d-Enantiomeric RTHLVFFARK-NH2: A Potent ... - ACS Publications

Subscriber access provided by Iowa State University | Library

Article 2

D-Enantiomeric RTHLVFFARK-NH: A Potent Multifunctional Decapeptide Inhibiting Cu -Mediated Amyloid #-Protein Aggregation and Remodeling Cu -Mediated Amyloid # Aggregates 2+

2+

Wei Liu, Xiaoyan Dong, and Yan Sun ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00440 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1

D-Enantiomeric RTHLVFFARK-NH2: A Potent Multifunctional

2

Decapeptide

3

Aggregation and Remodeling Cu2+-Mediated Amyloid β Aggregates

Inhibiting

Cu2+-Mediated

Amyloid

β-Protein

4 5

Wei Liu, Xiaoyan Dong and Yan Sun*

6

Department of Biochemical Engineering and Key Laboratory of Systems

7

Bioengineering of the Ministry of Education, School of Chemical Engineering and

8

Technology, Tianjin University, Tianjin 300354, China

1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

9

ABSTRACT:

10

Aggregation of amyloid β-protein (Aβ) into β-sheet-rich plaques is a general feature

11

of Alzheimer’s disease (AD). Homeostasis dysregulation of Cu2+ mediates Aβ to form

12

high cytotoxic aggregates, which causes cell damages by generation of reactive

13

oxygen species (ROS). To improve the inhibitory potency and explore the

14

multifaceted functions of our previously designed decapeptide, RTHLVFFARK-NH2

15

(RK10), we have herein reformulated the decapeptide into its D-enantiomer, rk10, and

16

the effects of chirality on Aβ aggregation, Cu2+-mediated Aβ aggregations and

17

aggregate-remodeling effects were investigated. The results revealed the following:

18

(1) The D-enantiomer presented enhanced inhibitory potency on Aβ fibrillogenesis in

19

comparison to RK10; rk10 and RK10 increased the cell viability from 60% to 91%

20

and 71%, respectively, at an equimolar concentration to Aβ. (2) The enantiomers were

21

chemically equivalent to Cu2+ chelation, ROS suppression and oxidative damage

22

rescue. (3) The D-enantiomer exhibited higher performance to inhibit Cu2+-mediated

23

Aβ aggregation, and more significantly attenuated the cytotoxicity caused by

24

Aβ42-Cu2+ complex than RK10. Cell viability was rescued from 51% to 89% and 74%

25

by co-incubating with rk10 and RK10 at 50 μM, respectively. Intracellular ROS levels

26

generated by Aβ42 and Aβ42-Cu2+ species were also remarkably decreased by treating

27

with rk10. (4) The enantiomers could remodel mature Aβ42-Cu2+ aggregates by Cu2+

28

chelation, and rk10 showed higher performance than RK10, as evidenced by the

29

enhanced cell viability from 57% to 86% by RK10 and to 96% by rk10. The

30

D-enantiomer also showed higher ability than RK10 on protecting the disrupted

31

species from reaggregation. Taken together, D-chiral derivatization of the decapeptide

32

resulted in a potent multifunctional agent in inhibiting Cu2+-mediated Aβ aggregation

33

and remodeling mature Aβ-Cu2+ species. To the best of our knowledge, this is the first

34

investigation on the chirality effect of a multifunctional peptide inhibitor on

35

Cu2+-mediated Aβ aggregation and on the remodeling effect of mature Aβ-Cu2+

36

aggregates. The work provided new insights into the critical role of chirality in the

37

multifaceted functions of peptide inhibitors against amyloid formation and its toxicity.

38 39

KEYWORDS: Alzheimer’s disease, amyloid β-protein, multifunctional inhibitor,

40

D-enantiomeric peptide, chirality, oxidative stress.

2


Page 2 of 38

Page 3 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41

INTRODUCTION

42

Alzheimer’s disease is one of the most prevalent and devastating neurodegenerative

43

diseases with an increasing incidence in recent years.1-3 The histopathological

44

hallmarks of AD are extracellular amyloid plaques and intracellular neurofibrillary

45

tangles in patients’ hippocampus.4 Amyloid β-protein (Aβ), the main component of

46

senile plaques, is highly related to neurons disfunction and memory deficits.5,6 Aβ is a

47

hydrophobic, intrinsically disordered, and aggregation-prone peptide consisting of

48

39−43 amino acid residues.7 Elevated levels of Aβ drive its aggregation into soluble

49

oligomer, protofibrils and finally mature fibrils.4,6 The intermediates of aggregation

50

have been accepted as the most toxic species to neurons.2,8,9 Therefore, Aβ cascade

51

hypothesis strongly supports exploration of inhibitors that can suppress Aβ

52

aggregation and reduce its cytotoxicity for development of potential drug candidates

53

for the treatment of AD.3,10-12 Meanwhile, investigations have revealed that the

54

homeostasis dysregulation of transition metal ions, especially Cu2+, not only mediate

55

Aβ aggregation into high-cytotoxicity species, but also produce reactive oxygen

56

species (ROS) including oxygen-free radicals (HO•) and hydrogen peroxide (H2O2)

57

via Fenton-type reactions, which cause severe oxidative stress.11,13,14 Therefore,

58

various metal-ion chelators have been studied for AD treatment.13,15,16 Considering

59

the complexity of AD onset (Aβ aggregation, metal dyshomeostasis, oxidative stress,

60

inflammation, and so on), the concept of “multifunctional inhibitor” has emerged. A

61

multifunctional inhibitor should possess two or more functions, such as inhibition of

62

Aβ aggregation into toxic species and chelation of transition-metal ions to arrest its

63

activities on ROS production as well as on enhancing the amyloid toxicity.12,17-19

64

Over the past decade, vigorous progress has been made in the design and

65

development of Aβ inhibitors and multifunctional inhibitors. These include

66

peptides,15,18,20-24

67

antibodies,33 and proteins.19,34,35 Small peptide-based inhibitors have attracted

