Study on the Efficiency and Interaction Mechanism of a Decapeptide

Briefly, 20 μL of stock MTT (5 mg/mL) mixed with 100 μL of DMEM medium was ..... These results were confirmed by TEM photos, as shown in Figure S6b...
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Study on the Efficiency and Interaction Mechanism of a Decapeptide Inhibitor of β‑Amyloid Aggregation Jing Liu, Wei Wang, Qian Zhang, Saihui Zhang, and Zhi Yuan* Key Laboratory of Functional Polymer Materials of Ministry of Education, and Institute of Polymer Chemistry, Nankai University, and Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071, China S Supporting Information *

ABSTRACT: This paper reports an active decapeptide inhibitor (RR: RYYAAFFARR) of β-amyloid (Aβ1−40) aggregation. Traditional inhibitors target the hydrophobic core of Aβ (Aβ16−20) and were designed based on the single hydrophobic interaction. RR was designed to target an extended region (Aβ11−23), which contains three important regions of Aβ1−40. RR exhibits stronger binding affinity for Aβ1−40 (KD = 1.10 μM) than the known β-sheet breaker LPFFD (KD = 156 μM). Our study shows that RR inhibited the fibrillation of Aβ1−40 by nearly 75% at an equimolar concentration, and that a 1:4 ratio of Aβ1−40/ RR almost completely inhibited fibrillation. The interaction mechanism was also investigated by changing the ionic strength or the structure of RR. The results revealed that RR binds to Aβ1−40 because of its strong affinity for Aβ11−23, which is mainly driven by hydrophobic and electrostatic interactions and hydrogen bonding.

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amyloid β peptide fibrillogenesis in vitro and prevents neuronal death induced by β-sheet fibrils in cell cultrue.22−24 Peptide inhibitors have also been designed to target the key hydrophobic core region at Aβ16−22 (KLVFFAE), which bind to the homologous sequence in Aβ and prevent its aggregation into amyloid fibrils. Building on the pioneering study of Tjernberg, various peptides and peptide derivatives have been synthetized as “Aβ-binding elements” based on the hydrophobic core sequence, including the methylation of amide groups,25−27 multivalency,28−30 conjugation of a functional group to a β-breaker motif,31−33 and α,α-disubstituted amino acids (α, α-AA) based on the KLVFF sequence.34 Although the studies that have targeted the hydrophobic core sequence have yielded encouraging results, some problems remain unresolved. First, some of the β-sheet breakers, require a large molar excess relative to Aβ to inhibit Aβ fibrillation.26,35,36 Second, some inhibitors merely affect the lag time of Aβ aggregation, delaying but not preventing it.19,30,37 In our view, the main cause of these phenomena is that the affinity of the inhibitors for the targeting interaction sites of Aβ was not strong enough. It is well-known that the interaction between proteins is based on the synergistic action of multiple weak interactions.38 Therefore, we speculated that if inhibitors can target several regions through multiple recognition and binding sites, the binding affinity between the inhibitor and Aβ would be enhanced and the inhibition of Aβ aggregation would be more

INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder associated with the loss of memory, cognitive decline, and behavioral disability, leading ultimately to dementia and death.1−3 Extracellular deposits of β-amyloid (Aβ) peptide, called “senile plaques”, are the main histopathological criterion for AD,4−7 and Aβ plays a central role in the pathophysiology of AD. Normally, Aβ exists as soluble monomers, but in AD patients, the peptide can self-assemble into insoluble neurotoxic aggregates with various morphologies, including dimers, oligomers, protofibrils, and mature fibrils.8,9 During the transitions between these forms, the conformation of Aβ changes from a random coil or α-helix to a β-sheet.10 Therefore, inhibiting the change of the secondary structure to β-sheet is a promising therapeutic approach to AD. It has previously been shown that the hydrophobic core KLVFF (Aβ16−20) of Aβ is the region that binds most efficiently to Aβ and that this region is essential for fibril formation.11−14 Consequently, numerous inhibitors targeting this region have emerged, and these can be divided into several categories, including small molecules,15 surfactants,16 chaperone proteins,17 peptides/peptide mimics,18 and nanoparticles.19 Among these inhibitors, short peptides can be considered lead compounds for the design of inhibitors of Aβ aggregation. The first pentapeptide inhibitor KLVFF (Aβ16−20), which was proposed by Tjernberg, could recognize and bind to full-length Aβ, preventing its aggregation into fibrils.20 Although the residues around KLVFF are considered to be a key region, they have been shown to form a β-sheet structure in isolation.14,21 Soto et al. then designed the β-sheet breaker LPFFD based on the Aβ17−20 (LVFF) sequence, and it was found that it inhibits © 2014 American Chemical Society

