Antagonistic Activity of Naphthoquinone-Based Hybrids toward

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Antagonistic Activity of Naphthoquinone-Based Hybrids toward Amyloids Associated with Alzheimer’s Disease and Type‑2 Diabetes Ashim Paul,§ Guru Krishnakumar Viswanathan,§ Satabdee Mahapatra,§ Gianfranco Balboni,¶ Salvatore Pacifico,∥ Ehud Gazit,§,† and Daniel Segal*,§,‡

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§

Department of Molecular Microbiology and Biotechnology, School of Molecular Cell Biology and Biotechnology, †Department of Materials Science and Engineering, Iby and Aladar Fleischman Faculty of Engineering, and ‡Sagol Interdisciplinary School of Neuroscience, Tel Aviv University, Ramat Aviv, Tel Aviv 6997801, Israel ¶ Department of Life and Environmental Sciences - Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, I-09124 Cagliari, Italy ∥ Department of Chemical and Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara 17-19, I-44121 Ferrara, Italy S Supporting Information *

ABSTRACT: Protein misfolding and amyloid formation are associated with various human diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Type-2 Diabetes mellitus (T2DM). No disease-modifying therapeutics are available for them. Despite the lack of sequence homology between the corresponding proteins, aromatic residues are recognized as common key motifs in the formation and stabilization of amyloid structures via π−π stacking. Thus, targeting aromatic recognition interfaces could be a useful approach for inhibiting amyloid formation as well as disrupting the preformed amyloid fibrils. Combining experimental and computational approaches, we demonstrated the anti-amyloidogenic effect of naphthoquinone-tryptophan-based hybrid molecules toward PHF6 (τ-derived aggregative peptide), Amyloid β (Aβ42), and human islet amyloid polypeptide (hIAPP) implicated in AD and T2DM, respectively. These hybrid molecules significantly inhibited the aggregation and disrupted their preformed fibrillar aggregates in vitro, in a dose-dependent manner as evident from Thioflavin T/S binding assay, CD spectroscopy, and electron microscopy. Dye leakage assay from LUVs and cell-based experiments indicated that the hybrid molecules inhibit membrane disruption and cytotoxicity induced by these amyloids. Furthermore, in silico studies provided probable mechanistic insights into the interaction of these molecules with the amyloidogenic proteins in their monomeric or aggregated forms, including the role of hydrophobic interaction, hydrogen bond formation, and packing during inhibition of aggregation and fibril disassembly. Our findings may help in designing novel therapeutics toward AD, T2DM, and other proteinopathies based on the naphthoquinone derived hybrid molecules. KEYWORDS: Alzheimer’s disease, Type 2 diabetes, aggregation, Aβ, τ-protein, IAPP

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INTRODUCTION Misfolding of certain proteins and their abnormal aggregation into amyloid depositions are associated with numerous human diseases, commonly referred to as proteinopathies. Notably, Amyloid-β (Aβ) and τ-protein,1 α-synuclein,2 and human islet amyloid polypeptide (hIAPP)3 are associated with Alzheimer’s disease (AD), Parkinson’s disease (PD), and Type-2 diabetes mellitus (T2DM), respectively.4 The growing body of evidence suggests that some of these proteinopathies might be associated with each other such that one disease can be a risk factor for the other. For example, AD patients have increased risk of developing T2DM or vice versa.5,6 No approved disease-modifying therapies are currently available for these debilitating diseases. The mechanism of amyloid formation is not fully understood. Increasing evidence suggests that the native nontoxic © XXXX American Chemical Society

monomers of these proteins may misfold and self-aggregate into β-sheet-rich soluble cytotoxic oligomers, which further elongate into pathogenic insoluble fibrillar assemblies.7 Therefore, targeting the oligomeric species and the higher order assemblies is an attractive disease-modifying strategy.8,9 Various strategies have been attempted for arresting or modulating the aggregation of amyloidogenic proteins, including antibodies,10 peptide inhibitors,11−13 small organic molecules,14,15 and nanoparticles.16,17 Some of these agents, including EGCG,18,19 Curcumin,20,21 quinone derivatives,22 and Genistein,23 were capable of modulating multiple amyloids with different efficiency. Received: February 28, 2019 Accepted: June 24, 2019 Published: June 24, 2019 A

DOI: 10.1021/acschemneuro.9b00123 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. Chemical structure of the naphthoquinone-based hybrid molecules.