68

extensive attention due to their high target affinity, easy synthesis, and favorable

69

biocompatibility.20,24 A series of peptide-based inhibitors such as D3 (rprtrlhrnr) and

small

molecules,14,17,25,26

nanoparticles,27-30

3


polymers,31,32


70

its derivatives are identified to be promising inhibitors and have entered Phase I

71

clinical trials.36-40 Numerous peptides have been designed to target the hydrophobic

72

core of Aβ sequence (Aβ16-20) and yielded encouraging results,20 such as β-sheet

73

breaker LPFFD,41 LVFFARK (LK7),29,42 and RTHLVFFARK (RK10).18 However,

74

there are still increasing demands on potent (multifunctional) inhibitors that work at

75

low concentrations.20

76

Recent studies have demonstrated that the aggregation of natural Aβ is sensitive to

77

chiral environment.43,44 For example, addition of the mirror image D-Aβ42 to the

78

natural L-Aβ42 solution enhanced the aggregation propensity of the protein, and

79

remarkably reduced the toxic soluble oligomers, leading to the alleviation of cell

80

damages caused by L-Aβ42 aggregation.9,45 Peptide inhibitors consisting of unnatural

81

D-enantiomers of the amino acids exhibited significantly stronger inhibition potency

82

than their L-enantiomers.25,46 This is considered due to the fact that natural Aβ amino

83

acid residues usually arrange in a specific orientation,43 so D-peptide inhibitors may

84

provide favorable conformations for binding to Aβ. Moreover, D-peptides are

85

resistant to proteolytic degradation and less immunogenic, and have high probability

86

to cross blood-brain barrier (BBB).40,47 All these factors play an dominant role in

87

practical clinical uses of peptide inhibitors. Hence, D-peptide-based inhibitors deserve

88

extensive explorations.

89

However, to the best of our knowledge, there is no research on the influences of

90

chirality of inhibitors on metal-mediated Aβ aggregation as well as on the

91

metal-mediated ROS production. Hence, we have herein reformulated our recently

92

designed bifunctional inhibitor RK10 into its D-enantiomer, rk10. RK10 was designed

93

by adding a chelating tripeptide RTH to LK7’s N-terminus, and showed high

94

specificity towards Cu2+-mediated Aβ aggregation.18 The D-enantiomer, rk10, is

95

expected to display higher inhibition potency, stronger ROS arresting activity on

96

Cu2+-mediated Aβ aggregation and better remodeling effect than its counterpart.

97

Therefore, the differences of the enantiomers in their inhibition performances were

98

extensively investigated by thioflavin T (ThT) fluorescence spectroscopy, dot blot 4


Page 4 of 38

Page 5 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


99

assay, atomic force microscopy (AFM), and cell viability assay, and the molecular

100

details about the inhibition mechanisms of the enantiomers were studied by NMR and

101

molecular docking. Then, intrinsic tyrosine (10Tyr) fluorescence quenching assay,

102

7-hydroxycoumarin-3-carboxylic

103

oxidative damage tests and isothermal titration calorimetry (ITC) measurement were

104

performed to assess the chelating property and antioxidant capability of the

105

enantiomers. Thirdly, the effects of the enantiomers on Cu2+-mediated Aβ42

106

aggregation were evaluated by the above biophysical and biological assays. Finally,

107

the remodeling effects of the enantiomers on mature Aβ42/Aβ42-Cu2+ aggregates were

108

explored, and the BBB permeation behaviors of the enantiomers were examined.

109

Results revealed that the chirality effect of the peptide-based inhibitor possesses a

110

critical position in Aβ and Cu2+-mediated Aβ aggregations.

acid

(CCA)

fluorescence,

protein/lipid/DNA

111 112

RESULTS AND DISCUSSION

113

D-Enantiomer Shows Higher Inhibition Efficiency. ThT fluorescence assay was

114

first used to characterize the inhibitory capability of the enantiomeric inhibitors.48

115

Both RK10 and rk10 inhibited Aβ42 aggregation in a concentration-dependent

116

manner, and the D-enantiomer always exhibited stronger inhibition potency than

117

L-enantiomer at the same concentrations (Figure 1A). For example, equimolar RK10

118

reduced the ThT fluorescence intensity to 53%, while its counterpart rk10 suppressed

119

the fluorescence intensity to 27%. At the same time, dot blot assay with OC (Aβ42

120

aggregate-specific) antibody was conducted to differentiate the inhibition efficiency

121

of the enantiomeric inhibitors (Figure 1B). It is seen from the figure that the dot blot

122

assay validated the ThT data. These results are also in good agreement with literature

123

data that the interactions between Aβ and peptide-based inhibitors display a

124

heterochiral stereoselectivity.46

125

D-Aβ42 possesses the ability to accelerate L-Aβ42 fibrillation,9 so it is interesting to

126

investigate the impact of the mirror-image inhibitors on Aβ aggregation kinetics. In

127

situ ThT fluorescence assay and the corresponding normalized lag time (Figure 1C, 5



128

1D) revealed that 2.5 μM rk10 (rk10:Aβ40 = 0.1:1) shortened the normalized Aβ40 lag

129

time to 0.80 ± 0.15, and 12.5 μM rk10 (rk10:Aβ40 = 0.5:1) decreased the normalized

130

lag time to 0.64 ± 0.14. So rk10 accelerated the nucleation of Aβ40 at the low

131

concentrations. This phenomenon is similar with the finding that the fibrillogenesis of

132

a racemic mixture of Aβ42 was 3 to 4 times accelerated.9 In contrast, 2.5 and 12.5 μM

133

RK10 increased the lag times to 1.29 ± 0.06 and 1.48 ± 0.22, respectively. Therefore,

134

it can be concluded that RK10 delayed Aβ40 nucleation. The corresponding final ThT

135

fluorescence intensity of Aβ40 incubated with RK10 and rk10 were 86% and 83% at

136

inhibitor:Aβ40 = 0.1:1, and 68% and 21% at inhibitor:Aβ40 = 0.5:1. Similar kinetic test

137

was conducted with Aβ42 (Figure S1 and Table S1), and the acceleration or

138

deceleration phenomenon was also observed at low inhibitor concentrations

139

(especially at 2.5 or 12.5 μM rk10 and at 2.5 μM RK10). The ThT fluorescence

140

indicates that the effects of the chiral inhibitors on Aβ aggregation were distinctly

141

different from each other.

