EGCG) Binary Modulators for Inhibiting

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Article Cite This: ACS Omega 2019, 4, 4233−4242

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Peptide−Polyphenol (KLVFF/EGCG) Binary Modulators for Inhibiting Aggregation and Neurotoxicity of Amyloid‑β Peptide Qunxing Huang,†,§,‡ Qiong Zhao,†,¶,‡ Jiaxi Peng,† Yue Yu,†,§ Chenxuan Wang,†,§ Yimin Zou,†,§ Yanlei Su,¶ Ling Zhu,†,§ Chen Wang,*,†,§ and Yanlian Yang*,†,§

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CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Key Laboratory of Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China § University of Chinese Academy of Sciences, Beijing 100049, P. R. China ¶ School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China S Supporting Information *

ABSTRACT: Alzheimer’s disease (AD) is known as a typical neurodegenerative disease, and the pathogenic hallmark is the aggregation and deposition of amyloid-β peptide (Aβ) fibrils and plaque on neuronal cells resulting in cell dysfunction and cell death. One effective approach to preventing and curing AD lies in tuning Aβ aggregation and inhibiting the neurotoxicity by using molecular modulators. Peptide breakers and antioxidants are widely used inhibitors to modulate Aβ aggregation and neurotoxicity, although less efficiency of single modulators hinders the practical application of these molecules. An integration of different molecular modulators is expected to make use of multiple interactions and modulating sites and therefore synergistically improve the capacity of modulators in inhibiting Aβ aggregation. In this work, the concept of a binary peptide−polyphenol binary modulator (Aβ-segment KLVFF and (−)-epigallocatechin-3-gallate, KLVFF/EGCG) is proposed, and the synergistic effect of the KLVFF/ EGCG modulator is demonstrated for efficient inhibition of Aβ aggregation and neurotoxicity. With the aid of thioflavin T fluorescence, circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy, the inhibitory effect on Aβ42 fibrillation was determined. Further liquid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations evidenced the affinity of the KLVFF/EGCG complex to the Aβ42 monomer. Furthermore, a tentative schematic mechanism is also proposed to illustrate the synergistic effect. Besides, the MTT assay and DCFH-DA (2′,7′dichlorodihydrofluorescein diacetate) test were performed to explore the reduction of Aβ42-induced neurotoxicity by treating with the KLVFF/EGCG complex. The binary inhibitors showed remarkable reduction in Aβ42-induced ROS generation. This work could be beneficial for the designing of potential therapeutic binary modulators and shed light on AD prevention.

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structural transformation from random coils into β-sheet structures, and then Aβ oligomers are formed with β-structurerich aggregation cores, followed by an assembly process from oligomers to protofilaments, and finally, to fibrils.8−13 The Aβ42 protofilament features a ″hairpin″ structure, two parallel β-strands with a turn in between forming a β-strand−turn−βstrand motif. Great efforts have been put into the inhibition of Aβ aggregation since the 1990s.2,4,14−17 The modulators were designed to block the formation of β-sheets to prevent the fibrilogenesis, or to destabilize the state of oligomers.18,19 One important strategy is to investigate the crucial regions in amyloid aggregation, and then synthesize the “key fragments” to interfere with the interaction of Aβ42.2,4,14−17,20,21 Tjernberg et al. were the first to synthesize 31 possible 10-

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, which is characterized by cognitive disorder and dementia in the old.1 Genetic, neuropathological, and transgenic animal model studies have implicated that the accumulation of amyloid-β peptide (Aβ) in the cerebral cortex is an important step in the development of AD pathology.2 Aβ peptides, which range from 39 to 43 residues in length, are cleaved from the membrane-bound amyloid precursor protein (APP). Aβ with 42 residues (Aβ42, shown in Scheme 1a) was found to have the highest percentage in amyloid plaques, and the diffusible nonfibrillar oligomers played a central role in the neurotoxicity.3−6 The assembling process was considered to be the core of AD pathology and led to a variety of closely linked pathological pathways such as metal dyshomeostasis, tau phosphorylation, inflammation, mitochondrial damage, oxidative stress, and the generation of reactive oxygen species (ROS), which eventually resulted in neuronal toxicity and cell death.7 In the aggregation process, Aβ peptide chains undergo © 2019 American Chemical Society

Received: October 14, 2018 Accepted: January 16, 2019 Published: February 26, 2019 4233