Received: December 6, 2013 Revised: January 21, 2014 Published: January 21, 2014 931

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Figure 1. Structure of peptide inhibitor (RR) designed for binding to the 11−23 region of Aβ. Black arrows indicate the amino acids in Aβ11−23 with which the different groups are designed to interact. The groups in RR are showed with different colors, and red represents the electrostatic interaction, green represents the hydrogen bond, and purple represents the hydrophobic interaction.

effective.39 On the basis of the studies described above, we designed a decapeptide inhibitor RR (RYYAAFFARR) with the help of computer simulation based on the structure of the Aβ11−23 region, which is expected to interact with Aβ1−40 in multiple ways, including via hydrophobic and electrostatic interactions and hydrogen bonding (Figure 1). The inhibitory effect of RR was evaluated with thioflavin T (ThT) fluorescence, circular dichroism (CD) spectra, transmission electron microscopy (TEM), and a cell viability assay. The binding affinity of RR for the Aβ1−40 monomer was also evaluated using the surface plasmon resonance (SPR) system. To further analyze the mechanism of the inhibitory effect and to test the region of interaction, SPR was used to examine the affinity and driving force between the Aβ11−23 and RR interaction. The results of this study should be helpful in designing new and effective peptide inhibitors to control the aggregation of Aβ as an AD therapy.

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MALDI-TOF Mass Spectrometry. The RR sample for MALDITOF was prepared by dissolving RR in PBS (10 mM, pH 7.4) and culturing in a shaking incubator at 37 °C. In order to prove the Aβ1−40, which was attached to the chip sensor surface, existed as monomeric form, the MALDI-TOF mass assay was performed. The way of preparation for the sample here was the same as that in the SPR assays. Thioflavin T (ThT) Fluorescence Assay. The Aβ1−40 fibrillation in the presence and absence of inhibitors was monitored by ThT fluorescence assay, which was carried out by a F-7000 Fluorescence spectrophotometer (Hitachi, Japan). The concentration of ThT store solution was 500 μM, and then the ThT solution was further diluted into phosphate buffer (pH 7.4) to reach a final concentration of 10 μM. A volume of 100 μL of Aβ1−40 with or without the inhibitor was put into 900 μL ThT solution, and then the mixture was blended briefly. All ThT fluorescence measurements were operated using a 1 × 1 cm2 quartz cuvette (excitation at 440 nm and emission at 485 nm). Each sample was measured three times, and then the data were averaged. Circular Dichroism Measurements. Circular dichroism (CD) spectra measurements were carried out using a Jasco J-715 CD Spectrometer (Jacso 715, Japan). The ellipticity was recorded between 260 and 190 nm in a 0.1 cm quartz cuvette with 10 mm path length at 37 °C. The bandwidth was 1.0 nm, and the scanning rate was 50 nm/ min with a continuous scanning mode. The background signal from the PBS buffer has been subtracted, and the data were averaged after being measured three times. Transmission Electron Microscopy. All experiments were performed by using TEM (FEI, Tecnai G2 F20, U.S.A.). To detect the effect of the peptide inhibitors on Aβ1−40 aggregation, TEM was used to observe the peptide morphology. Samples were prepared by placing a 15 μL droplet of sample solution on a carbon support Cucoated grid for 90 s, and then the excess fluid was wicked away. Subsequently, the samples were negatively stained with 1% tungstophosphoric acid for 90 s, the excess fluid was removed, and the grids were placed in a vacuum drier and dried at room temperature. Cell Viability Assays. Highly differentiated rat pheochromocytoma (PC-12) cells were cultured in a medium of 90% DMEM, and 10% fetal calf serum at 37 °C in an atmosphere of 5% CO2. For cell viability assays, cells were plated in 96-well plates at a density of 3 × 104 cells per well and maintained for 24 h. To evaluate the biological cytotoxicity of the RR itself, peptide solutions were prepared by dissolving the RR peptide in deionized water and diluting into DMEM medium to yield concentrations of 10−160 μM. Twenty-four hours after plating of cells, 150 μL of the RR solutions in DMEM were added into the 96-well plate. The cells were then incubated for 18 h with the inhibitor RR at 37 °C. Cell viability was determined by the thiazolyl-bule-tetrazolium-bromide