142

Then, the conformational transition of Aβ42 was assessed by circular dichroism

143

(CD) spectroscopy (Figure 1E). Freshly prepared Aβ42 monomer adopted a random

144

coil structure with a negative peak at 200 nm (Figure 1E, black curve). After

145

incubating for 48 h, Aβ42 transformed to β-sheet-rich structures with a negative peak

146

around 216 nm and a positive peak at 195 nm. Equimolar rk10 prevented the

147

conformational transformation from random coil to β-sheet structures. The presence

148

of equimolar RK10 showed a similar structural transition with the sample containing

149

Aβ42 alone. This confirmed the significantly higher inhibitory efficiency of rk10 than

150

RK10 in preventing the conformational transition of Aβ42.

151

Next, we used AFM imaging to observe the morphologies of Aβ40 and Aβ42

152

aggregates co-incubated with the enantiomeric inhibitors. The two enantiomeric

153

inhibitors exhibited little tendency to self-assembling (Figure S2). As illustrated in

154

Figure 1F, Aβ42 alone showed a twisted and elongated fibrillar network with a length

155

of several micrometers. With the increase of RK10 concentration, Aβ42 fibrils became

156

shorter and the amount of fibrils decreased, but there still existed amorphous 6


Page 6 of 38

Page 7 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


157

aggregates even at 100 μM. Co-incubating Aβ42 with rk10 showed more remarkable

158

morphological changes and rk10 could alter the pathway of Aβ42 aggregation at lower

159

concentrations. Similar results of Aβ40 incubated with the enantiomeric inhibitors

160

were obtained and illustrated in Figure S3. The results of AFM imaging are in

161

agreement with the above ThT fluorescence assays and CD spectra in the point that

162

Aβ aggregation was more susceptible to the D-enantiomeric peptide inhibitor.

163

Since neuronal toxicity is a natural consequence of Aβ fibrillation, we used

164

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity

165

assay with SH-SY5Y cells to assess the detoxification of the enantiomeric inhibitors.

166

Both RK10 and rk10 showed no significant cytotoxicity within the tested

167

concentration range (Figure S4). As illustrated in Figure 1G, Aβ42 aggregates reduced

168

cell viability to 60% of the control, similar to previous reports.35 RK10 and rk10

169

enhanced cell viability in a dose-dependent manner (Figure 1G). RK10 increased the

170

cell viability to 71% at 25 μM, and 90% at 100 μM. Its enantiomeric counterpart rk10

171

exhibited higher detoxification to Aβ42 species, resulting in 91% at 25 μM and up to

172

97% cell viability at 100 μM. In other words, the detoxification of 25 μM rk10 was

173

equivalent to that of 100 μM RK10, indicating that the inhibitory efficiency of rk10

174

was significantly higher than RK10.

175

From the above results, we could draw the conclusion that rk10 was superior over

176

its L-enantiomer as an inhibitor against Aβ aggregation and cytotoxicity. It is due to

177

its strong suppression on the conformational transition of Aβ to β-sheet-rich structure

178

(Figure 1E), which led to an off-pathway aggregation (Figure 1A, 1B, 1C, 1F) and

179

reduced toxicity of the Aβ species (Figure 1G).

180

The molecular interactions between the enantiomeric decapeptides and Aβ42 were

181

explored by isothermal titration calorimetry (ITC). As shown in Figure S5, the

182

titration data were not able to be fitted by a binding model. This might be due to the

183

complexity of this process including Aβ aggregation and the influence of RK10/rk10

184

on Aβ nucleation. NMR spectroscopy was then used to analyze the binding sites. The

185

1H

NMR signals of Aβ42 changed upon addition of the two enantiomers (Figure 2A); 7



Page 8 of 38

186

the amide protons of E22 (Figure 2B) and aromatic moieties of F19F20 (Figure 2C)

187

underwent remarkable changes, indicating the enantiomers interacted with these sites.

188

To further understand and visualize the interactions of the decapeptides with Aβ

189

monomers, docking studies were performed with monomeric Aβ40 (PDB ID 2LFM)

190

and Aβ42 (PDB ID 1IYT). As shown in Figures 3A and S6A, rk10 binds to the

191

hydrophobic region of Aβ40 (binding energy, -6.0 kcal/mol) through polar and

192

nonpolar interactions, but RK10 presents weaker binding energy with Aβ40 (-5.4

193

kcal/mol) (Figure S6A). Similar results were obtained in the molecular docking with

194

Aβ42 monomer (Figure 3B, 3C and Figure S6B); rk10 had higher binding energy (-4.5

195

kcal/mol) to Aβ42 than RK10 (-3.1 kcal/mol). The predicted conformations supported

196

the NMR data that the decapeptides bound to the hydrophobic region of the amyloid

197

protein. This result also confirmed the hypothesis that a D-enantiomer might provide

198

favorable conformations for binding to Aβ.45 Therefore, the D-enantiomer exhibited

199

higher inhibition efficiency than its enantiomeric counterpart.

