Analysis and Evaluation of the Inhibitory Mechanism of a Novel

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Bioactive Constituents, Metabolites, and Functions

Analysis and evaluation of the inhibitory mechanism of a novel ACE-inhibitory peptide derived from casein hydrolysate Maolin Tu, Hanxiong Liu, Ruyi Zhang, Hui Chen, Fengjiao Mao, Shuzhen Cheng, Weihong Lu, and Ming Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00732 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Journal of Agricultural and Food Chemistry

Analysis and evaluation of the inhibitory mechanism of a novel ACE-inhibitory peptide derived from casein hydrolysate

Maolin Tu a,b, Hanxiong Liu a, Ruyi Zhang a, Hui Chen a, Fengjiao Mao a, Shuzhen Cheng a,c, Weihong Lu b, Ming Du a,b,*

a

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China

b

Department of Food Science and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China

c

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China

Author contributions: Maolin Tu and Hanxiong Liu contributed equally to this study.

*Corresponding author Dr. Ming Du Tel: +86-411-86332275 Fax: +86-411-86323262 E-mail: [email protected].

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ACS Paragon Plus Environment


1

ABSTRACT

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Casein hydrolysates exert various biological activities and the responsible

3

functional peptides are being identified from them continuously. In this study, the

4

tryptic casein hydrolysate was fractionated by ultra-filtration membrane (3 kDa), and

5

the peptides were identified by CE-TOF-MS/MS. Meanwhile, in silico methods were

6

used to analyze the toxicity, solubility, stability, and affinity between the peptides and

7

ACE. Finally, a new angiotensin I-converting enzyme-inhibitory (ACEI) peptide,

8

EKVNELSK, derived from the αs1-casein (fragment, 35–42), was screened. The IC50

9

value of the peptide is 5.998 mM, which was determined by an HPLC method. The

10

Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against

11

ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform

12

molecular docking to propose the potential mechanisms underlying the ACEI activity

13

of the peptide. These results indicated that EKVNELSK is a new ACEI peptide

14

identified from casein hydrolysate.

15 16

Keywords: Casein, ACE-inhibitory activity, Peptide, In silico, CE-TOF-MS/MS,

17

Molecular docking,

18

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INTRODUCTION

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Cardiovascular disease (CVD) has been recognized the leading cause of death in

21

the worldwide. It is expected that there will be more than 23.6 million deaths in 2030

22

caused by CVD 1. Hypertension (blood pressure > 140/90), as one of the independent

23

risk factors for CVD, with characteristics of frequent, chronic, and age-related

24

disorders

25

can be an effective way to prevent CVD 5. To control the blood-pressure, the

26

angiotensin I-converting enzyme (ACE, EC 3.4.15.1) must be taken into consideration,

27

which plays a significant role in blood pressure regulation through both the

28

renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) in vivo. In the

29

RAS, the potent vasopressor angiotensin II (Ang II) can be released by the ACE

30

cleaving the C-terminal dipeptide from angiotensin I (Ang I) 6. In the KKS, ACE can

31

also inactivate the bradykinin (BK) by removing the two C-terminal dipeptides 7.

32

Therefore, more attentions have been gradually paid to find novel ACEI substances

33

for the sake of lowering blood pressure. In the recent years, pharmaceutical drugs

34

targeting ACE are shown evident in lowering high blood pressure. However, some

35

side effects have appeared in clinical treatments 8. To find safer drugs, increasingly

36

scholars have paid attention to food-derived ACE inhibitors, especially the peptides

37

which owner the natural advantages of safe and easily to be metabolized in bodies.

38

ACEI peptides have been continuously identified from milk, soybean, egg and so on.

39

2-4

, has been paid increasingly attention. Blood-pressure lowering treatment

Casein is an excellent source of bioactive peptides and has been widely used in 9-10

40

food industry as a functional ingredient

41

been identified from enzymatic hydrolysates of casein, especially ACEI peptides. For

42

example, peptide QSLVYPFTGPI (from β-casein) and ARHPHPHLSFM (from

43

κ-casein) showed potent ACEI activity in vitro 11.