biophysical assays for examining their effect on the aggregation of PHF6 (Ac-Val-Gln-Ile-Val-Tyr-Lys-NH2), a τ-derived amyloidogenic hexapeptide, which has been extensively used as a proxy for aggregation of the full-length τ protein.22,27 Subsequently, their efficacy toward aggregation of Aβ42 and hIAPP was tested. Modulation of PHF6 Aggregation by the Hybrid Molecules. The effect of the hybrid molecules on PHF6 aggregation was examined using Thioflavin S (ThS) assay. The intensity of ThS fluorescence upon binding to the cross-β structure of protein aggregates is a common measure for the quantity of amyloids formed.33 PHF6 alone aggregated and formed fibrils within 30 min (black, Figure 2a and Figure S7). In contrast, the intensity of ThS fluorescence was lower when PHF6 was incubated with increased doses of the hybrid molecules (PHF6:hybrid molecules5:1, 1:1, and 1:5), indicating inhibition of PHF6 aggregation in a dose-dependent manner, with maximum inhibition at 1:5 molar ratio. At 5-fold molar excess, NQTrp, NQTA, and NQTOL inhibited ∼67%, ∼75%, and ∼83% of PHF6 amyloid formation, respectively (Figure 2b). The secondary structure of aggregated PHF6, in the absence or presence of the hybrid molecules, was monitored by CD spectroscopy (Figure 2c and Figure S8). PHF6 fibrils alone (control) displayed a positive maximum near ∼194 nm and a negative minimum near ∼216 nm (Figure 2c), indicating βsheet conformation, as reported.30 Upon coincubation with increasing doses of the hybrid molecules, the intensity at ∼216 nm was reduced, indicating a decline in β-sheet content (Figure 2c and Figure S8). The morphology of PHF6 aggregates in the presence of the hybrid molecules was examined by TEM analysis (Figure 2d). Control PHF6 gave rise to long and dense fibrillar structures. However, in the presence of 5-fold molar excess of the hybrid molecules, the amount of fibrillar assemblies was markedly reduced, indicating significant inhibition of fibril formation. In diseased condition, the amyloidogenic protein exists in the form of mature fibrils and toxic oligomers, and therefore reducing such existing assemblies into nontoxic species becomes essential for drug development. To examine the ability of the hybrid molecules to disaggregate preformed fibrils of PHF6 in vitro, we first incubated PHF6 for 60 min, a duration which allowed them to form fibrils (based on ThS data in Figure 2a). Then, the hybrid molecules were added to the fibrillar assemblies and were further incubated for an additional 90 min. Kinetics of PHF6 fibrils disassembly was monitored by ThS fluorescence, which indicated a dosedependent reduction of ThS signal with time (Figure 2e,f and Figure S9), reflecting disaggregation of existing fibrils. At 5-fold molar excess, NQTrp, NQTA, and NQTOL were found to reduce existing PHF6 fibrils by ∼47%, ∼55%, and ∼58%, respectively (Figure 2f).

Despite the lack of similarity in the primary sequence between the various amyloidogenic proteins, certain common structural features were identified which play a pivotal role in the process of amyloid formation. Notably, aromatic amino acids were shown to facilitate the aggregation process and to stabilize the resultant fibrils through π−π stacking aromatic interactions in addition to extensive intermolecular hydrogen bonding between the self-assembled protein monomers.24,25 Thus, targeting the aromatic residues may be an effective approach for inhibiting the formation of amyloids associated with multiple proteinopathies.22,26−28 Quinones are useful modulators of aggregation of various amyloidogenic proteins.11 For example, we previously reported that quinone derivatives, naphthoquinone-tryptophan (NQTrp) and Cl-naphthoquinone-tryptophan (Cl-NQTrp), effectively inhibit the in vitro aggregation of various amyloids and disassemble pre-existing fibrils.22,27 They also reduced amyloid cytotoxicity and ameliorated AD symptoms in Aβ- and τ-expressing transgenic animal models.29,30 In these quinonebased molecules, tryptophan (Trp), the highest amyloidogenic aromatic coded amino acid,31 intercalates into the β-sheets of the aggregated proteins through π−π stacking aromatic interaction, whereas the naphthoquinone unit remains flexible to disturb the protein self-assembly process.29 Here, we report the synthesis, and characterization, of novel naphthoquinone-based hybrid molecules, NQTA and NQTOL (Figure 1), in which the naphthoquinone moiety is covalently linked with either tryptamine (TA) or L-tryptophanol (TOL), respectively. We demonstrate their “dual attributes”, i.e., inhibiting amyloid formation as well as disrupting the preexisting fibrils of Aβ, hIAPP, and the τ-derived PHF6 in vitro, comparable to NQTrp. These compounds efficiently modulated the toxicity of the tested amyloids toward neuronal cells in culture. Finally, combined molecular docking and molecular dynamic simulation provided mechanistic insights into the mode of action of these small molecules. Collectively, our results highlight these novel compounds as potential leads for developing therapeutics for AD, T2DM, and other proteinopathies.