200

Chelating Property of the Enantiomers and Protection from the Damage of

201

ROS. Aβ42 interacts with Cu2+ and leads to the extinction of

202

fluorescence, so

203

property of the enantiomers.49 The 10Tyr fluorescence intensity reduced to 54% upon

204

addition of 10 µM Cu2+, RTH/rth and RK10/rk10 restored the 10Tyr fluorescence in a

205

concentration-dependent manner (Figure 4A). On the other hand, the tested peptides

206

had no significant influence on the

207

the 1H NMR signals at the aromatic moiety of Tyr centered at 6.8 ppm changed up the

208

interactions as shown in Figure 2C, indicating that RK10/rk10 interacted with this

209

site. The above results signify that RTH, rth, RK10, rk10 sequestered Cu2+ from the

210

Aβ42-Cu2+ complexes and the two enantiomers had a similar behavior towards Cu2+

211

chelation and sequestration. This result is consistent with the conclusion that D- and

212

L-Aβ42 are chemically equivalent with respect to copper binding.45

10Tyr

10Tyr

intrinsic

fluorescence assays were performed to assess chelating

10Tyr

fluorescence of Aβ42 (Figure S7), although

213

Then, toxic ROS production catalyzed by Cu2+ was measured by CCA fluorescence

214

assays. The tripeptides RTH and rth showed similar abilities to suppress ROS 8


Page 9 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


215

generation (Figure S8). Rationally, RK10 and rk10 retained the metal chelation

216

property of the tripeptides and completely quenched the production of HO• at high

217

concentrations (Figure 4B). At the same time, the consumption of Asc validated the

218

CCA fluorescence results (Figure S9). These results confirmed the antioxidant

219

property of the enantiomeric decapeptides. In the presence of Aβ42, although Aβ42

220

suppressed the final Cu2+-Asc fluorescence (ROS level) to 16% (Figure 4C), Aβ42

221

itself could not quench the Fenton-type reactions because Aβ42 contains various amino

222

acids (e.g., 35Met) that possess complex redox properties.13 Further addition of RK10

223

or rk10 to the sample completely eradicated the ROS generation (Figure 4C),

224

indicating that RK10 and rk10 interacted with Aβ42-Cu2+ complex and stopped the

225

redox cycle.

226

The overproduction of ROS could cause oxidative damage of biomolecules such as

227

protein, lipids and DNA, and further trigger neuronal death, which is vital in the

228

pathology of AD.11 Therefore, the multifaceted protection effects of the enantiomeric

229

decapeptides were explored. Protein oxidation can lead to protein fragmentation and

230

cross-linking. The percentage of protein oxidation was detected and illustrated in

231

Figure 4D. RK10 significantly reduced protein oxidation to 59% at 50 μM

232

(Cu2+:RK10=1:0.5) and to 36% at 100 μM (Cu2+:RK10=1:1). Similar data were

233

obtained for rk10. So the enantiomeric decapeptides protect protein from oxidation.

234

ROS-induced lipid peroxidation is an important mechanism of neurodegeneration

235

in AD brain. The lipid peroxidation was measured by the TBARS assay. In the

236

presence of Cu2+, MDA was detected by UV spectrometer at 532 nm, implying that

237

phospholipid was damaged by ROS. From Figure 4E, co-incubating the enantiomeric

238

decapeptides decreased phospholipid peroxidation to 60%~68% at 100 μM

239

(Cu2+:Peptide=1:1), and to 11%~21% at 200 μM (Cu2+:Peptide=1:2). This indicates

240

the effective anti-oxidation of the enantiomeric multifunctional decapeptides.

241

Oxidation of nuclear and mitochondrial DNA have also been reported in AD. Thus,

242

in vitro experiment was designed to validate the DNA protection effect of RK10/rk10.

243

Plasmid DNA (pDNA) was treated with Cu2+-Asc and then analyzed by agarose gel 9



244

electrophoresis (Figure 4F). There existed supercoiled and relaxed forms in pDNA in

245

the absence of Cu2+-Asc, but the supercoiled form pDNA disappeared in the presence

246

of Cu2+-Asc, indicating that pDNA was damaged by ROS. Interestingly, the

247

enantiomeric decapeptides prevented pDNA from the damage of ROS.

248

In summary, the above in vitro anti-oxidative results indicate that RK10 and rk10

249

rescued protein, lipids and DNA from the oxidative damage of ROS, and their

250

protecting effects were similar.

251

Thus, the

10Tyr

fluorescence assay, CCA fluorescence measurement and

252

anti-oxidative experiments suggest that RK10 and rk10 retained the chelation property

253

of the tripeptides (RTH/rth), and the effects of the enantiopure counterparts on

254

stripping Cu2+ from Aβ42-Cu2+ species and suppressing the ROS generation were

255

similar. To further understand the interactions between the enantiomers and Cu2+, ITC

256

measurements were performed. The ITC data indicate that there was no significant

257

difference between the enantiomeric decapeptides on their binding affinities for Cu2+

258

(Figure S10, Table 1). The binding constant of Aβ to Cu2+ has been reported to be

259

0.974×106 M-1.13 Therefore, it would be strong enough for RK10/rk10 to isolate or

260

strip Cu2+ from Aβ42-Cu2+ complex because of their one order of magnitude higher

261

binding affinities. Notably, RK10/rk10 might not be able to sequester Cu2+ from

262

Cu-metalloproteins, because their binding affinities (3.2-3.6×107 M-1, Table 1) were

263

lower than Cu-metalloproteins (such as Cu-Zn superoxide dismutase (~1012 M-1)).

264

Therefore, the moderate affinity of RK10/rk10-Cu2+ might make it a good chelator for

265

AD treatment without interfering with the normal biological functions of Cu2+.

266

Enhanced Inhibition of Cu2+-Mediated Aggregation by the D-enantiomer. It

267

has been recognized that Cu2+ can accelerate the conformational transition of Aβ

268

monomers and facilitate the generation of more toxic aggregates.19 In this section, we

269

investigated the effects of the enantiomeric decapeptides on Cu2+-mediated Aβ42

270

aggregation (Figure 5). Cu2+ induced Aβ42 to aggregate into less β-sheet structures,

271

and the ThT fluorescence of Aβ42-Cu2+ aggregates decreased to 18% (Figure 5A).