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Scheme 1. (a) Amino Acid Sequence of Aβ42 and Schematic Illustration of Molecular Structures of Peptide (b) KLVFF and (c) Polyphenol EGCG

strand and interfere with the attachment of neighboring Aβ strands for assembly and aggregation and EGCG could bind to the Aβ backbone and maintain the random coil structure of Aβ, binary inhibitors might combine the different binding sites and lead to improved inhibition of aggregation. The strategies for combination of inhibitors are covalent conjugation or complex formation via weak interaction just by physical mixing. The covalently conjugated modulators not only combined the merits of single inhibitors but also provided convenience in drug delivery. Rajasekhar et al. designed covalently combined peptidomimetic inhibitors43 and hybrid modulators.44 The modulators demonstrated their abilities to prevent DNA damage and protein oxidation, which could efficiently reduce the Aβ toxicity. The complex formation by means of physical mixing is very convenient and versatile, which takes advantage of the rich chemistry of molecules, such as electrostatic and hydrophobic interactions between molecules that could possibly contribute to the formation of a complex. Furthermore, once the complex is formed, the increased steric hindrance could prevent Aβ folding and result in improved inhibition of aggregation. In previous study, Niu et al. reported hierarchical assemblies of KLVFF−terpyridine by scanning tunnel microscopy (STM). The formation of KLVFF−terpyridine complexes is confirmed by the hydrogen bonds between terpyridine and KLVFF, which synergistically enhanced the inhibition effect on the assembly of Aβ42.45 In the present work, we propose combining EGCG with KLVFF for formation of a complex to modulate the aggregation of Aβ42. The inhibitory effects were measured by AFM, TEM, liquid-state nuclear magnetic resonance(NMR), molecular dynamics (MD) simulations, dichroism (CD) spectroscopy, ThT fluorescence, MTT assay, and DCFH-DA (2′,7′dichlorodihydrofluorescein diacetate) test. On the basis of the results, a synergistic reaction was tentatively explained.

mers corresponding to amino acids 1−10 up to 31−40 of the Aβ40 molecules.2 The fragment Aβ12-21was found to have the strongest binding affinity with Aβ40 by means of surface plasmon resonance (SPR). They further dwindled the key residues to Aβ16−20 (KLVFF, shown in Scheme 1b). Balbach et al. proved that KLVFF adopted an antiparallel structure to that of Aβ42.21 Further 3D model20 and molecular dynamics9 simulations indicated that hydrophobic interactions act as a major factor in binding affinity. Although KLVFF has strong affinity to Aβ42, its efficiency in interfering fibrillation needs to be improved. As a result, a series of KLVFF derivatives including point mutations, additional residues at both ends, and branched KLVFF tetramers were designed to modulate Aβ aggregation,19,22−28 which demonstrated augments in interfering amyloid folding and a decrease in cytotoxicity. The use of non-peptide molecules is another approach to inhibiting Aβ fibrillation, among which polyphenols have drawn more and more attention in recent years.15,29−35 Their potential fibril-breaking characteristics are reported by Porat et al.33 Polyphenols feature aromatic structures and hydrogen bond donors and acceptors, which enable π−π interactions as well as hydrogen bonds between the polyphenol molecule and Aβ chain. Ehrenberg et al.15 have first reported the inhibition of Aβ fibrillation using (−)-epigallocatechin-3-gallate (EGCG, shown in Scheme 1c). Their study revealed that EGCG molecules could preferentially interact with unfolded Aβ monomers, oligomers, and fibrils. In addition, EGCG molecules could modulate aggregation of Aβ by randomly binding them to the backbone and maintaining its random coil structure, leading to nontoxic oligomers and hindering the formation of toxic oligomers.15 The molecular mechanism was further confirmed with thermodynamic analysis34,35 and nuclear magnetic resonance36 (NMR) by other groups. Further studies also found that EGCG can transform mature fibrils into random coil non-toxic oligomers,15 but its amyloid deformation efficiency is about 50% less at the interface compared to the bulk.37−42 Both KLVFF and EGCG have been reported as quite promising inhibitors, whereas the limited inhibitory efficiency hindered their practical applications. Because the peptide breaker, KLVFF, could bind to the confined region of the Aβ

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RESULTS AND DISCUSSION Modulation of Aβ42 Aggregation. The aggregation kinetics can be analyzed using the curves of thioflavin T (ThT) fluorescence intensity versus time. ThT associates rapidly with amyloid β-sheet structures,46 and the fluorometric technique allows the kinetics elucidation of the amyloid fibril assembly 4234

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binary KLVFF/EGCG as the modulator, the ThT fluorescence intensity (purple curve) was greatly reduced to a considerable low level, which is indicative of a synergistic inhibitory effect by the binary KLVFF/EGCG modulators. In view of the interesting synergistic effect caused by the binary KLVFF/EGCG modulator, CD, AFM, TEM, liquidstate NMR characterizations, and MD simulation were performed to further elucidate the mechanism of modulated Aβ aggregation. CD spectra measurements were performed to investigate the folding and conformational changes of Aβ42. After 48 h incubation, the spectrum curve of Aβ42 alone revealed characteristic changes as shown by the black curve in Figure 2a. The negative CD signal at 218 nm and the positive one at 197 nm were observed, indicating that the second-level structure is dominated by β-sheet structures, in agreement with the previous reports.8,29 In the next step, the folding pattern of Aβ42 with modulators was investigated. Because the modulator molecules may interfere with the CD signals, we obtained CD data of KLVFF, EGCG, and KLVFF/EGCG, and then set them as the corresponding zero baseline. CD data showed that modulators decreased conformational changes of Aβ42. With the addition of KLVFF (shown by the blue curve), the negative CD signal at 218 nm shifts to 210 nm, suggesting the co-existence of α-helix and β-sheet structures. When EGCG acts as a modulator, the CD spectrum is remarkably different from the former one (shown by the purple curve), implying that EGCG interacts with Aβ42 in an absolutely different mode from KLVFF. A strong negative CD signal around 192 nm is observed, demonstrating that the secondlevel structure tends to convert to a random coil. Finally, under the modulation by the binary KLVFF/EGCG modulator (shown by the green curve), two weak positive CD signals at 195 and 206 nm are observed, together with a broad valley in the range of 220−230 nm (the absolute signal value is still above zero). Such a CD spectrum indicates the formation of rigid β-turn structures for a small portion of Aβ42, which represents irregular packing of peptide chains with good structural stability. The appearance of β-turns demonstrates that some Aβ42 monomers aggregate into non-ordered structures. As these unstructured aggregates usually have low toxicity or are non-toxic,10 these observations might indicate a decrease in neurotoxicity of Aβ42 upon the addition of the binary modulator. KLVFF is a key segment of Aβ42 that could recognize core segment 16−20 of Aβ42, and EGCG is a small molecular