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RESULTS AND DISCUSSION The designed hybrid molecules, naphthoquinone-tryptamine (NQTA) and naphthoquinone-tryptophanol (NQTOL), were synthesized by reacting naphthoquinone with tryptamine (TA) and L-tryptophanol (TOL), respectively, using the reported protocol (Figure 1).32 The synthesized hybrid molecules were characterized by HPLC, mass spectrometry, and 1H NMR spectroscopy (see Supporting Information: Figure S1−S6). To investigate the efficacy of the hybrid molecules (NQTrp, NQTA, and NQTOL) in modulating the aggregation of amyloidogenic peptides in vitro, we first used various B


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Figure 2. (a) Time- and (b) dose-dependent ThS fluorescence for inhibition of amyloid formation by PHF6 (50 μM) in the absence (black) and presence of the hybrid molecules (NQTrp, NQTA, and NQTOL). (c) CD spectra and (d) TEM images for the inhibition of PHF6 fibrils in the absence and presence of 5-fold molar excess of the hybrid molecules. (e) Time- and (f) dose-dependent ThS fluorescence for disaggregation of the preformed fibrils of PHF6 (50 μM) in the absence and presence of the hybrid molecules. (g) CD spectra and (h) TEM images for the disaggregation of preformed fibrils of PHF6 in the absence and presence of 5-fold molar excess of the hybrid molecules. Experiments were performed in MOPS buffer (pH 7.2) at 25 °C.

TEM analysis (Figure 2h) revealed that in the absence of the hybrid molecules, the preformed PHF6 assemblies have typical fibrillar morphology, whereas in their presence, the density of fibrillar assemblies was markedly reduced. Essentially no fibrillar aggregates were observed in the presence of 5-fold molar excess of NQTOL, yet some amount of small broken fibrils was observed in the presence of a similar concentration of NQTrp and NQTA. Collectively, results of the various assays for PHF6 are in agreement and indicate that the hybrid

In CD spectra (Figure 2g and Figure S10), untreated PHF6 fibrils generated by 60 min preincubation (control) displayed a positive maximum near ∼197 nm and a negative minimum near ∼218 nm, indicating β-sheet rich conformation. Then, hybrid molecules were added and coincubated for additional 90 min, which resulted in a reduction of the CD signal at ∼218 nm in a dose-dependent manner, indicating a decrease in βsheet content. C


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Figure 3. (a) Time- and (b) dose-dependent ThT fluorescence for inhibition of amyloid formation by Aβ42 (10 μM) in the absence (black) and presence of the hybrid molecules (NQTrp, NQTA, and NQTOL). (c) CD spectra and (d) TEM images for the inhibition of Aβ42 fibrils in the absence and presence of 5-fold molar excess of the hybrid molecules. (e) Time- and (f) dose-dependent ThT fluorescence for disaggregation of the preformed fibrils of Aβ42 (10 μM) in the absence and presence of the hybrid molecules. (g) CD spectra and (h) TEM images for the disaggregation of preformed fibrils of Aβ42 in the absence and presence of 5-fold molar excess of the hybrid molecules. Experiments were performed in PBS (pH 7.4) at 37 °C.

in a dose-dependent manner (Figure 3b and Figure S11). At the end point of the assay, a 5-fold molar excess of the hybrid molecules reduced ThT signal by 79−88% compared to untreated Aβ42, indicating robust inhibition of aggregation (Figure 3a,b). CD spectra of untreated Aβ42 (control) showed β-sheetrich conformation, evident by a positive peak near ∼196 nm and a negative band near ∼215 nm (Figure 3c and Figure S12). Upon coincubation with the hybrid molecules, intensity of the negative band (∼216 nm) was reduced in a dose-

molecules are efficacious inhibitors of PHF6 aggregation as well as disrupting preformed PHF6 fibrils. Modulation of Aβ42 Aggregation by the Hybrid Molecules. Next, the efficacy of the hybrid molecules toward in vitro aggregation of Aβ42 peptide, a major culprit of AD, was measured. ThT fluorescence analysis indicated that Aβ42 (10 μM) alone readily formed amyloid fibrils, as evident by the rapid increase of the ThT signal until a plateau was reached by 15 h (Figure 3a). The hybrid molecules (Aβ42:hybrid molecule5:1, 1:1, and 1:5) lowered ThT fluorescence level D