MATERIALS AND METHODS

Materials. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, 99%) and thioflavin T (ThT, 75%) were purchased from Heowns (China). Aβ1−40 peptide (95.52%), Aβ11−23 (Ac-EVHHQKLVFFAED-NH2; 98%), RR (Ac-RYYAAFFARR-NH2; 98%), LPFFD (99.16%), RR-1 (Ac-RYYAAGGARR-NH2; 98%), and RR-2 (Ac-RGGAAFFARRNH2; 98%) were purchased from GL Biochem (Shanghai) Ltd. The cell lines PC-12 were purchased from Cell Bank of Chinese Academy of Science. The other reagents were commercially available and used without purification. Peptide Pretreatment. Aβ1−40 was disaggregated by treatment with HFIP as described previously.40 To disrupt any aggregation, Aβ1−40 was dissolved in HFIP to 1 mg/mL, and the solution was incubated at 37 °C for 1 h. To prepare the fresh Aβ1−40 monomer solution, HFIP was evaporated off under N2 and the resulting white powder was lyophilized overnight. Finally, the peptide was stored in freezer at −20 °C until ready for use. For the preparation of Aβ1−40 solution, NaOH (10 mM) solution was used to dissolve the Aβ1−40, sonicating the solution for 1 min, and the stocking solution was diluted in PBS buffer for different concentrations. Preparation of Aβ1−40 Solutions. In order to avoid the possible influence by bacteria and obtain the reliable and reproducible result, all vials, pipet tips, DI water, and PBS used in this study, except SPR, were autoclaved and all the peptides were processed by UV irradiation for 1 h. To prepare the 20 μM Aβ1−40 solution, the Aβ1−40 in NaOH (10 mM) stocking solution was diluted in PBS buffer (10 mM, pH 7.4). For each inhibitor (RR, RR-1, RR-2, LPFFD), we incubated Aβ1−40 (20 μM) with 20, 40, and 80 μM of inhibitors in PBS buffer, shaking at 37 °C at various time intervals as long as 10 days. 932

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(MTT) assay. Briefly, 20 μL of stock MTT (5 mg/mL) mixed with 100 μL of DMEM medium was added into the 96-well plate, and the plate was incubated at 37 °C. Four hours later, 120 μL of DMSO was added after the medium-MTT solution was removed. Finally, absorbance intensity at 490 nm was measured with an ELISA reader. At least three independent experiments with five replicates were carried out, and the results were averaged. To test the inhibitory effect of the peptide inhibitor RR on Aβ1−40induced toxicity, solutions of Aβ1−40 and RR at three different ratios (1:1, 1:2, 1:4) were prepared, and Aβ1−40 alone was regarded as the control experiments. In this part of the experiment, the solutions of Aβ1−40 with or without RR were preincubated for 12 h in DMEM. Cell viability was determined by the MTT assay as described above. Surface Plasmon Resonance (SPR) Assays. The SPR experiments were all performed on a Biacore 3000 system (Biacore, Uppsala, Sweden) using CM5 sensor chips (provided by Z. F. Luo, University of Science and Technology of China). It was reported that different surface chemistry of matrix can dramatically influence the adsorption amount of Aβ on the surface.41 Therefore, in our study, we chose the CM5 sensor chips consistently. On the CM5 sensor chip, the gold surface is covered with a matrix of carboxymethylated dextran, a flexible unbranched carbohydrate polymer forming a thin surface layer approximately 100 nm thick. The buffer used to immobilize Aβ11−23 and Aβ1−40 through amide bond formation was 10 mM sodium acetate buffer (pH 5.0). The running buffer was 10−50 mM PBS (pH 7.4) containing 50−200 mM sodium chloride, and 10 mM PBS buffer (50 mM sodium chloride) was used unless otherwise noted. The regeneration buffer was 50 mM sodium hydroxide and sodium dodecyl sulfonate (SDS) solution. Prior to use, all buffer was filtered by using a 0.22 μm anatop filter and all sample solutions for SPR assays were centrifuged before used. Immobilization of Aβ11−23 peptide onto the CM5 chip followed the standard amide bond forming conditions as described elsewhere.42 Briefly, the carboxymethyl dextran matrix of the sample flow channel and reference flow channel were activated by the 1:1 mixture of EDC (200 mM) and NHS (50 mM). The Aβ11−23 (100 μg/mL) solution was then injected into the activated sample flow channel, and the free and unreacted NHS esters in the sample channel were capped with ethanolamine. The reference flow channel was activated by the same method, and was blocked directly, instead of attaching any peptide. Immobilization of Aβ1−40 monomer, which was freshly prepared by 10 mM NaOH solution at 5 mg/mL and performed onto the CM5 surface until reached a final change in response units (RU) of 1000, followed the same protocol as Aβ11−23 peptide. In the end, the sensor surface was cleaned by the regeneration buffer, which also could disassemble the possibly formed Aβ assemblies during the immobilization. All SPR kinetic experiments were performed at 298 K with 5−7 different concentrations of inhibitor peptide solutions in PBS running over the Aβ-immobilized chip surfaces at a constant flow rate of 30 μL/min, which could minimize the mass transfer effect. The running time of inhibitor peptide was 90 s, followed by a 300 s dissociation phase. The chip surface was regenerated twice using SDS and NaOH solution. Data from the reference flow channel without Aβ immobilization were subtracted from raw data. The data were processed by Biaevaluation software (Biacore), and the dissociation constant (KD) fitted using the 1:1 langmuir binding model unless otherwise noted. Molecular Docking. In the docking, the Silicon Graphics SGI INDIGO 2 workstation was used. The structure of RR was built by SYBYL 6.91 program package, and minimized by molecular dynamics and molecular mechanics. In the Protein Data Bank, there are several possible structure of Aβ1−40 are available, and the structure with PDB code 1BA443 was chosen for the docking because it was resolved in an environment with a pH closer to water. In addition, the structure of Aβ (pdb ID 1BA4) was obtained in the water/SDS micelles, which could simulate a water-membrane interface. Aβ1−40 is soluble in micelles, which simulates to some extent a cell membrane environment.43 In all cases the conformation with the lowest energy was applied as the initial molecular conformation. Aβ1−40 docking to RR with different