. To date, lots of bioactive peptides have

3



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To identify the bioactive peptides more effectively, some in silico methods have

45

been employed in recent studies. Quantitative structure-activity relationship (QSAR)

46

method, which has been widely applied to elaborate the relationships between the

47

peptides’ primary structures and the ACEI activity

48

been used to study the active mechanisms of the inhibitors against the receptors, such

49

as ACE

50

peptides by analyzing the physicochemical characteristics of the peptides. For

51

instance,

52

https://web.expasy.org/compute_pi/, has the function of predicting the molecular

53

weight (MW) and Iso-electric point (PI) of the potential bioactive peptides. ProtParam

54

tool can be used to predict instability index of peptides, available at

55

https://web.expasy.org/protparam/. The toxicity of the peptides can be predicted by

56

the online server ToxinPred, available at http://crdd.osdd.net/raghava/toxinpred/.

13

12

. Molecular docking also has

and thrombin Online servers have been used to screen the bioactive

the

online

server

Expasy-Compute

pI/Mw,

available

at

57

The aim of this study is to screen and identify a novel ACEI peptide from casein

58

hydrolysate and elaborate its active mechanisms. The peptides from casein

59

hydrolysates were determined by CE-Q-TOF-MS/MS. Toxicity, solubility, and

60

stability of the peptides were predicted by in silico methods. A novel ACEI peptide

61

was identified and subsequently synthesized. The IC50 value and the inhibition mode

62

of the peptide were investigated. Molecular docking analysis also were used to

63

elucidate the potential mechanism of ACEI activity of the peptide.

64 65

MATERIALS AND METHODS

66

Materials and chemicals

67 68

ACE (from rabbit lung), Hippuryl-histidyl-leucine (HHL), HPLC grade acetonitrile (ACN), and trifluoroacetic acid

(TFA) were purchased from

4


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Sigma-Aldrich Co. (St. Louis, MO, USA). Casein and trypsin were obtained from

70

Solarbio (Beijing, China). All other chemical reagents were of analytical grade.

71

Preparation of casein hydrolysates

72

Bovine casein solution (50 mg mL-1) was prepared by dissolving bovine casein

73

powder in deionized water. Then, 0.1 M NH4HCO3 buffer was used to adjust the pH

74

of the casein solution to 8.0. The hydrolysis reaction was started by the adding trypsin

75

with 2500 U g-1 of enzyme concentration. Other hydrolysis parameters were set as

76

120 min of hydrolysis time and 37 °C of temperature. Heating the samples in boiling

77

water (10 min) to quench the reaction before adjusting the pH to 7.0 (after the samples

78

cooled to below 25 °C). Subsequently, centrifuged the samples at 8000×g for 20 min

79

at 4 °C. Then, the supernatant was fractionated by Amicon Ultra ultrafiltration tube

80

(Millipore, [MWCO] = 3 kDa) according to the molecular weight and the fraction

81

with peptides’ molecular weight no more than 3 kDa was collected and freeze-dried

82

before storage at -80 °C.

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Determination of ACEI activity

84

The assay of in vitro ACEI activities of the casein hydrolysate and the peptide

85

were conducted by an HPLC method as previously reported 14.

86

Identification of peptides by CE-Q-TOF-MS/MS

87

CE analysis was conducted on a CESI 8000 Plus System (AB SCIEX, Inc.,

88

Redwood City, CA, U.S.A.). The uncoated fused-silica capillary (91 cm of total

89

length, i.d. 30 µm) was coupled to Q-TOF-MS/MS (impact II, Bruker Daltonic GmbH,

90

Bremen, Germany) through electrospray ionization source (ESI). In the process of

91

capillary separation, 10% acetic acid solution and 100 mM ammonium acetate were

92

used as the background electrolyte (BGE) buffer and the conductive liquid,

93

respectively.

The

washing

and

injection

of

the

5


sample

were

operated


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hydrodynamically. Before loading each sample, the bare fused capillary was

95

conditioned by forwarding with 0.1 M NaOH for 3 min, followed by 3 min with 0.1

96

M HCl and 4 min with ultrapure water and 3 min with BGE for reversing and

97

followed by 4 min forward with BGE. Subsequently, the samples were loaded on

98

using under 20 psi pressure (1 min), then followed with BGE (30 s, 20 psi). The

99

separation was then conducted electrodynamically (1 h) by applying a voltage of +20

100

kV after injection. The CE-ESI-Q-TOF-MS was set as the following parameters:

101

150 °C of dry temperature, 3.0 L min-1 of dry gas speed. Moreover, the top 20 intense

102

ions were collected in the MS spectra over an m/z range of 50–2200. Finally, Data

103

Analysis 4.0 software (Bruker Daltonic GmbH, Bremen, Germany) was used to

104

process the MS data. MASCOT searching engine (Matrix Science, London, UK) was

105

adopted to identify the sequence of the peptides in the sample.