272

Addition of the tripeptides gradually restored the ThT fluorescence to 100%, so the 10


Page 10 of 38

Page 11 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


273

tripeptides inhibited Cu2+-induced Aβ42 aggregation by chelating Cu2+. However, the

274

tripeptides were incapable of inhibiting Aβ42 aggregation. In contrast, RK10 and rk10

275

kept the ThT fluorescence intensity around 11% to 19%, demonstrating the

276

enantiomeric decapeptides inhibited not only Cu2+-induced Aβ42 aggregation by

277

chelating Cu2+, but also the self-aggregation of Aβ42 after stripping the copper ions

278

from the Aβ42-Cu2+ complexes.

279

Cu2+-mediated Aβ42 aggregation led to the formation of amorphous aggregates

280

(Figure 5B), which was in line with previous reports.19 RK10 gradually changed the

281

morphology of Cu2+-Aβ42 aggregates from amorphous to fibrillar structures.

282

Meanwhile, neither amorphous nor fibrillar structured aggregates were observed in

283

the presence of 100 μM rk10. This result is considered due to the different inhibition

284

efficiency of the enantiomeric decapeptides after isolating Cu2+ from the Aβ-Cu2+

285

complexes, as demonstrated in Figure 1F. The results of AFM imaging suggest that

286

the D-enantiomer possesses stronger inhibition efficiency on Cu2+-mediated Aβ42

287

aggregation than its L-enantiomeric counterpart as do in the inhibition of Aβ42

288

aggregation (Figure 1A, 1F).

289

Moreover, the detoxification of the enantiomers on Aβ42-Cu2+ aggregates were

290

assessed by the MTT assay. Aβ42 aggregates into more toxic species induced by Cu2+.

291

The viability of SH-SY5Y cells reduced to 51%, and the enantiomeric tripeptide

292

RTH/rth failed to provide protection to the cells against Aβ42-Cu2+ toxicity (Figure

293

5C). RK10 enhanced the cell viability to 68%, 74%, and 83% at the molar ratios of

294

RK10:Aβ42 at 1, 2 and 4, respectively. Interestingly, the rk10 showed significantly

295

higher detoxification than RK10; it rescued the cell viability to 74%, 89%, and 91% at

296

rk10: Aβ42 at 1, 2 and 4, respectively. Remarkably, the biological effect of 50 μM

297

rk10 was more significant than 100 μM RK10 on alleviating Cu2+-mediated Aβ

298

cytotoxicity. The detoxification study further confirmed the advantage of the

299

D-enantiomer over its L-enantiomeric counterpart on Cu2+-mediated Aβ42

300

aggregation.

301

Excessive ROS formations from the redox of Aβ42-Cu2+ aggregation and the 11



Page 12 of 38

302

self-aggregation

303

2′,7′-dichlorofluorescin diacetate (DCFH-DA) was then used as a fluorescence probe

304

to study the intracellular oxidant stress.17,19 As illustrated in Figure 6, SH-SY5Y cells

305

treated with Aβ42 aggregates showed 167% ROS generation, and Cu2+ dramatically

306

promoted the ROS production to 216%. The ROS generation data are similar with

307

those reported in literature.19 Intriguingly, equimolar RK10 decreased the ROS

308

production caused by Aβ42-Cu2+ species to 148%, and equimolar rk10 attenuated ROS

309

level to 114%. This result was in good agreement with the MTT assays (Figure 5C)

310

and confirmed the efficient antioxidant property of rk10 in vivo. Considering the

311

higher inhibition potency of rk10 than RK10 on Aβ aggregation, the excellent

312

antioxidant and detoxification characteristics in cellular level might be due to its

313

enhanced interactions with Aβ in the presence of Cu2+. In other words, although rk10

314

possesses similar chelation property with RK10, it could suppress more ROS

315

generated by Aβ aggregation than RK10, so the cytotoxicity induced by Aβ42-Cu2+

316

was more significantly alleviated by rk10.

of

Aβ42

cause

mitochondrial

damage

and

cell

death.17

317

Remodeling Mature Aβ42-Cu2+ Aggregates and Preventing Reaggregation. The

318

removal of existing amyloid plaques is another therapeutic target in AD treatment and

319

this property is considered as the merit of a potential therapeutic agent,19,50 so the

320

influences of the enantiomeric inhibitors on mature Aβ42 species (25 μM) were

321

assessed. On the Aβ42 fibrils formed in the absence of Cu2+ ions, the final ThT

322

fluorescence intensity showed no significant decrease and the morphology of Aβ42

323

fibrils remained unchanged (Figures S11 and S12), implying that the inhibitors were

324

unable to disrupt mature Aβ42 aggregates. This result is in accordance with our

325

previous work dealing with bifunctional agents with a metal-chelating capability.19

326

Then, the effects of the enantiomeric inhibitors on aged Aβ42-Cu2+ aggregates were

327

investigated. As mentioned above in the discussion on the results shown in Figure 4B,

328

Aβ42-Cu2+ aggregates displayed amorphous structures. After co-incubating the

329

Aβ42-Cu2+ aggregates with the enantiomeric tripeptide RTH/rth, the morphology of

330

Aβ42-Cu2+ species transformed into mixed fibrillar and amorphous structures (Figure 12


Page 13 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


331

S13), which indicates that the chelator disaggregated the mature Aβ42-Cu2+ aggregates

332

in part or all by chelation, and the disaggregated species remodeled into fibrils in part

333

in the absence of Cu2+ ions, as previously reported.50 The decapeptides also exhibited

334

the ability to modulate mature Aβ42-Cu2+ species (Figure 7A). By treating Aβ42-Cu2+

335

species with RK10, the morphology of the aggregates transformed into fibrils, plaques

336

and less visible amorphous structures at high concentrations (Figures 7A and S14).