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Figure 4. (a) Time- and (b) dose-dependent ThT fluorescence for inhibition of amyloid formation by hIAPP (10 μM) in the absence (black) and presence of the hybrid molecules (NQTrp, NQTA, and NQTOL). (c) CD spectra and (d) TEM images for the inhibition of hIAPP fibrils in the absence and presence of 5-fold molar excess of the hybrid molecules. (e) Time- and (f) dose-dependent ThT fluorescence for disaggregation of the preformed fibrils of hIAPP (10 μM) in the absence and presence of the hybrid molecules. (g) CD spectra and (h) TEM images for the disaggregation of preformed fibrils of hIAPP in the absence and presence of 5-fold molar excess of the hybrid molecules. Experiments were performed in PBS (pH 7.4) at 37 °C.

dependent manner, indicating a decline in β-sheet content. In TEM analysis, Aβ42 alone showed clear, dense fibrillar assemblies (Figure 3d). However, no such fibrils were observed in the presence of 5-fold molar excess of any of the three compounds tested.

The effect of the hybrid molecules on preformed Aβ42 fibrils was investigated by first allowing incubation of Aβ42 for 20 h to form fibrils (based on ThT results from Figure 3a), and then adding either various doses of the hybrid molecules (Aβ42:hybrid molecules5:1, 1:1, and 1:5) or none (control), for an additional 20 h incubation. Kinetics of disaggregation of E


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Table 1. Percentage of Dye Leakage from LUVs upon Treatment with the Oligomers or Fibrils of PHF6, Aβ42, and hIAPP in the Presence or Absence of the Hybrid Molecules Untreated LUVs Untreated LUVs Oligomers Mature fibrils NQTrp treated fibrils NQTA treated fibrils NQTOL treated fibrils

PHF6

Untreated LUVs

∼8%

Aβ42

∼8% ∼35% ∼17% ∼14% ∼15% ∼12%

hIAPP

∼7% ∼42% ∼16% ∼15% ∼13% ∼11%

the preformed fibrils was monitored by ThT fluorescence (Figure 3e,f, and Figure S13) which revealed that the fluorescence intensity remained in plateau when Aβ42 was untreated, indicating that fibrils existed during the assay (Figure 3e). However, in the presence of the hybrid molecules, ThT signal dropped in a dose-dependent manner reflecting a reduction of preformed fibrils by as much as ∼57%, 48%, and 44%, for NQTOL, NQTA, and NQTrp, respectively (Figure 3e,f). This reduction of preformed Aβ42 fibrils was corroborated by CD analysis. Untreated fibrils exhibited a negative band at ∼216 nm and a positive maximum at ∼198 nm, indicating β-sheet-rich conformation, whereas in the presence of the hybrid molecules, the intensity at ∼218 nm was reduced in a dose dependent manner, indicating a substantial reduction of β-sheet content (Figure 3g and Figure S14). TEM analysis of the preformed Aβ42 fibrils indicated that in the absence of the hybrid molecules, there were ample long and thin fibrils (Figure 3h). Such fibrils were markedly less common, and few amorphous like structures were apparent in the presence of 5-fold molar excess of the tested molecules, indicating a significant reduction of Aβ42 fibrils. Taken together, these results indicate that the hybrid molecules substantially disrupted preformed fibrils of Aβ42. Modulation of hIAPP Aggregation by the Hybrid Molecules. Amyloid assemblies of hIAPP are the major cause for the pancreatic β-cell dysfunction and pathogenesis of T2DM.3,34 Examination of the effect of the hybrid molecules toward hIAPP fibrillization using ThT fluorescence revealed that the ThT signal reached a plateau in 10 h, indicating completion of fibrillization (control), whereas in the presence of the hybrid molecules (hIAPP:hybrid molecule5:1, 1:1, and 1:5), fluorescence intensity decreased in a dose-dependent manner (Figure 4a,b, and Figure S15), signifying substantial reduction of fibrils formed. In CD spectra, hIAPP alone showed a positive maximum near ∼197 nm and a negative minimum near ∼218 nm, indicating β-sheet conformation. The intensity at ∼218 nm was reduced upon coincubation with different doses of the hybrid molecules, indicating reduction in β-sheet content (Figure 4c and Figure S16). hIAPP aggregates, which in the absence of the hybrid molecules exhibited overt thin fibrillar morphology in TEM analysis, were essentially eliminated at a 5-fold molar excess of any of the three compounds tested (Figure 4d) in agreement with the ThT and CD results. We investigated further the ability of the hybrid molecules to disrupt preformed fibrils of hIAPP in vitro. To that end, hIAPP (10 μM) was incubated alone for 15 h to generate fibrils (as determined in black curve, Figure 4a). Subsequently, different concentrations of the hybrid molecules, or none, were added into the fibrillar assembly for an additional 15 h of coincubation, and their effect on the preformed fibrils was