106

Screening of the potential ACEI peptide

107

Some properties of the peptides are crucially important for the further studies of

108

the peptides especially drug-oriented, such as the toxicity, solubility, and stability.

109

Therefore, those factors have been taken into consideration for screening the potential

110

ACEI peptides. The ToxinPred server was used to analyze toxicity of the peptides,

111

available at http://crdd.osdd.net/raghava/toxinpred/. The solubility of the peptide was

112

evaluated

113

www.innovagen.com/proteomics-tools. The instability index of the peptide was

114

predicted by using ProtParam Tool (http://web.expasy.org/protparam/). In additional,

115

the affinities of the peptides against the ACE were compared by evaluating the

116

molecular docking results, which were conducted on the Discovery Studio R2

117

software (Neotrident Technology Ltd., Beijing, China).

118

Chemical synthesis of peptides

by

the

online

Innovagen

6


server,

available

at

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Selected peptide was synthesized by a solid-phase method at Cellmano Biotech

120

Limited Corporation (Hefei, China), which was with purity of 98.55% and verified by

121

HPLC.

122

Assay of ACEI kinetics

123

The inhibition pattern of the peptide, EKVNELSK, was explored by using

124

Lineweaver-Burk plots of 1/V versus 1/HHL. The concentrations of the HHL were set

125

as 0.5, 1, 2 and 3.5 mM, meanwhile, the concentrations of the peptide were set as 0,

126

4.228 and 8.456 Mm. The Vmax and Km were calculated, respectively, as the Y and

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X-axis intercepts of the primary plot.

128

Molecular docking analysis

129

Discovery Studio 2017 R2 software has been used to further evaluate the active

130

mechanism of the ACEI peptide through molecular docking technology according to

131

the reported methods

132

PDB ID: 1O8A) was downloaded from RCSB Protein Data Bank. The 3D-structures

133

of the peptide was produced by DS 2017 R2, and then minimized it by given the

134

CHARMm force field. Meanwhile, ACE molecular was treated by the procedures of

135

cleaning, preparing, removing waters and adding hydrogen. Finally, partial flexibility

136

program CDOCKER was chosen to perform the docking program with special

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binding sites (coordinates x: 36.189, y: 43.643 and z: 55.175) and receptor radius (16

138

Å). The results of molecular docking were evaluated based on the -CDOCKER

139

energy scores, interaction site, and interaction force types with ACE.

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Statistical analysis

141

15

, with some modifications. ACE crystal structure (Human,

All experiments were conducted in triplicate and the data were presented as the

142

mean ± SD in this study. Origin 8.0 (OriginLab Corporation, Northampton, MA, USA)

143

software was adopted to process the data. 7



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RESULTS AND DISCUSSION

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ACEI activity and peptide composition of casein hydrolysate

147

In this study, the ACEI activity of the casein hydrolysate with the molecular

148

weight no more than 3 kDa, has been determined by an HPLC method. As shown in

149

Fig.1, the ACEI activity of the mixture became significantly from the concentration of

150

0.05 mg mL-1, and the IC50 value of the casein hydrolysate was 0.4424 mg mL-1. To

151

analyze the component of the active mixture, CE-ESI-Q-TOF-MS/MS was used to

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identify the peptides (Table 1). There is a total of 32 peptides were identified from the

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casein mixture and 14, 11, 5, and 2 peptides were obtained from αs1-, αs2-, β- and

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κ-CN, respectively. Although the mixture has been fractionated by ultrafiltration tube,

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it’s not hard to find that there are also two peptides with molecular weight more than

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3 kDa, which may attribute to the fact that the ultrafiltration tube can’t separate the

157

mixture completely. As casein hydrolysate mixture with potent ACEI activity, the

158

identified peptides can be considered as the potential ACE inhibitors.