337

Addition of rk10 to Aβ42-Cu2+ aggregates changed the morphology from amorphous

338

to twisted fibril structures at 25 μM, and only very small amount of amorphous

339

aggregates could be observed at 50 and 100 μM rk10. More importantly, it has been

340

reported that the disaggregated Aβ42-Cu2+ could reaggregate to β-sheet-rich structures

341

and toxic forms,50 implying the inhibiting potency of an inhibitor plays a dominant

342

role in the later period of disaggregation. Therefore, in situ ThT fluorescent assays

343

were conducted to analyze the process of disaggregation and reaggregation. The

344

fluorescent intensity of the RTH/rth-treated Aβ42-Cu2+ aggregates increased in the

345

following co-incubation (Figure S15), demonstrating that removal of Cu2+ from

346

Aβ42-Cu2+ species led to the formation of β-sheet-rich structures. With 12.5 μM

347

RK10, the ThT fluorescence intensity dropped slightly in the first 3 h, and gradually

348

recovered to the control (Aβ42-Cu2+ aggregates) level (Figure 7B, line 2). This result

349

implies that RK10 could disaggregate Aβ42-Cu2+ aggregates, but a low concentration

350

of RK10 was incapable of preventing reaggregation of the disrupted aggregates due to

351

its moderate inhibition efficiency. By contrast, no obvious increase of fluorescence

352

intensity was observed in the samples treated by the D-enantiomer at the same

353

concentration. This evidently indicates the stronger inhibitory effect of rk10 than

354

RK10 in suppressing the reaggregation of the disaggregated species after removal of

355

Cu2+ from Aβ42-Cu2+ species.

356

The remodeling effect of the enantiomers on mature aggregates was further

357

confirmed by MTT assays (Figure 7C). Mature Aβ42 aggregates reduced the cell

358

viability to 70% of control, and the effects of RK10 and rk10 on the cytotoxicity of

359

the mature Aβ42 aggregates were negligible. This result is consistent with ThT assays 13



360

(Figure S11) and AFM imaging (Figure S12). In contrast, Aβ42-Cu2+ aggregates

361

reduced the cell viability to 57%, and equimolar RK10 and rk10 treated Aβ42-Cu2+

362

aggregates increased cell viability to 86% and 96%, respectively. Cell viability assays

363

indicate that both the enantiomeric inhibitors were capable of dissociating preformed

364

Aβ42-Cu2+ aggregates and alleviating the toxicity of the aggregates. Similarly with the

365

above results about inhibition of aggregation and ROS production, the D-enantiomer

366

showed higher potency on remodeling mature Aβ42-Cu2+ species than its

367

L-enantiomeric counterpart.

368

Taken together, the above results indicate that the D-enantiomeric decapeptide rk10

369

possessed higher inhibition efficiency on Aβ aggregation than its L-counterpart. To

370

achieve a similar effect on rescuing SH-SY5Y cells, the effective concentration of

371

rk10 was only 1/4 of RK10 (Figure 1G). In the presence of Cu2+, the D-enantiomer

372

exhibited higher suppression to Cu2+-mediated Aβ aggregation, and protected cultured

373

cells from Cu2+-mediated Aβ cytotoxicity at lower concentrations than RK10 (Figure

374

5C). Intercellular test revealed that rk10 suppressed more ROS generated by Aβ

375

aggregation as well as Cu2+-mediated Aβ42 aggregation than RK10 (Figure 6).

376

Moreover, rk10 showed higher performance in remodeling mature Aβ42-Cu2+

377

aggregates by Cu2+ chelation, and higher ability on protecting the disrupted species

378

from reaggregation (Figure 7). By contrast, the enantiomers showed similar

379

anti-oxidative effects on rescuing protein, lipids, and DNA from the oxidative damage

380

of ROS (Figure 4). Therefore, the superiority of D-enantiomer over L-enantiomer was

381

on its enhanced inhibition towards Aβ aggregation, Cu2+-mediated Aβ aggregation,

382

intracellular ROS, as well as remodeling effects.

383

Finally, passive BBB permeability of the enantiomeric decapeptides were evaluated

384

by parallel artificial membrane permeation assay (PAMPA).51 According to the

385

criteria established by Di et al,51 under our experimental condition, samples with

386

permeability (P) values greater than 3.8×10-6 cm/s are regarded to be able to cross the

387

BBB, while those less than 1.7×10-6 cm/s exhibit low BBB permeation (Table S2,

388

Figure S16, Table S3). The P values of RK10 and rk10 were determined to be 14


Page 14 of 38

Page 15 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


389

1.16×10-6 and 1.24×10-6 cm/s, respectively (Table S4). This indicates that the

390

enantiomeric decapeptides were of poor BBB permeation with the PAMPA-BBB

391

model. To enhance the BBB permeability of the inhibitors, one way that can be

392

considered is to conjugate the peptide to a cell-penetration peptide (CPP) or to

393

nanoparticles coupling with such a CPP.52

394 395

CONCLUSIONS

396

In this work, a D-enantiomeric decapeptide, RTHLVFFARK-NH2, was

397

systematically investigated for its effect on Aβ and Cu2+-mediated Aβ aggregations by

398

comparison with its L-enantiomeric counterpart. Both the enantiomers possess

399

multifunctionality in inhibiting Cu2+-mediated Aβ aggregation, arresting ROS

400

production, remodeling mature Aβ-Cu2+ aggregates and attenuating related

401

cytotoxicity, but the D-enantiomer is obviously superior over the L-enantiomer in the

402

following aspects. (1) The D-enantiomer shows significantly higher inhibition

403

efficiency on Aβ aggregation. (2) The enantiomers are chemically equivalent with

404

respect to copper binding, ROS suppression, and oxidative damage rescue, but the

405

D-enantiomer exhibits more excellent ability on inhibiting Cu2+-mediated Aβ

406

aggregation, alleviating the corresponding cytotoxicity, and attenuating ROS damage

407

generated by Aβ42 and Aβ42-Cu2+ aggregates. (3) Both the enantiomeric decapeptides

408

could remodel mature Aβ-Cu2+ species as high affinity Cu2+ chelators, but it was the

409

D-enantiomer that could protect the disrupted aggregates from reaggregation and

410

showed higher detoxification on the Aβ-Cu2+ complexes. The findings would help

411

design of more potent multifunctional peptide inhibitors.