process as well as the testing of agents that might modulate their assembly or disassembly.47−50 As shown in Figure 1. To

Figure 1. Aggregation dynamics of Aβ42 (42 μM) with and without modulators by ThT fluorescence. Modulators: KLVFF, EGCG, and KLVFF/EGCG binary modulator. The molar ratios of Aβ42 to modulators are maintained at 2:1.

prevent ThT self-fluorescence, baseline corrections using ThT in the same concentration were applied to eliminate the influence. KLVFF and EGCG molecules may also interfere with fluorescence intensity, so deductions of baselines from KLVFF or EGCG in the same concentration were applied to corresponding curves. The typical aggregation process of the amyloid peptide contains three continuous stages, namely, lag stage, elongation stage, and equilibrium stage. The β-sheet contents were shown by variation in ThT fluorescence intensity. In the elongation phase, the amyloid aggregates undergo conformational arrangements to form β-sheet structures. In sigmoidal fitting curves, the time it takes to increase the β-sheet contents by half is recorded as t1/2. The self-aggregation kinetics of Aβ42 is represented by a typical sigmoidal curve (black curve), which implies that Aβ42 monomers can be efficiently converted into amyloid aggregates in 12 h. The ThT signal levels off after 12 h suggesting that equilibrium in amyloid aggregation has been reached. With individual KLVFF or EGCG as modulators, the fluorescence signals are both observed to be reduced, demonstrating that both KLVFF and EGCG could inhibit Aβ42 aggregation. KLVFF (red curve) decelerated the aggregation of Aβ42 by increasing t1/2 from 4.4 to 5.4 h. By comparison, the inhibitory effect of EGCG (blue curve) was better than that of KLVFF, which demonstrated that the aggregation of Aβ42 is inhibited by increasing t1/2 from 4.4 to 6.0 h. More interestingly, with

Figure 2. (a) CD spectra of Aβ42 (42 μM) and with KLVFF, EGCG, and the KLVFF/EGCG binary modulator. The molar ratios of Aβ42 to modulators are maintained at 2:1. Incubation time: 48 h. (b, c) CD spectra curves of KLVFF and KLVFF/EGCG. Incubation time: (b) 1 h and (c) 48 h. The unit of the y axis is [θ] = ° cm2 dmol−1. 4235

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Figure 3. (upper) AFM and (lower) TEM images of the Aβ42 (42 μM) aggregates with and without modulators after incubation at 37 °C for 48 h. (a, e) Aβ42 alone; (b, f) Aβ42/KLVFF; (c, g) Aβ42/EGCG; and (d, h) Aβ42/KLVFF/EGCG.

Figure 4. Structural changes of Aβ42 revealed by 1H NMR. (a) Conformational changes of Aβ42 assemblies with KLVFF/EGCG binary modulators. (b) Emergence of new peaks when KLVFF and EGCG were mixed together. The molar ratio of Aβ42:/KLVFF/EGCG was maintained 2:1:1. Incubation time: 48 h.

the previous literature.40 In addition, the comparison of CD spectra of KLVFF/EGCG and Aβ42/KLVFF/EGCG (green curve in Figure 2a) confirms that the final β-turn structures are located on the spectra of Aβ42 rather than KLVFF, which revealed that the formation of structurally stable aggregates is a result of the co-contributions from KLVFF and EGCG. The morphology of Aβ42 aggregates with or without modulators was probed by AFM and STM (as shown in Figure 3). Each sample was measured in at least five different locations to guarantee the reliability. The self-assembled Aβ42 aggregates were mainly nanofibrils and a small amount of irregularly shaped particles (Figure 3a,e). With KLVFF alone as the modulator, the morphology of Aβ42/KLVFF aggregates had changed into mainly small particles along with a small amount of nanofibrils (Figure 3b,f). The result showed that KLVFF could partially inhibit the Aβ42 fibrillation. The agglomerated particles with irregular shapes were also observed, which resulted from the entanglement of peptide molecules indicating the less effectiveness of the inhibitors. With EGCG alone as the modulator, Aβ42 aggregates into well-dispersed particles while a small amount of nanofibrils and particle clusters could also be observed (Figure 3c,g). These phenomena demonstrated that EGCG can efficiently interrupt Aβ42 aggregation, although the inhibitory effect was still needed to be improved. Because Figure 2a shows that EGCG as the modulator led to random coil structures in solution, the generation of aggregates might be ascribed to the self-assembly