159

Screening of the potential active ACEI peptides

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In the present casein hydrolysate, the ACEI peptides have reported were

161

explored

162

(http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) and the published literatures.

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As labelled in Table1, there are six peptides (FFVAPFPEVFGK 16, ALNEINQFYQK

164

17

165

activity, with IC50 values of 18 µM, 264 µM, 600 µM, 11.9 µM, 60 µM, 15 µM,

166

respectively. Therefore, those six peptides must be partly of the active factors, which

167

made the casein mixture exhibit high ACEI activity.

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, AMKPWIQPK

by

18

searching

, FALPQYLK

19

the

, NMAINPSK 17, AVPYPQR

BIOPEP

20

) showed ACEI

The analysis of the human ACE-lisinopril complex has laid a solid foundation 8


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for the related studies of ACE inhibitors. There is a central active site groove that

170

extends for about 30 Å in the structure of ACE, which is the active site buried with

171

lisinopril

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peptides with large spatial structures are less likely to be bioactive. Moreover, from a

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cost point of view, the peptides with fewer amino acids are more meaningful for

174

exploring the drug-derived bioactive peptides. Therefore, the present study was aimed

175

to identify a novel ACEI peptide with the number of amino acids less than ten.

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Twelve peptides with the characteristics of the novel and the sequences consist of less

177

than ten amino acids (Table 2). Toxicity is one of the fundamental factors should be

178

taken into consideration for the various products of foods and drugs. Consequently,

179

the identified ACEI peptides in this study were assessed for potential toxicity in silico

180

using ToxinPred 22. As shown in Table 2, all of the peptides showed no toxicity.

21

. Consequently, due to the limited space of the ACE active site, the

181

Solubility is also an important characteristic of the peptides, which will influence

182

the absorption, distribution, and elimination of the peptides in the human body 23. The

183

aqueous solubility of the potential ACEI peptides was evaluated by the online tool

184

Innovagen, one peptide (VLPVPQK) showed poor water solubility (Table 2).

185

Meanwhile, the stability has also been used as an evaluation index to screen

186

potential ACEI peptide, which was accessed by the online tool ProtParam. In this

187

program, if the instability index is smaller than 40, which is predicted as stable; when

188

the instability index value above 40, it is predicted as may be unstable. As shown in

189

Table 2, only 5 (EGIHAQQK, EKVNELSK, NRLNFLK, NRLNFLKK, FFSDKIAK)

190

out of 12 peptides were predicted as stable. Subsequently, molecular docking analysis

191

was adopted to evaluate the affinity of the peptides against ACE. The peptide

192

EKVNELSK against ACE acquired highest -CDOCKER ENERGY (kcal mol-1) score

193

(185.54), which means this peptide has the highest affinity with ACE (Table 3). 9



194

Hence, the peptide EKVNELSK is with higher probability to have the function of

195

inhibiting the ACE activity. To confirm the ACEI activity of the peptide,

196

EKVNELSK was synthesized for further study.

197

IC50 determination of the ACEI peptide activity

198

The HPLC method was adopted to evaluate the IC50 value of the peptide

199

EKVNELSK. Fig. 2 showed the ACE inhibitory activity of the peptide at various

200

concentrations. The regression equation, Y= (1.113E-2) X2+4.57673X+22.14678

201

(R2=0.98312), was used to evaluate the IC50 value of the peptide EKVNELSK. The

202

results showed that the peptide was a novel ACE inhibitor with an IC50 value of 5.998

203

mM. Although it is seemly that the peptide EKVNELSK showed week in vitro ACE

204

inhibitory activity. However, as the hypertension pathophysiology is very complicated

205

that related to a series of system activities 24, it is hard to judge the in vivo effect of the

206

peptide. Some studies showed that the relationship between in vitro ACE inhibition

207

and antihypertensive activity is not apparent

208

peptide KVLPVPQ with an IC50 value of 1000 µM, which is much higher than

209

YPFPGPIPN (IC50 of 15 µM). But, the peptide KVLPVPQ showed significant blood

210

pressure-lowering effect in vivo, which is 4.5 times that of YPFPGPIPN 25. Therefore,

211

the novel ACEI peptide, EKVNELSK, can be regarded as one of the blood

212

pressure-lowering inhibitor candidates and its effect in vivo of this peptide should be

213

further studied.