412 413

METHODS

414

Materials. Synthetic Aβ42, Aβ40 were purchased from GL Biochem (Shanghai,

415

China). Decapeptide RK10/rk10 and tripeptide RTH/rth were all obtained with a

416

purity of >95% from ZiYu Biotechnology (Shanghai, China. The LC-MS

417

characterization data are shown in Figure S16 and Figure S17). The C-terminus of the 15



Page 16 of 38

418

decapeptide was amidated. 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), ThT, MTT,

419

BSA, HEPES were purchased from Sigma-Aldrich (St. Louis, MO, USA).

420

Coumarin-3-carboxylic acid (CCA), L-ascorbic acid (Asc) were received from J&K

421

Scientific Ltd. Porcine brain lipid was purchased from Avanti Polar Lipids.

422

Dulbecco’s modified Eagle’s medium /Ham's Nutrient Mixture F-12 (DMEM/F12)

423

and fetal bovine serum (FBS) were obtained from Gibco Invitrogen (Grand Island,

424

NY, USA). Human neuroblastoma SH-SY5Y cells were received from the Cell Bank

425

of the Chinese Academy of Sciences (Shanghai, China). Anti-amyloid fibrils OC

426

antibody

427

chemiluminescence kit were received from Beyotime, China. Nitrocellulose

428

membranes were purchased from PALL Gelman Laboratory. All other chemicals

429

including metal chloride salts were analytical reagent grade and used without further

430

purification.

was

obtained

from

Merck

Millipore.

DCFH-DA

and

ECL

431

Thioflavin T (ThT) Binding Fluorescence. The measurement of ThT fluorescence

432

intensity was used to quantitatively assess cross-β structures.48 Briefly, 200 μL sample

433

was mixed with 2 mL ThT solution (25 μM in HEPES buffer). The excitation and

434

emission wavelengths were 440 and 480 nm, respectively. The fluorescence intensity

435

of inhibitor and buffer was subtracted as background, final data were normalized with

436

Aβ only samples.

437

We used in situ ThT assay to monitor Aβ40/Aβ42 aggregation kinetics with or

438

without decapeptides. A volume of 200 μL samples containing 25 μM Aβ40/Aβ42

439

monomers, 25 μM ThT and different concentration of the enantiomeric decapeptides

440

were added into 96-well plate. The aggregation kinetics was measured using a

441

fluorescence plate reader (TECAN Austria GmbH, Grödig, Austria) at 10 min reading

442

intervals and 5 s shaking before each read. Data for Aβ40 were averaged from six

443

replicates and fitted by the sigmoidal Boltzmann model Eq.(1), and the lag time (Tlag)

444

was calculated from Eq. (2).

445

ymax - y0

y = y0 + 1 + exp( -k(t - t1/2))

16


(1)

Page 17 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2

446

Tlag = t1/2－

k

(2)

447

where y, y0, ymax, k, t1/2 represent fluorescence intensity at t, minimum fluorescence

448

intensity, maximum fluorescence intensity, apparent first-order aggregation constant,

449

the time required to reach half the maximum fluorescence intensity, respectively. Data

450

for Aβ42 were averaged from six replicates and the lag time is defined as the time

451

required to reach half the maximum of fluorescence intensity.

452

Dot Blot Assay. In the assay, 20 μL aliquots of peptide-treated Aβ42 samples were

453

spotted on nitrocellulose membranes, and the membranes were blocked with 10%

454

skimmed milk in 20 mM Tris-buffered saline (pH 7.4) with 0.01% Tween 20 (TBS-T)

455

for 1 h at room temperature. Then, the membranes were washed three times with

456

TBS-T. The membranes were incubated with the primary antibody OC (1:3000) (Aβ42

457

aggregates-specific antibody) for 1h at room temperature. After rinsing with TBS-T,

458

the membranes were incubated with the secondary antibody (anti-rabbit IgG, 1:

459

40000) conjugated with horseradish peroxidase for another 1h, and detected using

460

ECL chemiluminescence kit.

461

Circular Dichroism (CD) Spectroscopy Measurements. CD spectra were

462

collected with a JASCO J-810 spectrometer using a 1 mm path length quartz cell at

463

37 °C. The parameters were controlled as 1 nm bandwidth, and 100 nm/min scanning

464

speed with a continuous scanning model. Data were averaged from there independent

465

samples.

466

Atomic Force Microscopy (AFM) Imaging. AFM was used to monitor the

467

morphologies of Aβ species. After the protein aggregated, aliquots of 50 μL of each

468

sample were placed on a freshly cleaved mica substrate. After incubation for 5 min,

469

the substrate was rinsed with 8 mL DI water and dried by N2 stream before

470

measurement. Images were acquired in the tapping mode on an atomic force

471

microscope (Primitive Nano Inc., Guangzhou, China) under ambient conditions.

472

MTT Cell Viability Assay. The human neuroblastoma SH-SY5Y cells were

473

cultured in DMEM/F12 medium with 10% FBS, and 1% penicillin-streptomycin

474

antibiotic (100 U/mL penicillin and 100 μg/mL streptomycin sulfate) at 37 °C in an 17



475

atmosphere of 5% CO2. For MTT cell viability assays, the cells were seeded for 24 h

476

in a 96-well plate at density of 8×103 cells/well. Then different kinds of testing

477

samples that have been pre-incubated for 18 h were introduced to the cells and treated

478

for additional 24 h. The final concentration of Aβ42 in the cells was 5 μM. After that,

479

10 μL of MTT solution (5.5 mg/mL in HEPES buffer) was added into each well and

480

incubated for another 4 h. Afterwards, the plate was centrifuged at 1500 rpm for 10

481

min to discard the medium and 100 μL DMSO was added to lyse cells. Finally, the

482

absorbance at 570 nm was measured by a multimode microplate reader (TECAN

483

Austria GmbH, Grödig, Austria). Data were obtained from the average of six

484

replicates in this part.