inhibitor that could stabilize the random coil structures of Aβ42; thus, the understanding of the interactions between KLVFF, EGCG, and Aβ42 might be helpful to reveal the mechanism of the inhibitory effect. CD spectra measurements were performed. The CD curve of KLVFF is shown by a blue line in Figure 2b. After 1 h incubation, KLVFF showed β-sheet characteristics in which a positive peak at 193 nm and a negative peak around 216 nm were observed. The structural changes should be attributed to the self-assembly of KLVFF driven by hydrogen interactions and hydrophobic interactions. After incubation for 48 h, with regard to the KLVFF curve shown by a blue line in Figure 2c, the obvious positive CD signal around 200 nm evidences the formation of β-sheets and α-helices. This phenomenon also supports the hypothesis that the secondary structures of Aβ42 aggregates are partially due to the assembly of KLVFF. KLVFF/EGCG structures are shown by purple curves. The CD spectrum at 1 h exhibited the characteristic signals of β-turns, demonstrating that EGCG can rapidly interact with KLVFF and remodel its assembly pathway. After 48 h, the β-turn structures disappear according to Figure 2c, which might be because the amino residues on short KLVFF chains are insufficient to stabilize β-turns. By further comparison, it is found that the CD spectra of KLVFF/ EGCG and Aβ42/EGCG (purple curve in Figure 2a) are quite analogous to each other. This is because EGCG forms hydrogen bonds at random sites on the full length of Aβ42, rather than merely on the KLVFF segment, as supported by 4236

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Figure 5. (a) 1H−1H 2D spectra of KLVFF (red) and mixed with EGCG (green) (b) 1H−15N HSQC spectra of 15N-labeled Aβ42 alone (blue) and mixed with KLVFF (red) and KLVFF/EGCG binary modulators (green). (c) Binding model between Aβ42 (the shaded molecular structure) and the highlighted KLVFF/EGCG binary modulators. The hydrogen bonds between KLVFF (cyan) and EGCG (green) molecules and Aβ42 are noted with dashed lines, which indicates the synergistic effect in the binary modulator.

monomers from assembling (Figures 1 3), the Aβ42 monomer was applied as the starting template to calculate the binding energy with inhibitor molecules. The molecular structure of Aβ42 and KLVFF in aqueous solution51,52,54 and the binding sites of peptide KLVFF on Aβ4240 were based on previously reported structures. The docking results are shown in Figure 5c. The binding interactions between Aβ42 and KLVFF (as shown in Figure 5c) were mainly on Ala2, Glu3, and Ala21 with a binding energy of −7.5 cal/mol, and those between EGCG and Aβ42 were mainly on Ala21 and Glu22 with a binding energy of −5.5 cal/mol. The binary inhibitors interact with the Aβ42 molecule through hydrogen bonds, and the increased steric hindrance effect of this binary complex compared with the individual ones eventually leads to the prohibited Aβ42 aggregation. In the previous work, Niu et al. demonstrated that terperidine molecules could bind with KLVFF by hydrogen bonding. The binary complex could contribute both peptide− organic and peptide−peptide interactions in the amyloid aggregation process, preventing folding of β-sheet structures.43 In this work, the KLVFF/EGCG binary inhibitors are also demonstrated to be more efficient than a single inhibitor. According to the above, the synergistic modulation of Aβ42 aggregation by KLVFF and EGCG was tentatively illustrated (Figure 6). The assembly of randomly distributed Aβ42 molecules with random coil structures turns into ordered nanofibrils with β-hairpin structures (Figure 6a).8−13 When KLVFF is added, it can selectively bind to one key segment (Aβ16−20) on Aβ42 (Figure 6b).17,20 Due to the blockage of the Aβ16−20 region, the ordered packing of the β-sheets is consequently inhibited. However, KLVFF has little influence on the other segments, especially on other key segments, such as Aβ33−42. As shown in Figure 6c, EGCG is expected to bind to any site on the full length of Aβ42 randomly, including the amide group on the main chain and the amino/carboxyl groups on the side chain or terminals. In this way, Aβ42 tends to aggregate into amorphous structures, whereas the nonselective modulation of EGCG might be less effective when the amino