conformations was performed using the Autodock 4.0 program. During the docking, RR was chosen as the flexible ligand and Aβ1−40 was fixed. A total of 100 different docking runs were performed to find the best binding conformation based on the binding energy (Ebinding), which was deduced from the following expressions.

E binding = Ecomplex − EAβ − E RR where Ecomplex was the binding energy of Aβ1−40 with RR, EAβ and ERR represented the energy of Aβ1−40 and RR, respectively.

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RESULTS AND DISCUSSION Design of Inhibitors. The full Aβ molecule is generally divided into five regions: the N-terminus,44,45 GAG-binding sites,46 hydrophobic core,47,48 turn region or “salt bridge” region,49−51 and the C-terminus.52,53 It has been confirmed that the process of Aβ aggregation is controlled by specific amino acids and peptide regions,51 and large numbers of studies have demonstrated that various Aβ peptide regions contribute differently to Aβ aggregation. The study of Meredith54 found that the “salt bridge” which forms between Asp23 and Lys28 because of their opposite charges strongly favors the nucleation and fibrillation of Aβ1−40. The imidazole rings of His13 and His14 also offer hydrogen bond sites. On the basis of these studies and the structure of the Aβ11−23 region,43 we expanded the conventional target of the hydrophobic core and designed the decapeptide inhibitor RR (shown in Figure 1). Two tyrosines were included in the sequence of RR to potentially form hydrogen bond with His13 and His14 via the phenolic hydroxy. The hydrophobic interaction with the hydrophobic core region was established with two phenylalanines. The two arginines at the C-terminus were targeted by the “salt bridge” and were expected to form electrostatic interactions with Glu22 and Asp23 via the two positive charges on the arginine moieties. The alanine was introduced as the spacer between the functional amino acids, and is expected to enhance the inhibitory capacity of RR via its methyl group. The length of the spacer was screened using a molecular docking method. During the docking process, the functional amino acids in the ligand sequence were kept constant, but the length of the spacer between the interaction sites was varied. The length of spacer and the potentially most effective structure of RR were determined based on the principle of lowest energy and spacing matching (Figure S1): two alanines between tyrosine and phenylalanine (YYAAFF), and one alanine between phenylalanine and arginine (FFARR). RR has multiple weak interaction sites, which recognizes and interacts with the corresponding residues of Aβ1−40. Multiple arginines were also introduced into the structure of RR to increase the repulsion between peptides, to prevent the aggregation of RR itself. This was confirmed with MALDI-TOF mass spectrometry (Figure S2a) and CD (Figure S2b). Affinity of Inhibitors and the Aβ1−40 Monomer Assessed by SPR. For an effective inhibitor, there is a relationship between its affinity for Aβ and its inhibition of Aβ aggregation and toxicity.55 In our design, the inhibitor RR has multiple interaction sites and should exhibit great affinity for Aβ. So the SPR system was used to study the affinity between RR and Aβ1−40, and in the experiments, monomeric Aβ1−40 was immobilized on the CM5 chip surface, as suggested by the MALDI-TOF MS result (Figure S3). To avoid the possibility of Aβ assemblies on the chip, we immobilized the monomeric Aβ1−40 to the chip surface, which could manipulate the density 933