214

Inhibition mode of the peptide EKVNELSK

24

. Such as, β-casein derived ACEI

215

The ACE inhibition pattern of the peptide EKVNELSK was evaluated by using

216

the Lineweaver-Burk plot. As shown in Fig. 3, when the peptide concentration

217

increased, the Vm values decreased, which indicated that the existing of the peptide

218

EKVNELSK probably hindered the substrate bind to ACE active site. Meanwhile, the 10


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higher of the peptide concentrations, the higher of Km values in the reactions, which

220

implied that the higher concentration of substrate is essential for the ACE catalytic

221

reaction. Moreover, the peptide EKVNELSK is a mixed-type ACE inhibitor. Similarly,

222

the peptide MLLCS has been identified from the hydrolysates of Styela plicata

223

peptides WVYY and WYT derived from the hemp seed proteins

224

mixed inhibition pattern against ACE. The mixed-type mode of ACE inhibition of the

225

peptide EKVNELSK indicated that the peptide binds to both the active and nonactive

226

sites of ACE, which consequently reduces the catalytic activity of ACE 27.

227

Interaction of the peptide and ACE

27

26

,

also showed the

228

The docking study of the peptide (EKVNELSK) at the ACE active site was

229

performed by Discovery Studio 2017 R2 software. As shown in Table 3, the value of

230

peptide EKVNELSK from -CDOCKER_Energy indicated that the peptide

231

EKVNELSK has the highest strength of the affinity against ACE in the five potential

232

ACEI peptides. The most stabilized poses of the peptide EKVNELSK against ACE

233

were obtained with the values of electrostatic energy (Eele), Van der Waals energy

234

(Evdw), and potential binding energy (Epot) of -675.211, -14.8202 and -631.721 kcal

235

mol-1, respectively. The hydrogen bonds formed between the peptide and ACE played

236

a vital role in inhibiting the ACE activity by stabilizing the structure of the

237

non-catalytic enzyme-peptide complex 15, 27. Table 4 and Fig. 4 showed that totally of

238

15 H-bonds have formed between ACE and peptide EKVNELSK, which contains

239

nine (LYS118, ASP121, GLU123, SER219, ASP358, TYR360, VAL399, ARG402,

240

ARG522) kinds of ACE residues. The residues GLU123 and ARG522 also formed

241

H-bonds with the ACEI peptides (WG and PRY), as reported by Fu et al.

242

Meanwhile, three kinds of electrostatic also formed between EKVNELSK and ACE at

243

the ACE residues of LYS118, GLU411, and ARG522. Moreover, the electrostatic 11


15

.


244

interaction of ZN701 and hydrophobic interactions of ALA89, ALA125, PRO519,

245

ARG124, ILE88) with ACE also contributed to the stabilization of the

246

EKVNELSK-ACE complex. Therefore, peptide EKVNELSK is a novel and effective

247

casein-derived ACE inhibitor.

248 249

ACKNOWLEDGEMENTS

250

We are grateful to Dr. Fang Luo of the NeoTrident Technology Ltd. (Beijing,

251

China) for the molecular docking instruction and Qin Zhang of the Dalian Polytechnic

252

University for helpful discussion.

253 254 255 256

FUNDING This study was financially supported by the National Natural Science Foundation of China (31371805).

257 258 259

Notes All authors declare that they have no conflicts of interest.

260 261

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Figure Captions Fig. 1. Total ion current (TIC) chromatogram and ACEI activity of bovine casein hydrolysate (molecular weight no more than 3 kDa). Hydrolysis was digested by trypsin at 37 °C for 2 h, with 50 mg mL-1 of protein and 2500 U g-1 of the enzyme as the substrate. Fig. 2. The ACEI activity of the peptide EKVNELSK. The ACEI activity of the peptide was determined by the HPLC method. The IC50 value was determined by the regression equation: Y= (1.113E-2) X2+4.57673X+22.14678 (R2=0.98312). Fig. 3. Lineweaver-Burk plot of ACE inhibition by the peptide EKVNELSK. The ACE activities were measured in the absence or presence of the peptide EKVNELSK (■, control; ●, 4.228 mM; ▲, 8.456 mM). 1/[S] and 1/[V] represent the reciprocal substrate concentration and velocity, respectively. Fig. 4. The molecular docking simulation of the peptide EKVNELSK with ACE (PDB: 1O8A). (A), (B) and (C) represent the general overview, local overview, and 2D-diagram of docking pose of peptide EKVNELSK, respectively.