induced by solvent evaporation during the drying process. With the binary KLVFF/EGCG modulator, the morphology of Aβ42 KLVFF/EGCG was observed to be amorphous, and there were no observable nanofibrils (Figure 3d and 3h), which demonstrated a very efficient inhibitory effect. These results are in good accordance with the ThT fluorescence and CD spectra results. It was reasonable to infer that KLVFF and EGCG can efficiently inhibit Aβ42 aggregation in a synergistic way. It is essential to provide evidence that the KLVFF/EGCG modulators act synergistically to prevent Aβ42 from selfassembling. In the following experiments, liquid-state nuclear magnetic resonance spectroscopy (NMR) measurements were used to detect new binding sites of assemblies. As shown in the blue box in Figure 4a, when Aβ42 monomers were mixed with KLVFF/EGCG and incubated for 48 h, new peaks emerged other than those of Aβ42 or KLVFF/EGCG, indicating that Aβ42 and the binary modulator interacted with the binding sites. The KLVFF/EGCG complex was also explored. As shown in Figure 4b, 1H NMR data revealed that new signal peaks emerged after 48 h incubation. For further identification of binding sites on Aβ42, 2D NMR spectra were obtained. First, we use 1H−1H nuclear Overhauser effect spectroscopy (NEOSY) to investigate the chemical shift of KLVFF residues. As shown in Figure 5a, a slight chemical shift of KLVFF residues could be observed after adding EGCG (molar ratio = 1:1) The evidence confirmed that KLVFF and EGCG may form a complex. We explored chemical shift of Aβ42 by 1 H−15N heteronuclear single quantum coherence (HSQC). As shown in Figure 5b, moderate changes of the chemical shift of Aβ42 (blue) was clearly observed after addition of KLVFF (red) and the KLVFF/EGCG complex (green), which confirmed the interaction between Aβ42 and KLVFF and between Aβ42 and the complex. In addition, molecular dynamics computations were performed using Autodock plugin PyMOL software to explore binding affinity between binary modulators and Aβ42. On the basis of previous experiments where binary inhibitors could prevent Aβ42 4237

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of all modulators were much lower than that of Aβ42. In particular, the combination of KLVFF and EGCG did not lead to higher toxicity compared to Aβ42, which is important for controlling the side effects of the binary modulators. Then, MTT assays were applied to test the effect of modulators on Aβ42 cytotoxicity. The concentration ratio of Aβ42 (42 μM) to KLVFF and EGCG is 2:1:1. To prevent signal interference from modulator molecules, the MTT numerical values of modulators without cells are tested and then subtracted accordingly. As shown in Figure 7a, With KLVFF alone as the modulator, cell viability was increased from 51 to 59%, which could be inferred that KLVFF alone could not efficiently lower Aβ42 cytotoxicity. With EGCG alone as the modulator, cell viability increased up to 62%, which was slightly better than the case of KLVFF. This results agreed with the hypothesis that EGCG could inhibit the formation highly toxic oligomers, as proposed by Ehrnhoefer et al.15 In our work, KLVFF/EGCG binary modulators allowed the highest cell viability up to 79% at **P < 0.001, exhibiting the remarkable synergistic effect. In view of that, cell viability of the KLVF/EGCG complex increased up to 88%, and the binary inhibitor showed a good decrease in Aβ42 cytotoxicity. Extensive oxidative stress could disrupt the integrity of the mitochondria, which may further lead to cell death. Aβinduced oxidative injury and inflammation are widely recognized in neuropathology.55−57 In recent years, polyphenols have been proved to be efficient in modulation of oxidative stress. Further exploration in modulators to reduce Aβ42-derived oxidative stress will provide details of Aβ42 neurotoxicity. First, we performed DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) test for H2O2 generation to study oxidative stress-related toxicity from Aβ42 and modulators. As shown in Figure S2b, DCFH-DA fluorescence increases significantly with Aβ42 monomer concentration implying that reactive oxygen species (ROS) generation was likely induced by Aβ42 monomers. There was no direct evidence that KLVFF inhibitor concentrations were related to ROS generation. Moreover, EGCG could efficiently reduce ROS generation, which was in accord with previous studies. The KLVFF/EGCG complex showed a promising reduction of ROS because of the EGCG component. Then, DCFH-DA fluorescence experiments were performed to reveal modulation of Aβ42-induced ROS generation by inhibitors. Then, in further experiments, SH-SY5Y cells (same generation) were cultured and treated with Aβ42 monomers (42 μM). As shown in Figure 7b, DCFH-DA fluorescence intensity decreased as the KLVFF concentration increased. It can be inferred that KLVFF may reduce oxidative stress by interacting with Aβ42. EGCG showed its efficiency in reducing oxidative stress, which was confirmed by a previous study.34 Moreover, KLVFF/ EGCG binary inhibitors led to good reduction of Aβ42induced ROS content. The results are better than those from the single components, indicating the synergistic effect. The synergistic modulation of binary inhibitors to increase SHSY5Y cell viabilities is shown in Figure 7c. The KLVFF/EGCG complex shows the highest cell viability increase among the Aβ42 monomer-treated samples. The inhibitors were likely to reduce cytotoxicity by interacting with Aβ42 and reducing ROS generation. The contributions of ROS reduction to viability improvement with KLVFF, EGCG, and the KLVFF/ EGCG complex were calculated to be 16, 54, and 51%.The strategy of designing peptide breaker/polyphenol binary