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Figure 2. SPR sensorgrams of RR (a) and LPFFD (b) at various concentrations flowing over Aβ1−40 immobilized on the sensor chip. RR (or LPFFD) was injected for 3 min at a flow rate of 30 μL/min, followed by a 300 s dissociation phase. The chip was then regenerated with 50 mM NaOH at a flow rate of 30 μL/min, initially for 60 s with 0.05% SDS and then for 90 s without SDS.

Figure 3. β-Sheet structure of Aβ1−40 (20 μM) was monitored using ThT fluorescence in the absence and presence of different concentrations of RR. The samples were incubated at 37 °C for 10 days with agitation.

examined whether RR inhibits the aggregation of Aβ1−40 effectively in the next section. Effects of RR on Aβ ThT Fluorescence. A ThT fluorescence assay was used to quantify fibril formation based on the specific binding of the ThT dye to β-sheet structures.57 The fluorescent signal is proportional to the amount of β-sheet structure, so we used ThT fluorescence assays to characterize the fibrillation of Aβ1−40. The results (Figure 3) of the ThT binding assay of Aβ1−40, in the absence or presence of different concentrations of RR, showed that RR strongly and effectively inhibited β-sheet aggregation of Aβ1−40 in a dose-dependent manner. In particular, equimolar mixtures of Aβ1−40 (0.087 μg/ μL, 20 μM) and RR (0.027 μg/μL, 20 μM) reduced the relative change in the ThT fluorescent signal by nearly 75%. Incubation of Aβ1−40 and RR (0.110 μg/μL, 80 μM) in a molar ratio of 1:4 resulted in a 91% reduction in the fluorescent signal, which did not increase during incubation for 10 days. It was reported that incubation of Aβ1−40 (0.5 μg/μL, 115 μM) for 7 days in the presence of equimolar (0.074 μg/μL, 115 μM) or 20-fold molar excess (1.5 μg/μL, 2.3 mM) of LPFFD resulted in 53.5 and 84.1% inhibitions of fibrillogenesis, respectively.22 The ThT fluorescent assay indicated that RR is a more potent inhibitor of Aβ1−40 aggregation. So the results of the ThT fluorescence experiments are compatible with the relative affinities measured with SPR assays.

of the Aβ molecules on the sensor surface and minimize its aggregation. As the RR working solutions flowed over the chip surface, RR rapidly bound to the Aβ1−40 monomer immobilized on the chip surface, and then gradually dissociated from the surface with a dissociation constant (KD) of 1.10 μM (Figure 2a). The SPR experiment revealed that RR interacted specifically with the immobilized target peptide, the Aβ1−40 monomer. We also tested the affinity of a known β-sheet breaker LPFFD for the Aβ1−40 monomer (Figure 2b), and the KD value was 156 μM. The amount of RR that bound to Aβ1−40 was much higher than the amount of LPFFD (at same concentration) that bound to Aβ1−40 (Figure S4). It is well-known that LPFFD is an effective Aβ1−40 inhibitor, as demonstrated by Soto,22 and it was also reported that it displays a special binding affinity for Aβ1−40.56 Thus, our SPR assays showed that RR had a higher binding affinity for Aβ1−40 than LPFFD, which preliminarily verified the advantage of synergy of the multiple weak interactions. Man Hoang Viet56 provided theoretical support for the correlation between the binding affinity and the inhibitory ability: stronger binding between the inhibitor and Aβ correlates with more effective inhibition. Christopher W. Cairo55 also demonstrated with SPR assays that ligands with greater affinity for Aβ effectively altered its aggregation and inhibited its cell toxicity. Our results show that RR binds to the Aβ1−40 molecule with strong binding affinity. Therefore, we 934

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Figure 4. Typical CD spectra of aggregated Aβ1−40 (20 μM) in the absence and presence of different concentrations of RR (a) 20, (b) 40, and (c) 80 μM, cultured at 37 °C for 7 days with agitation.