Untreated LUVs

∼37% ∼18% ∼14% ∼11% ∼10%

monitored by ThT fluorescence, CD spectroscopy, and TEM analysis. In the presence of the hybrid molecules, ThT signal declined in a dose-dependent manner (Figure 4e,f, and Figure S17) with maximum reduction, relative to untreated hIAPP fibrils by ∼66%, 61%, and 53% in the presence of 5-fold molar excess of NQTOL, NQTA, and NQTrp, respectively. This disruption of the preformed hIAPP fibrils was reflected also in the CD results (Figure 4g and Figure S18), where the negative minimum band at ∼216 nm and positive maximum at ∼196 nm represented β-sheet-rich conformation of untreated hIAPP fibrils. The intensity at ∼216 nm was reduced with increasing doses of the tested molecules, indicating a reduction in β-sheet content. The morphology of the preformed hIAPP fibrils in the presence of hybrid molecules monitored by TEM corroborated the ThT and CD results. Amorphous structures were evident in the presence of 5-fold molar excess of the hybrid molecules, whereas untreated preformed hIAPP assemblies exhibited typical fibrillar morphology (Figure 4h). Taken together, these results indicate an efficient disaggregation of the preformed hIAPP assemblies by all three hybrid molecules. Hybrid Molecules Reduce Amyloid-Induced Dye Leakage from LUVs. The soluble oligomers or protofibrils of amyloidogenic proteins (including τ-protein, Aβ42, and hIAPP) are reported to be more toxic than the corresponding mature fibrils, presumably due to the formation of pores in the cell membrane.35−38 To examine whether the disrupted preformed fibrillar assemblies by the hybrid molecules can reduce the toxic oligomeric species, we performed carboxyfluorescein entrapped large unilamellar vesicles (LUVs) leakage assay.39,40 Prior to the leakage assay, dye-entrapped LUVs were prepared, and their formation was confirmed by TEM analysis (Figure S19). For the leakage assay, untreated LUVs were examined as a reference for spontaneous leakage of the dye and were compared to LUVs incubated with either the corresponding oligomeric species or the mature fibrils (Table 1 and Figure S20). The oligomeric species for this experiment were generated by incubating PHF6 (50 μM), Aβ42 (10 μM), or hIAPP (10 μM) alone for 10 min, 5 h, and 5 h, respectively, whereas fibrillary species were obtained by incubating them for 3, 40, and 30 h, respectively (based on the results of ThT assays, Figures 2e, 3e, and 4e). Triton X-100 was used for induction of complete dye release from the LUVs, and its fluorescence value was considered as 100% dye leakage. The percentage of dye leakage by individual species was measured according to eq 1 %Leakage =

(observed fluorescence − initial fluorescence) (total fluorescence − initial fluorescence) × 100%

F

(1) DOI: 10.1021/acschemneuro.9b00123 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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at 5-fold molar excess NQTOL, NQTrp, and NQTA restored viability up to 86 ± 3%, 76 ± 1%, and 85 ± 2%, respectively, in comparison to the viability of cells exposed to untreated PHF6 fibrils (52 ± 6%) (Figure S22a-c). Similar experiments were performed using fibrils of Aβ42 on SH-SY5Y and hIAPP on HEK-293 cell lines (Figure S22d-i). Here too, the hybrid molecules significantly ameliorated the cytotoxic effects induced by Aβ42 and hIAPP fibrils, and NQTOL appeared to be the most effective among them. Atomistic Insights into the Mode of Action of the Hybrid Molecules. In an attempt to gain atomistic insights into the interaction of the hybrid molecules with the monomers of PHF6, Aβ42, and hIAPP, and to delineate a plausible molecular mechanism resulting in inhibition of their aggregation, we performed molecular docking. For Aβ42, we used the available structure of its fragment (Aβ10-35, PDB ID 1HZ3). Molecular docking was performed using the Autodock 4.2 package as described previously.44 Best docking poses and binding energies of the hybrid molecules with the monomers of these peptides are shown in Table 2 and Figure 5. Strong binding energies reflected the tight association of the hybrid molecules with the peptide monomers (Table 2).