Figure 6. Schematic illustration of Aβ42 aggregation in the presence of single and binary KLVFF/EGCG modulators. (a) Aβ42 molecules with random coil structures self-assembled into fibrils with β-sheet structures. The KLVFF sequences in the Aβ42 molecules are highlighted in light blue color. (b) KLVFF segments recognized the corresponding KLVFF sequence in the Aβ42 molecules, inhibited the Aβ42 aggregation, and resulted in reduced β-sheet structures. (c) Binding of EGCG molecules onto Aβ42 resulting in the Aβ42 aggregation inhibition. (d) Recognition of the KLVFF segments combined with the corresponding sequence in Aβ42 and the binding of EGCG lead to the synergistically enhanced inhibitory effect, possibly increasing the steric hindrance because of the formation of the bulky KLVFF/EGCG complex.

acid residues are very hydrophobic with less hydrogen bond donors and acceptors. Simultaneous utilization of KLVFF and EGEG is therefore expected to integrate the advantages of the two. The synergistic effect can be explained in Figure 6d: KLVFF inhibits the compact packing of Aβ16−20 segments due to molecular recognition, whereas EGCG prevents the formation of hydrogen bonds among Aβ chains due to the occupation of active sites on the full length of Aβ42. Furthermore, KLVFF and EGCG are efficiently connected, the complex can recognize Aβ16-20 because of hydrophobic interactions, and the steric hindrance effect may inhibit the formation of β-sheets. Modulation of Aβ42 Neurotoxicity. To investigate the effect of modulators on Aβ42 neurotoxicity, SH-SY5Y neuroblastoma cells were employed as a benchmark model to evaluate cell viability. In the following experiments, all data were derived from 5 parallel control groups to guarantee the reliability. First, half maximal inhibitory concentration (IC50) values of both modulators and Aβ42 were tested. The results are shown in Figure S1.The IC50 values are determined to be 88, 71, and 42 μM for KLVFF, EGCG, and Aβ42, respectively (Figure S1). Then, we chose the concentration of Aβ42 (42 μM) and maintained molar ratios of Aβ42/KLVFF/ EGCG=2:1:1. In this concentration, KLVFF (21 μM), EGCG (21 μM), and KLVFF/EGCG (21 and 21 μM) showed high cell viabilities of 93, 95, and 88%, respectively (Figure S2a), The results demonstrated that the toxicity values 4238

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Figure 7. Effect of modulator type on the cytotoxicity of Aβ42. (a) Reduced Aβ42 cytotoxicity (42 μM) to SH-SY5Y cells in the presence of KLVFF, EGCG, and the KLVFF/EGCG complex. The molar ratio of Aβ42/KLVFF/EGCG was maintained at 2:1:1.All the cell viability was monitored by using an MTT assay. (b) DCFH-DA fluorescence revealed the reduced ROS generation induced by Aβ42 (42 μM). The concentration in the x axis refers to the complex concentration with a molar ratio of KLVFF/EGCG maintained at 1:1. (c) Cell viability increase in the presence of KLVFF (21 μM), EGCG (21 μM), and the KLVFF/EGCG (21 and 21 μM) complex. Green parts stand for the inhibitory effect with Aβ42 (42 μM), and white parts stand for ROS reduction. Data are presented as mean ± SEM. n = 5, *P < 0.05, **P < 0.02, ***P < 0.001.

helpful for designing potential therapeutic binary inhibitors or even drugs for AD prevention and treatment.

inhibitors could be a general approach for developing a new method for AD prevention or treatment.

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CONCLUSIONS In this study, the concept of a binary peptide/polyphenol binary modulator was proposed. With KLVFF as a peptide breaker and EGCG as a polyphenol modulator, the KLVFF/ EGCG binary modulator showed a synergistic effect in inhibiting the aggregation of Aβ42. With the aid of ThT, CD, TEM, and AFM, the strong inhibitory effect on amyloid fibrillation was verified. By liquid-state NMR measurements and MD simulation, molecular interactions between Aβ42 and binary inhibitors were tentatively proposed, which could possibly explain the synergistic effect: KLVFF could selectively bind to the key segment of Aβ42 chains, and the abundant hydroxyl groups of EGCG allow formation of hydrogen bonds with the main chain and pendant side groups of Aβ. The KLVFF/EGCG binary modulator could synergistically enhance the inhibition effect compared to KLVFF alone or EGCG alone possibly because the increased steric hindrance of the KLVFF/EGCG complex improves the inhibition of Aβ42 fibril extension. As a consequence, the KLVFF−EGCG binary modulator efficiently decreased Aβ-induced neurotoxicity at relatively low concentration of modulators. The KLVFF/ EGCG complex was likely to reduce cytotoxicity by interacting with Aβ42 and reducing ROS generation. This work could be