485

Isothermal Titration Calorimetry (ITC). ITC measurements were performed on

486

a VP-ITC microcalorimeter (MicroCal, Northampton, MA) at 37 °C to detect the

487

binding affinities of the enantiomeric inhibitors with Aβ42 and Cu2+. For minimized

488

perturbations caused by air bubbles, all samples needed to be degassed by

489

centrifugation at 6000 rpm, 30 °C for 10 min before titration. Measurements were

490

conducted in 20 mM HEPES buffer (100 mM NaCl, pH = 7.4). Stirrer speed was kept

491

at 307 rpm. Each injection was 10 μL. To detect the binding of the inhibitors to Aβ42,

492

an RK10/rk10 solution at 200 μM was injected into a cell containing 20 μM Aβ42

493

monomer to achieve a complete binding isotherm. Similar experiment was conducted

494

with 2.5 mM copper chloride to 0.5 mM RK10/rk10 (10 mM glycine was contained in

495

the HEPES buffer for stabilizing Cu2+).13,18 The resulting dilution were determined

496

and subtracted from the integrated data before curve fitting. The data were fitted and

497

analyzed by one-site binding model of the MicroCal version of ORIGIN 7.0 software

498

to obtain ΔH, TΔS and association constant (K).

499

NMR Spectroscopy. 1H NMR experiments were carried out in 10 mM Tris buffer

500

containing 150 mM NaCl (pH 7.4) with 10% D2O. Samples containing 0.1 mM Aβ42

501

were mixed with the enantiomeric decapeptides (0.2 mM). NMR data were

502

immediately acquired on a Bruker 600 MHz spectrometer.

503

Molecular Docking. Molecular docking was accomplished using AutoDock Vina 18


Page 18 of 38

Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


504

(http://vina.scripps.edu).53,54 The three-dimensional crystal structures of Aβ40 and

505

Aβ42 monomers were retrieved from protein data bank (PDB ID 2LFM and 1IYT,

506

https://www.rcsb.org).55,56 Using AutoDock Vina, binding calculations were made

507

between receptor Aβ monomer and the enantiomeric decapeptides. RK10/rk10 was

508

chosen as the flexible ligand and Aβ monomer was fixed. The Lamarckian Genetic

509

Algorithm was used for the calculations. All other parameters were set as default. The

510

docking poses were captured using VMD tools.

511

Tyrosine Fluorescence Spectroscopy Assay. The intrinsic fluorescence of 10Tyr at

512

Aβ42 can be quenched by binding with Cu2+. Chelators can sequester Cu2+ from

513

Aβ42-Cu2+ complex, resulting in fluorescence recovered. Tyrosine fluorescence were

514

measured to assess the effects of chelating agents on Aβ42 /Aβ42- Cu2+ complex.23

515

Aβ42 (25 μM) with or without Cu2+ (10 μM) were incubated with different kinds of

516

chelating agents for 20 min at 37 °C. Tyrosine fluorescence intensity were measured

517

by

518

excitation/emission of 274/299 nm, respectively.

fluorescence

spectrometer

(PerkinElmer,

LS-55,

MA,

USA)

at

an

519

Measurement of HO• in vitro. Hydroxyl radical is one of the unstable

520

intermediates generated by Fenton-type reactions. The production of hydroxyl radical

521

was measured with coumarin-3-carboxylic acid (3-CCA) as the fluorescent probe.

522

3-CCA could be oxidized to 7-hydroxycoumarin-3-carboxylic acid (7-OH-CCA) by

523

HO•. Thus the time-dependent production of HO• was measured by monitoring

524

7-OH-CCA fluorescent signal at 452 nm upon excitation at 395 nm (slit width = 5.0

525

nm). The concentration of 3-CCA, Cu2+, Asc, Aβ42 were 500 μM, 10 μM, 500 μM, 25

526

μM, respectively for all measurements. Asc should be added into the testing sample at

527

last before experiments.

528

Ultraviolet Measurement of Ascorbic Acid. The samples for UV test consist of

529

10 μM Cu2+, 500 μM Asc and different concentration of chelating agents. A volume

530

of 1 mL testing sample was injected into quartz pool with l=0.5 cm. The consumption

531

of Asc was recorded by an ultraviolet spectrometer at 265 nm.

532

Multifaceted Anti-Oxidation Assays. Protein oxidative damage was measured by 19



533

co-incubating bovine serum albumin (BSA) with 2,4-dinitrophenylhydrazine

534

(DNPH).17 Lipid peroxidation was assessed by the TBARS test.57 DNA damage was

535

detected by agarose electrophoresis.17 Detailed experimental methods are provided in

536

the Supporting Information.

537

Measuring Intracellular ROS Levels. The effects of the enantiomeric

538

decapeptides on Aβ42-Cu2+ complex induced oxidative stress was performed with

539

SH-SY5Y cells by DCFH-DA fluorescence assay.19 According to manufacturer’s

540

protocol, SH-SY5Y cells were seeded in a 96-well plate at a density of 8×103

541

cells/well, Aβ42 samples were added after 24 h, and co-incubated for 24 h. Then, the

542

cells were washed with HEPES buffer twice and incubated with FBS-free medium

543

containing 10 μM DCFH-DA for 30 min at 37 °C. The cells were washed twice and

544

fluorescence intensity at 535 nm was recorded with excitation wavelength at 488 nm

545

using microplate reader.

546 547

In Vitro BBB Permeation Assay. This section is available in the Supporting Information.

548

Data Analysis. Data are shown as mean ± standard deviation. One-way ANOVA

549

with Tukey’s post hoc test was employed to assess the statistically significant, and p

d-Enantiomeric RTHLVFFARK-NH2: A Potent ... - ACS Publications

Recommend Documents