Freshly prepared PHF6 oligomers (aged 10 min), mixed with the LUVs, caused rapid leakage of dye until 100 min (∼35% leakage, Table 1), after which dye release was saturated (red, Figure S20a,d). The mature PHF6 fibrils caused ∼17% leakage from the LUVs (Table 1 and blue, Figure S20a,d). Spontaneous release of the dye from the untreated LUVs was minimal (∼8% leakage) and was saturated after 70 min. These results confirmed that PHF6 oligomers caused more dye leakage and subsequently more pore formation in LUVs than mature fibrils, and hence are likely to be more toxic. Disaggregation of the preformed PHF6 fibrils by the hybrid molecules resulted in 12−15% leakage which was considerably lower than dye release caused by the PHF6 oligomers (35%). This outcome indicated that the hybrid molecules disassembled PHF6 fibrils into a species which caused negligible disruption to the artificial LUV membrane. A comparable analysis of Aβ42 or hIAPP oligomers (both aged 5 h) with LUVs revealed rapid dye release until 400 min (∼42% or ∼37%, Table 1) and saturation after 500 min (Figure S20). The mature fibrils (aged 40 and 30 h for Aβ42 and hIAPP, respectively) caused lesser leakage from LUVs than oligomeric species. These results indicate that, as for PHF6, Aβ42/hIAPP oligomers were more deleterious to membrane than the mature fibrils. The hybrid molecules disaggregated preformed Aβ42 or hIAPP fibrils causing a low level of dye release (10−15%), which was less than the corresponding oligomers induced leakage, in agreement with the findings obtained for PHF6. Collectively, the results of LUV leakage assays corroborate the results of the other in vitro experiments mentioned above, indicating that the hybrid molecule disassembled fibrils of the three tested amyloids into structures which were nondestructive toward membranes. Reduction of PHF6, Aβ42, and hIAPP Induced Cytotoxicity by the Hybrid Molecules. Next, we examined the effect of the hybrid molecules toward cytotoxicity engendered by PHF6, Aβ42, and hIAPP amyloids. To that end, we first studied whether the hybrid molecules are cytotoxic by incubating them at different concentrations (1− 100 μM) with human neuroblastoma SH-SY5Y or with human embryonic kidney (HEK-293) cell lines and evaluating cell viability by XTT assay. As shown in Figure S21, at 1 and 5 μM, there were no toxic effects from any of the hybrid molecules toward either cell line, and even at 100 μM, the cell viability remained as high as ⩾80% compared to the untreated control cells (Figure S21a-f). In parallel, the cytotoxic effects of PHF6, Aβ42, and hIAPP incubated with the cells was determined as a control, in the absence of the hybrid molecules. Toxicity of different doses of PHF6 and Aβ42 fibrils was monitored toward SH-SY5Y cells and of hIAPP fibrils toward HEK-293 cells. As shown in Figure S21g-i, a dose-dependent decrease of cell viability was observed for all three amyloids reaching ∼50% reduction (IC50) of cell viability at 50 μM of PHF6, 15 μM of Aβ42, and 15 μM of hIAPP, as compared to untreated control cells, in agreement with previous reports.41−43 These IC50 values of each amyloid were used for the amelioration of amyloidinduced toxicity experiments. To evaluate the effect of the hybrid molecules on PHF6 induced cytotoxicity, SH-SY5Y cells were treated for 24 h with 50 μM of PHF6 fibrils alone or together with various concentrations (1−250 μM) of the hybrid molecules (Figure S22). The hybrid molecules reduced the toxicity of PHF6 fibrils significantly in a dose-dependent manner. For example,

Table 2. Predicted Binding Energies of Hybrid Molecules with Monomers of PHF6, Aβ Fragment, and hIAPP Binding energy (kcal mol−1) Peptide

NQTrp

NQTA

NQTOL

PHF6 (PDB ID 2ON9) Aβ fragment (PDB ID 1HZ3) hIAPP (PDB ID 5MGQ)

−5.2 −6.61 −7.47

−5.6 −7.1 −6.98

−5.6 −7.21 −7.27

Figure 5. Best docking poses of the hybrid molecules with monomers of PHF6, Aβ fragment (Aβ10−35), and hIAPP. Peptides are represented as cartoons with lines, and ligands are represented as sticks.