EXPERIMENTAL SECTION Materials and Methods. Aβ42 and KLVFF were purchased from Shanghai Science Peptide Biological Technology Co., Ltd. 15N-labeled Aβ42 was purchased from Xi’an Ruixi Science Peptide Biological Technology Co., Ltd. (−) Epigallocatechin-3-gallate (EGCG), dimethyl sulfoxide (DMSO), hexafluoroisopropanol (HFIP), thioflavin T (ThT), and acetonitrile were purchased from J&K Scientific Co., Ltd. RPMI-1640 medium, phosphate-buffered saline (PBS), and fetal bovine serum (FBS, filtered using a 0.1 μm filter three times) were purchased from Thermo Fisher Scientific Inc. Methylthiazolyldiphenyltetrazolium bromide (MTT) and penicillin/streptomycin antibiotics were purchased from Beijing Solarbio Science & Technology Co., Ltd. All chemicals were of reagent grade or higher. Aβ42 and KLVFF were dissolved in HFIP to a concentration of 1 mg/ mL, respectively. Then, a certain amount of Aβ42 or KLVFF solution (according to the desired concentration) was added into 1.5 mL microcentrifuge tubes and dried under vacuum at −20 °C. The HFIP-removed peptide samples were temporarily stored at −20 °C before use. EGCG powder was dissolved in DMSO to obtain a 100 mM homogeneous solution. ThT solution (1 mM ) was prepared by first dissolving ThT powder 4239

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Transmission Electron Microscopy. Transmission electron microscopy (TEM) imaging was carried out on carboncoated 200-mesh copper grids that were glow-discharged (S200, Beijing Daji Tech. Co.) for 3 min and dried. TEM samples were prepared by negative staining with 10 μL of 1% uranyl acetate dehydrate (Beijing Puyihua Scientific Co.) for 30 s and dried using nitrogen gas. At each step, the excess moisture was maintained by draining along the periphery using a piece of filter paper. Dried grids were examined using TEM (Hitachi HT7700 Compact-Digital TEM) operated at 80 kV. Nuclear Magnetic Resonance Spectroscopy. NMR spectra for characterizing interactions with Aβ42 were recorded on a Bruker Ascend 600 (Bruker, USA) operating at 600 MHz (1H), equipped with a cryoprobe and z gradients by using Bruker TopSpin 3.5 software. HSQC NMR spectra were recorded at 25 °C by using eight scans and 1024 complex points per increment and 256 increments in the 15N dimension. Four testing solutions were prepared (denoted as “Aβ42”, “Aβ42/KLVFF”, “Aβ42/EGCG”, and “Aβ42/KLVFF/ EGCG”), with H2O and D2O (90 and 10%, respectively) as solvent. Aβ42 samples were prepared at a concentration of 420 μM, and the molar ratio of Aβ42/KLVFF/EGCG was maintained at 2:1:1. Then, the solutions were applied to NMR tubes. The peaks due to water were suppressed using Originlab 9.0. MTT Assay. SH-SY5Y neuroblastoma cells (supplied by Cell Resource Center, Chinese Academy of Medical Sciences & Peking Union Medical College) were used for cell viability measurement. SH-SY5Y neuroblastoma cells were maintained in a medium prepared by adding 15% FBS and 1% penicillin/ streptomycin antibiotics into RPMI-1640 essential medium and grown in a 5% CO2 atmosphere at 37 °C. Cells were harvested from flasks and plated in 96-well plates (Costar 3599, Corning, USA) with approximately 5 × 103 cells per 100 μL of medium per well. Plates were incubated at 37 °C for 12 h to allow cells to attach. Different compositions of drugs (consisting of one or more components among Aβ42, KLVFF, and EGCG) were added into individual wells, and additional fresh medium was supplemented to ensure that the final medium volume for each well reached 200 μL, including the control culture (with cells) and the free medium (without cells). The plates were then incubated at 37 °C for an additional 48 h. Cell viability was determined using an MTT toxicity assay with addition of 20 μL of 5 mg/mL MTT to each well. After incubation for 4 h at 37 °C, a 100 μL aliquot of DMSO was added into each well to completely dissolve the generated formazan crystals. The absorbance at 490 nm was measured using a microplate absorbance reader (Tecan infinite M200, Switzerland). Averages from six replicate wells were used for each sample and control, and each experiment was repeated five times. Cell viability was calculated by dividing the relative absorbance of wells containing modulators (corrected by subtracting out the average absorbance of the free medium) by the relative absorbance of wells for control culture (corrected by subtracting out the average absorbance of the free medium). Measurement of Intracellular ROS Level. SH-SY5Y neuroblastoma cells (supplied by Cell Resource Center, Chinese Academy of Medical Sciences & Peking Union Medical College) were used for cell viability measurement. SHSY5Y neuroblastoma cells were maintained in a medium prepared by adding 15% FBS and 1% penicillin/streptomycin antibiotics into RPMI-1640 essential medium and grown in a