Figure 5. TEM images of Aβ1−40 (0.087 μg/μL, 20 μM) incubated with RR or LPFFD for seven days: (a) Aβ1−40 alone, (b) RR 20 μM (0.027 μg/ μL), (c) RR 40 μM (0.054 μg/μL), (d) RR 80 μM (0.110 μg/μL), (e) LPFFD 80 μM (0.05 μg/μL). Scale bars: 200 nm.

Effects of RR on Aβ Secondary Structure. The secondary conformational change in Aβ from an α-helix and/ or random coil to a β-sheet is regarded as the key step in the aggregation process.58 Therefore, CD measurements were made to determine the influence of RR on the secondary structural changes in Aβ1−40 during incubation. The CD spectrum of freshly prepared Aβ1−40 alone showed an obvious minimum at 200 nm, indicative of the main random coil

conformation (Figure 4). After culture for 7 days, the minimum at 200 nm disappeared and the CD spectrum of Aβ1−40 displayed a broad minimum ellipticity at 217 nm, which is a typical characteristic of β-sheet structures, as shown in Figure 4. The CD curve of Aβ1−40 in the presence of equimolar RR also showed an upward shift of ellipticity above 217 nm toward zero (Figure 4a), reflecting a reduction in the β-sheet content because of the binding of RR and Aβ molecules. When Aβ1−40 935

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was incubated with RR in a molar ratio (Aβ1−40/RR) of 1:2 (Figure 4b), the ellipticity above 217 nm almost disappeared. Furthermore, the CD curve of Aβ1−40 incubated with RR for 7 days (in a molar ratio of Aβ1−40/RR of 1:4) did not change compared with that of freshly prepared samples (Figure 4c). These results indicate that RR inhibits the change in the conformation of Aβ1−40 to β-sheet, which was just the reflection of the strong binding force between Aβ1−40 and RR. It is worth noting when the molar ratio of Aβ1−40 and RR was 1:4, RR almost stabilized the initial conformation of Aβ1−40. Effects of RR on Aβ Aggregation Morphology. To confirm the inhibitory effect of RR on Aβ1−40 aggregation, TEM was used to study the morphology of the aggregates that formed in the absence and presence of RR. When Aβ1−40 (0.087 μg/μL, 20 μM) was incubated alone for 7 days, the sample formed mature fibrils (Figure 5a), which were approximately 1−10 nm wide and tens to hundreds of nanometers long. In the presence of 1−4 mol equiv of RR, the morphology of Aβ1−40 changed and the fibril content decreased dramatically (Figure 5b−d). When Aβ1−40 was incubated in the presence of 4-fold molar excess of RR (0.110 μg/μL, 80 μM), the aggregation of Aβ1−40 was strongly inhibited by RR, and only amorphous aggregates were visible but without any ordered fibrillar aggregates on the entire copper grid (Figure 5d). While treated with LPFFD (0.05 μg/ μL, 80 μM) under the same conditions for 7 days resulted in much shorter, dispersed fibrils (Figure 5e). The TEM images of the morphology confirmed that RR had more effect on Aβ1−40 aggregation than LPFFD, as suggested by the ThT fluorescence assay. Effects of RR on Aβ Cytotoxicity. The toxicity of Aβ lies in its soluble oligomeric forms and is related to its selfassembly. Therefore, we studied the effect of RR as a peptide inhibitor of Aβ1−40-induced toxicity by measuring cell viability. First we examined the toxicity of RR because it was designed to reduce the cytotoxicity of amyloid β fibrils. The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure S2c) showed that RR itself had little toxicity for neuronal PC-12 cells at concentrations of up to 160 μM. Figure 6 shows that RR protected PC-12 cells from Aβ1−40induced toxicity, but to different extents. Cell viability after Aβ1−40 (20 μM) treatment was approximately 65% that of the control, whereas incubation of Aβ1−40 with RR strikingly enhanced cell viability. Furthermore, RR in a molar ratio of 1:1 (Aβ1−40/RR) was sufficient to significantly prevent Aβ1−40induced toxicity and Aβ1−40 aggregation. The viability of PC-12 cells increased to about 95% when Aβ1−40 was incubated with RR in a molar ratio of 1:4 (Aβ1−40/RR), which was almost equal to the control. Because Aβ neurotoxicity is related to the formation of the β-sheet structure,59−61 the MTT assay results suggest that RR is an effective inhibitor of Aβ1−40, supporting the results of the ThT fluorescence and CD assays. Interaction Mechanism of RR and Aβ. In our study, the peptide inhibitor RR was designed based on the structural sequence of Aβ11−23. Therefore, RR should recognize this special region when it interacted with Aβ1−40, and should display similar affinity for Aβ11−23 and Aβ1−40. Then we examined the affinity of RR for Aβ11−23 and Aβ1−40, which theoretically should be similar if our design was correct. Kinetic curves of the interaction between Aβ11−23 and RR were constructed (Figure 7a) and the dissociation constant (KD) was 4.30 μM. From the dissociation constants (Figure 7b), we determined that the affinity of RR for Aβ1−40 (KD = 1.10 μM)