Interestingly, NQTOL exhibited relatively high binding energy with the PHF6, Aβ fragment, and hIAPP peptide monomers. This data is in agreement with the outcomes from the in vitro assays portraying the enhanced ability of NQTOL to inhibit aggregation of the amyloidogenic peptides. Putative interaction sites of the hybrid molecules with PHF6, Aβ fragment, and hIAPP monomers (Table S1 and Figure S23) revealed that the hybrid molecules interacted with the peptide G


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Figure 6. Geometric parameter analysis of PHF6 fibril−hybrid molecules system: Formation of hydrogen bonds between PHF6 fibrils with (a) NQTrp, (b) NQTA, and (c) NQTOL at 20 ns. MD trajectories obtained after 20 ns simulation of the PHF6 fibril in the presence or absence of hybrid molecules: (d) Central axis view and (e) Longitudinal axis view.

reports, the present study revealed that the hybrid molecules interacted with various residues of Aβ fragment including Glu11, Val12, His13, His14, and Lys16, in addition to Leu17, Val18 located in the core hydrophobic region (16KLVFF20),48 and this possibly rendered their inhibitory effects toward Aβ42 aggregation. To further validate the results of the Aβ fragment, we also performed a docking study of the hybrid molecules with Aβ40, and the predicted binding energies (Table S2) between them were in a similar pattern as with the Aβ fragment. We observed that the hybrid molecules interacted with the different residues of Aβ40, His6, Asp7, Ser8, His13, Gln15, Lys16, and Val18 (Table S3 and Figure S24) through hydrogen boding and hydrophibic interaction, which were also very similar to that observed for Aβ fragment and agreed with previous reports.46,47 Small molecules like lipoic acid, ascorbic acid, curcumin, resveratrol, and genistein were reported to inhibit the aggregation of hIAPP by interacting with its hydrophobic residues, such as Leu12, Phe15, His18, Phe23, Ala25, Leu27, and Tyr37.23,49,50 Corroborating these reports, the docking results reported herein suggest that the hybrid molecules predominantly interacted with Asn residues of hIAPP (Asn14, Asn21, and Asn22) and formed hydrophobic contacts with Arg11 and Ser19 in addition to the previously reported Leu12, Phe15, and His18. Together, these interactions facilitated the inhibition of hIAPP aggregation.

monomers via hydrogen bonds, predominantly with the polar/ charged residues (Glu, Gln, His, Lys, and Asn) and also with hydrophobic residues (Val, Leu, Ile, and Tyr). Hydrophobic contacts were also observed with Val, Ile, Tyr, Arg, Ser, Lys, His, Phe, Leu, Gln, and Asn residues of the monomeric peptides. It is appreciated that protein fibrillar aggregates are governed by hydrogen bonding as well as hydrophobic and π−π interactions, and disturbing these interactions may eventually inhibit amyloid formation.24,29 In line with this statement, we have previously reported the importance of hydrogen bond between the Val residues and π−π stacking of Tyr residue in the formation of β-sheet structures between PHF6 dimers and, in turn, its effect on the fibril formation.27 In the present study, we found that the hybrid molecules formed hydrogen bonds with Gln2, Ile3, Val4, and Tyr5 and also exhibited hydrophobic contacts, which may inhibit aggregation of the PHF6 peptide. Vitamin-K3 was found to interact with Ser8, Glu11, Val12, Gln15, Lys16, and Phe19 residues of Aβ42, which resulted in inhibition of its aggregation.45 Among these residues, Lys16 was identified as key residue in Aβ42 because of its active interaction with the Vitamin-K3 via hydrogen bonding. Likewise, the interaction of inhibitors such as EGCG, Myricetin, Curcumin, and Pyrazinamide with Val12, Gln15, and Lys16 residues of Aβ42 also resulted in their antiamyloidogenic property.46,47 In agreement with these previous H