into DMSO and then diluting the obtained 100 mM solution to 1 mM using PBS. Both EGCG solution and ThT solution were stored at 4 °C before preparing testing solutions. ThT Fluorescence Assay. ThT fluorescence assay was performed to determine the dynamic formation of amyloid-like aggregates, because the fluorescence emission of ThT dye is known to be significantly strengthened when ThT binds to βsheet aggregate structures. Four testing solutions were prepared (denoted as “Aβ42”, “Aβ42/KLVFF”, “Aβ42/ EGCG”, and “Aβ42/KLVFF/EGCG”), with 2 mM PBS as solvent and 0.2% DMSO for assisting dissolution. The concentrations of Aβ42, KLVFF, EGCG, and ThT for each testing solution (if any) were fixed at 42, 21, 21, and 10 μM, respectively. A control solution composed of 2 mM PBS and 0.2% DMSO was also prepared for reference. Each testing solution and control solution was pipetted into the well of a 96-well plate (black plate, Costar 3925, Corning, USA), with a piece of tin foil as a cover. The plate was loaded into a microplate absorbance reader (Tecan infinite M200, Switzerland) to survey the variation of ThT fluorescence strength, with excitation at 450 nm and emission at 480 nm. Each reading represented the average of three values determined using a time scan after subtracting out the fluorescence contribution from free ThT, as obtained from the control testing result. Each sample was repeated five times to grantee its reliability. Circular Dichroism Spectroscopy. Circular dichroism (CD) spectroscopy was employed to elucidate the variation of peptide chain conformation in solution caused by modulators. Four testing solutions were prepared (denoted as “Aβ42”, “Aβ42/KLVFF”, “Aβ42/EGCG”, and “Aβ42/KLVFF/ EGCG”), with triple-distilled water as the solvent and 1% acetonitrile for assisting dissolution. The concentrations of Aβ42, KLVFF, and EGCG for each testing solution (if any) were fixed at 42, 21, and 21 μM, respectively. Four Aβ42-free control solutions were also prepared for reference, corresponding to the four testing solutions. Each testing solution and control solution was incubated in a constant-temperature oscillator at 37 °C for 48 h. CD measurements were made on a J-810 spectropolarimeter (JASCO, Japan) by transferring a 300 μL aliquot from the incubated testing solution to a 1 mm-pathlength CD cuvette. CD spectra were recorded in the spectral range of 190−260 nm, at 1 nm bandwidth and 5 nm/min scan speed, subtracting out the contribution from the corresponding control solutions. Each final spectrum represented the average of three spectra scanned for the same sample. Each sample was repeated five times to grantee its reliability. Atomic Force Microscopy. Atomic force microscopy (AFM) was utilized to probe the three-dimensional (3D) structure of the air-dried samples. Four testing solutions were prepared the same way as that for the CD measurements. After an incubation procedure that was exactly the same as that in preparing CD samples, 10 μL of testing solution was dropped onto the surface of a freshly cleaved mica. The remaining liquid was removed 20 min later, and nitrogen gas was applied to dry the mica surface carefully. Tapping mode AFM measurements were then performed on a Nanoscope IIID AFM instrument (Bruker, USA) under ambient conditions. Commercial silicon tips (Bruker, USA) with a nominal spring constant of 40 N/m and resonant frequency of 300 kHz were used in all experiments. Each sample was scanned in at least 5 different locations to guarantee its reliability. 4240

DOI: 10.1021/acsomega.8b02797 ACS Omega 2019, 4, 4233−4242

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5% CO2 atmosphere at 37 °C. Cells were harvested from flasks and plated in 96-well plates (Costar 3599, Corning, USA) with approximately 1 × 104 cells per well. Intracellular ROS levels were measured using the H2O2-sensitive DCFH-DA fluorescence.53 After drug treatments, SH-SY5Y cells were washed with PBS and stained with DCFH-DA (40 μM) for 30 min in the dark. All samples were centrifuged, and the supernates were removed. Then, the cells were lysed with PBS containing 1% Triton X-100. Finally, the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.52 SH-SY5Y cells were cultured with the medium as the control group, and the intracellular ROS levels were expressed as percentage of control. Averages from six replicate wells were used for each sample and control, and each experiment was repeated five times. Results of quantitative studies were expressed as mean ± SEM of at least five independent experiments. Differences were assessed using one-way analysis of variance (ANOVA) for repeated measurements followed by Fisher’s least-significantdifference test when appropriate. Statistical analysis data were obtained using IBM SPSS Statistics 24.0 (Armonk, NY). Molecular Dynamics Prediction. Molecular structures of Aβ42 and KLVFF in aqueous solution were based on works of researchers.51,52 Binding sites of Aβ42 and KLVFF were built according to previous work.43 Binding affinity was calculated by utilizing molecular graphic program PyMOL Autodock plugin and the algorithm DSSP.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02797. IC50 values of Aβ42, KLVFF, and EGCG in SH-SY5Y cells and cytotoxicity and ROS generation of Aβ42 and different modulators, KLVFF, EGCG, and the KLVFF/ EGCG complex (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C.W.). *E-mail: [email protected] (Y.Y.). ORCID

Ling Zhu: 0000-0003-3818-6093 Yanlian Yang: 0000-0002-5607-5509 Author Contributions ‡

These authors contributed equally.

Funding

This work was supported by the National Natural Science Foundation of China (nos. 21773042, 21673055, and 31600803), the Beijing Natural Science Foundation (no. 2162044), and the National Key Research and Development Program of China (2016YFF0203803). Notes

The authors declare no competing financial interest.

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