Figure 6. Viability of PC12 cells treated with Aβ1−40 (20 μM) alone or with different concentrations of RR for 18 h. Cell viability was measured with MTT assays and is shown as a percentage of the untreated cells (control; *P < 0.05).

was a little higher than its affinity for Aβ11−23. However, the KD values of RR for both Aβ11−23 and Aβ1−40 were in the same order of magnitude. Moreover, in the subsequent study, when we changed the ionic strength, the type of ions, and the structure of the peptide inhibitor, Aβ11−23 exhibited dissociation constants and trends with the inhibitors similar to those of Aβ1−40. From these results, we infer that the Aβ11−23 segment is the main binding region for RR in its interaction with Aβ1−40, which is consistent with our original design. To confirm the existence of hydrophobic interaction in the binding of RR to Aβ, the two phenylalanines of RR were replaced with two glycines, producing the new peptide RR-1. With the lack of these two phenylalanines, a distinct difference was observed in the interaction between Aβ1−40 and RR or RR1. The binding curves for RR-1 and Aβ1−40 are shown in Figure 8a. The mode of the interaction between RR-1 and Aβ1−40 is the typical “fast binding−fast dissociating” mode because their affinity is weak; this is also true for Aβ11−23 (Figure S5a). The KD value (KD = 102 μM) for RR-1 binding to Aβ1−40 was much higher than that of RR, fitted using steady-state affinity analysis. We also examined the inhibitory effect of RR-1 on the aggregation of Aβ1−40 using ThT fluorescence and TEM. Compared with RR, RR-1 showed less obvious inhibition of Aβ1−40 aggregation (Figure 8c). The TEM images of RR-1 incubated with Aβ1−40 for seven days showed amyloid β fibrils, but fewer than when Aβ1−40 was cultured alone (Figure S6a). In other words, the inhibitory effect of RR decreased markedly when its two phenylalanines were substituted with two glycines. Therefore, we infer that hydrophobic interactions play an important role in facilitating the interaction between RR and Aβ1−40, that hydrophobic interactions are one of the key interactions involved in this binding, and that the two phenylalanines of RR are key binding sites. When we designed the structure of RR, two tyrosines were introduced for hydrogen bonds. However, the factors that influence hydrogen bonds are very complex and difficult to explain. Therefore, we altered the structure of RR by removing these two tyrosines and studied the change in its inhibitory capacity. RR-2 was homologous to RR with the two tyrosines of RR replaced with two glycines. The lack of these two tyrosines, 936

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Figure 7. (a) SPR sensorgrams of RR at various concentrations flowing over Aβ11−23 immobilized on the sensor chip. RR was injected for 3 min at a flow rate of 30 μL/min, followed by a 300 s dissociation phase. The chip was then regenerated with 50 mM NaOH at a flow rate of 30 μL/min, initially for 60 s with 0.05% SDS and then for 90 s without SDS; (b) comparison of the affinities of Aβ11−23 and Aβ1−40 for RR.

Figure 8. SPR sensorgrams of RR-1 (a), RR-2 (b) at various concentrations flowing over Aβ1−40 monomer immobilized on the sensor chip. RR-1 was injected for 3 min at a flow rate of 30 μL/min, followed by a 300 s dissociation phase. The chip was then regenerated with 50 mM NaOH at a flow rate of 30 μL/min, initially for 60 s with 0.05% SDS and then for 90 s without SDS; (c) β-sheet structure of Aβ1−40 (20 μM) was monitored using ThT fluorescence in the absence and presence of RR, RR-1, and RR-2 at the same concentrations (80 μM); (d) Effects of ionic strength on the kinetic constants for the interaction between Aβ1−40 and RR.

