectrometer (Bio-Rad, Hercules, CA, USA) purged with a continuous flow of dry air. Sixty-four interferograms were accumulated in each case at a spectral resolution of 1 cm−1. The FTIR absorbance spectra were blank- subtracted and baseline-corrected to reveal the amide I region (1600–1700 cm−1). For each spectrum, the sharp water peaks in the amide I and flanking regions (1580–1800 cm−1) were eliminated by using a multiple Gaussian function to fit only the experimental data that were devoid of such peaks in this region and replotting the spectrum as the best fitted function. All spectra were normalized to the same amide I region area. Additional smoothing was not carried out.

4.7. Atomic Force Microscopy

Int. J. Mol. Sci. 2019, 20, 5558 14 of 18

4.6. FTIR Spectroscopy Samples were incubated for 48 h to promote aggregation as described above in the presence of 0.32 µM compounds 1–6, centrifuged at 13,000 g for 10 min and resuspended in D O to a final volume × 2 of 72.8 µL, to reach a final concentration of 27.8 mg/mL. For each sample, 40 µL was deposited onto an Omni-Cell transmission cell with a 25 µm spacer (Specac, Orpington, UK), and Fourier-transform infrared (FTIR) spectra were recorded using a FTS-40 FTIR spectrometer (Bio-Rad, Hercules, CA, USA) purged with a continuous flow of dry air. Sixty-four interferograms were accumulated in each 1 case at a spectral resolution of 1 cm− . The FTIR absorbance spectra were blank-subtracted and 1 baseline-corrected to reveal the amide I region (1600–1700 cm− ). For each spectrum, the sharp water 1 peaks in the amide I and flanking regions (1580–1800 cm− ) were eliminated by using a multiple Gaussian function to fit only the experimental data that were devoid of such peaks in this region and replotting the spectrum as the best fitted function. All spectra were normalized to the same amide I region area. Additional smoothing was not carried out.

4.7. Atomic Force Microscopy HEWL samples pre-incubated for 48 h under aggregating conditions with or without 0.32 µM compounds 1–6 were diluted 100 times in ﬁltered deionized water. A 5 µL aliquot of each of the resulting samples was placed on freshly cleaved mica at room temperature. After a few minutes, the mica was slowly washed with 100 µL of deionized water and dried with N2. Each image was acquired in a non-contact mode at a scan speed of 30 µm/s, loop ﬁlter of 3 Hz and force of 200 nN with a Dual Scope Probe Scanner (Veeco, model Auto probe, CP-Research, CA, USA) with an area of 5 5 µm2. × Conical shape silicon tips (MikroMasch NSC16) with a resonance frequency of 150 kHz and a nominal constant of 40 N/m were used. Fifteen images were collected for each compound.

4.8. Cell Cultures Authenticated human neuroblastoma SH-SY5Y cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and they tested negative for mycoplasma contaminations. SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), F-12 Ham with 25 mM HEPES and NaHCO3 (1:1) supplemented with 10% fetal bovine serum (FBS), 1.0 mM glutamine and 1.0% penicillin and streptomycin solution. Cells were maintained in a 5.0% CO2 humidified atmosphere at 37 ◦C and grown until 80% confluence for a maximum of 20 passages.

4.9. 3-(4,5-Dimethylthiazol)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction Test HEWL samples pre-incubated for 48 h under aggregating conditions in the absence or presence of 0.32 µM compounds 1–6 were centrifuged and the pellets were dried under N2 to remove any compounds and DMSO, dissolved in cell culture medium at a ﬁnal HEWL concentration of 2 µM (monomer equivalents), and added to SH-SY5Y cells seeded in 96-well plates for 24 h at 37 ◦C. The cell medium was then removed and the MTT solution (0.5 mg/mL in RPMI) was added to the cells for 4 h. The medium was then aspirated, and the formazan product was solubilized with cell lysis buﬀer (20% SDS, 50% N, N-dimethylformamide, pH 4.7) for 1 h, as previously described [62].

4.10. Measurement of Caspase-3 Activity HEWL aggregates were formed in the absence or in the presence of 0.32 µM of compounds 1–6 as described in the previous section and then added to the cell culture medium of SH-SY5Y cells seeded on glass coverslips for 24 h at 2 µM (monomer equivalents). Caspase-3 activity was then analyzed by using FAM FLICATM caspases 3 & 7 solution (caspase 3 & 7 FLICA kit FAM-DEVDFMK, Immunochemistry Technologies, LLC, Bloomington, MN, USA). Cell ﬂuorescence was analyzed by the TCS SP8 scanning confocal microscopy system (Leica Microsystems, Mannheim, Germany) equipped with an argon laser source, as previously reported [62]. A series of 1.0 µm thick optical sections (1024 1024 pixels) was × Int. J. Mol. Sci. 2019, 20, 5558 15 of 18 taken through the cell depth for each sample using a Leica Plan Apo 63 oil immersion objective × for ﬂuorescence measurement at 488 nm. The confocal microscope was set at optimal acquisition conditions, e.g., pinhole diameters, detector gain, and laser powers. Settings were maintained constant for each analysis.

4.11. Statistical Analysis All data were expressed as means standard error of mean (S.E.M). ANOVA followed by ± Bonferroni’s post comparison test was used to compare diﬀerent values.

5. Conclusions In conclusion, we have provided experimental evidence of different effects of various substituents of the architecture of the 1,3,5-triarylbenzene molecule on the inhibition of the self-assembly of HEWL. We showed that –OH, –OCH3, and –CH3 groups as electron-donor groups positioned on aromatic rings have a low efficacy, whereas –F and –NO2 as electron-withdrawing groups have great efficacy in inhibiting amyloid formation by HEWL. Based on the results of this study and others, it is apparent that small aromatic compounds and functional groups appended on these aromatic molecules can have a profound effect on their ability to inhibit amyloid formation. These results may ultimately find applications in the development of potential amyloid inhibitors and, thus, possible use of these compounds as a therapeutic approach in the treatment of amyloid deposition diseases.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/22/ 5558/s1. Author Contributions: Conceived and designed the experiments: H.R., F.C. Performed the experiments: H.R., R.T., A.B., and F.B. Analyzed the data: F.C., H.R., R.T., and C.C. Contributed reagents/materials/analysis tools: F.C., C.C., and H.R. Wrote the paper: H.R. and F.C. All authors revised the text and figures and agreed on their content. Funding: This work was supported by a grant from the Research Council of the University of Payam Noor (49103/7). Acknowledgments: We also thank the Iran National Science Foundation (INSF) and Hakim Sabzevari University for partial financial support of this work. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

HEWL hen egg white lysozyme ThT thioflavin T HPA/NCP H6P2W18O62/nanoclinoptilolite CR Congo red AFM atomic force microscopy FTIR Fourier-transform infrared spectroscopy MTT 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide NMR nuclear magnetic resonance TLC thin layer chromatography DMSO dimethyl sulfoxide mp melting point DMEM Dulbecco’s modified Eagle’s medium PBS phosphate buffered saline

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