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ACS Chemical Neuroscience Disassembly of Preformed Amyloids by the Hybrid Molecules: A Molecular Dynamics Approach. Molecular dynamics (MD) simulation, using PHF6 fibril as a model, was performed for gaining atomistic insights into a plausible mechanism by which the hybrid molecules disrupt preformed fibrils (Figure 6), leading to a reduction in amyloid content. To that end, a PHF6 fibril system was built using 42 peptides in predefined β-sheet conformation, as described previously.27,41,51 Several in vitro and in silico studies have shown that the PHF6 peptide fibrils are made up of highly ordered cross-β structures.52−54 Also, we have previously demonstrated that the β-sheets between the PHF6 peptide pairs in fibrillar architecture were formed owing to the hydrogen bonds between the main-chains of Val1-Gln2, Ile3-Gln2, Ile3-Val4, and Tyr5-Val4. Among these amino acids, Val was found to be the key residue in maintaining the β-sheet conformation between the two-peptide pairs.27 In the present study, MD simulation indicated that the hybrid molecules form hydrogen bonds predominantly with Val residue of the PHF6 (Figure 6ac). Specifically, NQTrp molecule M2 formed one hydrogen bond with Val1 (bond length: 2.6 Å), and M3 formed two hydrogen bonds with Val1 (bond length: 2.4 Å, 3.3 Å),27 NQTA molecule M2 formed two hydrogen bonds with Val1 (bond length: 3.2 Å, 2.7 Å), and M3 formed three hydrogen bonds with Val4 (bond length: 2.3 Å, 2.3 Å, 3.5 Å) and NQTOL molecule M1 formed one hydrogen bond with Tyr5 (bond length: 2.9 Å), M2 formed one hydrogen bond with Tyr5 (bond length: 3.3 Å), and M3 formed three hydrogen bonds with Val4, Tyr5, and Ace group (bond length: 2.4 Å, 2.5 Å, 3.0 Å) at 20 ns. The observed higher affinity of NQTOL toward PHF6 could be the cause for an apparently enhanced efficacy of NQTOL observed in the experimental assays compared to the other compounds tested. Grounded on these results, we hypothesize that interaction of the hybrid molecules with these hydrogen bond forming residues of the PHF6 peptide might disrupt the existing peptide−peptide interaction in the β-sheet-rich fibrillar arrangement, eventually disassembling the preformed aggregates. MD simulation trajectories were analyzed to understand whether the hybrid molecules could cause ruptures in the strands of PHF6 fibril. The fibrillar architecture of the control fibril (PHF6) simulated in the absence of the hybrid molecules27 was intact without any hint of disassembly (Figure 6d). The central axis view of the control fibril revealed that the β-sheets were tightly packed and bonded well to each other. However, when simulated in the presence of the hybrid molecules, the conformation of the fibril changed significantly and acquired an unusual arrangement when viewed from the central axis comprising loosely packed β-sheets, which were parting away from each other (Figure 6d). When visualized from the longitudinal axis, the control PHF6 fibril had a uniform twist with well-defined pitch length, whereas in the presence of the hybrid molecules, overt breaks in the fibrillar strand were apparent in places where the hybrid molecules interacted with the peptide fibril (Figure 6e).

molecules appeared to be more efficient modulators toward the slowly aggregating peptides (Aβ42 and hIAPP) than the fast-aggregating peptide (PHF6). This is plausibly due to the slow rate of primary nucleation of peptide molecules allowing adequate time for interaction by the inhibitor molecules.55,56 These hybrid molecules were also found to be nontoxic toward the neuroblastoma (SH-SY5Y) and kidney (HEK-293) cell lines and ameliorate the cytotoxicity caused by PHF6, Aβ42, and hIAPP aggregates. Molecular docking revealed that the hybrid molecules displayed significant interactions with the peptide monomers facilitating inhibition of aggregation. Additionally, molecular dynamics simulation provided a plausible mechanism for disassembly of preformed fibrils arbitrated by the hybrid molecules. It is noteworthy that in all in vitro assays as well as in the computational studies, NQTOL appeared to be superior over the other hybrid molecules tested. Collectively, these results indicate that the hybrid molecules are anti-amyloidogenic and may be useful as leads for developing therapeutics for proteinopathies.

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METHODS

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ASSOCIATED CONTENT

The characterization data of the novel naphthoquinone-based hybrid molecules, supporting biophysical/theoretical data and experimental/ theoretical methods are provided in the Supporting Information. S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.9b00123. Materials and Methods; characterization including spectra, chromatography, kinetics, microscopy, cell viability, interaction diagrams (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: ++972-3-640-9835. Fax: ++972-3-640-9407. ORCID

Ashim Paul: 0000-0003-2182-8248 Ehud Gazit: 0000-0001-5764-1720 Author Contributions

D.S. and A.P. conceived the project and wrote the manuscript. G.B. and S.P. synthesized the hybrid molecules and conducted the characterization. A.P. performed all the in vitro experimental work and analyzed the results. G.K.V. and S.M. performed the in silico work and analyzed the results. G.K.V. wrote the in silico part. E.G. and all the authors read and corrected the manuscript and approved the manuscript. Funding

This work was supported by the RoseTrees Trust (to D.S). G.K.V. thanks the TATA trusts for the postdoctoral scholarship. Notes

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The authors declare no competing financial interest.

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CONCLUSIONS The present work demonstrated the generic effect of a novel group of naphthoquinone-based hybrid molecules (NQTrp, NQTA, and NQTOL) to inhibit aggregation of amyloids and disaggregate the preformed fibrillar assemblies of PHF6, Aβ42, and hIAPP in vitro into innocuous species. The hybrid

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Antagonistic Activity of Naphthoquinone-Based Hybrids toward

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