The same phenomenon was observed when RR-2 interacted with Aβ11−23 (Figure S5b). What is more, as shown in Figure 8c, the incubation of Aβ1−40 with RR-2 in a molar ratio of 1:4 reduced the fluorescence intensity by 48.6%. These results were confirmed by TEM photos, as shown in Figure S6b. These results indicate that the two tyrosines of RR are essential to and participate in the interaction between RR and Aβ1−40, possibly involving hydrogen bonds or hydrophobic interactions because of the phenol moieties.

which were originally designed to form hydrogen bonds with His13 and His14 of Aβ, also affected the interaction between the inhibitor and Aβ. Figure 8b shows the kinetic curves of the interaction between RR-2 and Aβ1−40 determined with SPR system. The interaction between RR-2 and Aβ1−40 belongs to the typical “fast binding−fast dissociating” mode of binding, and the KD was 89.3 μM when fitted using a steady-state affinity analysis. From these results, the affinity for Aβ1−40 decreased after the two tyrosines were removed from the structure of RR. 937

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Figure 9. Interaction model of RR docked to Aβ1−40 using autodock 4.0: red represents the electrostatic interaction, green represents the hydrogen bond, and purple represents the hydrophobic interaction.

The interaction between the arginines of RR and the “salt bridge” region played an important role in our design of RR in this study. Therefore, to further probe the driving force and mechanism behind the binding of RR to Aβ, we used SPR to investigate the effect of ionic strength on their binding. Typical SPR kinetic curves for the interaction between RR and Aβ1−40 at a series of different NaCl concentrations in PBS are shown in Figure S7. The SPR results for the binding of RR to Aβ1−40 at different concentrations of NaCl are shown in Figure 8d. In NaCl concentrations ranging from 0 to 200 mM, the value of −lg KD clearly increased as the salt concentration increased (i.e., the ionic strength increased). The change in the affinity between RR and Aβ11−23 with increasing ionic strength is shown in Supporting Information, Figure S8. It is well-known that increasing the salt concentration can reduce or even completely shield electrostatic interactions. Therefore, our data show that electrostatic interactions also play an important role in the binding of RR to Aβ1−40, as in the binding of RR to Aβ11−23 (Figure S8). Moreover, the arginines in RR are considered to be binding sites. Figure 9 shows a possible intercation pattern for the binding observed in the molecular docking study. The docking results indicate that RR binds the residues 11−23 of the Aβ1−40 molecule with multiple weak interactions, including hydrophobic and electrostatic interactions and possibly hydrogen bonding. Therefore, the model we propose to explain the inhibitory effect of RR on Aβ1−40 aggregation involves the initial recognition of Aβ11−23 by RR, after which RR binds to the full Aβ1−40 molecule through multiple weak interactions (as shown in Scheme 1), including hydrophobic and electrostatic interactions and hydrogen bonds. And the multiple weak

interactions favor RR bind to Aβ1−40, and at last interfere and inhibit the aggregation of Aβ1−40.

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CONCLUSIONS A novel peptide Aβ inhibitor, RR, is designed based on the structure of the Aβ11−23 sequence, which involves three important subregions of Aβ1−40: the GAG-binding sites, hydrophobic core, and “salt bridge” region. The results confirmed that RR binds to the Aβ1−40 molecule via multiple weak interactions, including hydrophobic and electrostatic interactions and presumably hydrogen bonding. RR exhibits strong binding affinity for Aβ1−40 (KD = 1.10 μM) and effectively inhibits the aggregation and fibrillation of Aβ1−40 according to the results of a ThT fluorescence assay, CD, and TEM. RR also markedly reduced the cytotoxicity induced by Aβ1−40 self-assembly and significantly enhanced the viability of PC12 cells according to an MTT assay. The results of this study suggest that utilizing the synergy of multiple weak interactions is a promising strategy for the design and development of novel effective inhibitors of Aβ.

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ASSOCIATED CONTENT

S Supporting Information *

The screen process of the spacers between different functional amino acid sites, characterization of RR, TEM images of Aβ1−40 cultured with RR-1 and RR-2, and SPR kinetic curves of different inhibited peptides (RR-1, RR-2, and LPFFD) binding to Aβ (Aβ1−40 or Aβ11−23) under different conditions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

Scheme 1. Schematic Representation of the Possible Mode of Interaction between Aβ1−40 and RR

ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (20634030, 51003048, 51273094), State Key Fundamental R&D Project (2011CB606202), and PCSIRT (IRT1257) for support of this work. We also thank Zhao-Feng Luo, University of Science and Technology of China, for his technical guidance in SPR tests.

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