17



Page 18 of 27

Table 1 Peptides Profile of Casein Hydrolysate Identified by CE-ESI-Q-TOF-MS/MS. No. 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

Protein αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN

αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN β-CN

m/z meas. 416.1951 455.7382 1070.1953 473.7646 1159.0664 692.8659 669.3396 1118.6130 880.4720 753.8928 460.7463 790.9140 624.9996 634.3531 684.3493 549.8074 490.2843 623.8211 509.7609 817.4429 437.7254 452.7708 344.8805 451.2648 624.3294 415.7288

z 2 2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 3 3 2 2

Mr. calc. 830.3770 909.4668 3206.5859 945.5131 2315.1296 1383.7227 1336.6735 2234.2283 1758.9377 1505.7773 919.4804 1579.8206 1870.9788 1266.6972 1366.6881 1097.6055 978.5538 1245.6353 1017.5090 1632.8835 873.4378 903.5290 1031.6240 1350.7732 1246.6517 829.4446

Scores 53.99 25.78 63.49 47.88 76.75 37.63 63.12 28.98 94.33 67.13 21.25 39.49 40.56 67.12 60.79 32.44 30.38 87.48 49.19 69.10 38.44 27.29 23.17 34.90 51.98 28.95

Start K K K K K R K K K R K K R K K K K K K K K K K K K

AA Sequence EDVPSER EGIHAQQK EGIHAQQKEPMIGVNQELAYFYPELFR EKVNELSK EPMIGVNQELAYFYPELFR FFVAPFPEVFGK HIQKEDVPSER HPIKHQGLPQEVLNENLLR HQGLPQEVLNENLLR LHSMKEGIHAQQK PFPEVFGK VPQLEIVPNSAEER YKVPQLEIVPNSAEER YLGYLEQLLR ALNEINQFYQK AMKPWIQPK FALPQYLK ITVDDKHYQK LTEEEKNR LTEEEKNRLNFLK NMAINPSK NRLNFLK NRLNFLKK RNAVPITPTLNR TKLTEEEKNR AVPYPQR

18


End Y E Q D Q E Y F F E L L L F T T A L K E K I E L D

Length 7 8 27 8 19 12 11 19 15 13 8 14 16 10 11 9 8 10 8 13 8 7 8 12 10 7

ACE activity

ACE inhibitor

ACE inhibitor ACE inhibitor ACE inhibitor

ACE inhibitor

ACE inhibitor

Page 19 of 27


27 28 29 30 31

β-CN β-CN β-CN β-CN κ-CN

507.2634 436.7798 437.2458 390.7523 478.2653

2 2 2 2 2

1012.5164 871.5491 872.4789 779.4905 954.5175

40.11 33.53 33.89 33.29 35.83

K R K K R

32

κ-CN

1052.5454

3

3153.6063

20.63

R

HKEMPFPK INKKIEK VKEAMAPK VLPVPQK FFSDKIAK SPAQILQWQVLSNTVPAKSCQAQPTTM AR

19


Y F H A Y

8 7 8 7 8

H

29


Page 20 of 27

Table 2 Toxicity, Solubility and Stability of the Potential ACEI Peptides. Number of

Solubility in

residues

water

EDVPSER

7

2

EGIHAQQK

3

No.

Peptide sequence

ToxinPed

Instability index

1

Good

Non-Toxin

unstable (118.63)

8

Good

Non-Toxin

stable (37.64)

EKVNELSK

8

Good

Non-Toxin

stable (-1.86)

4

PFPEVFGK

8

Good

Non-Toxin

unstable (68.01)

5

LTEEEKNR

8

Good

Non-Toxin

unstable (114.33)

6

NRLNFLK

7

Good

Non-Toxin

stable (-25.03)

7

NRLNFLKK

8

Good

Non-Toxin

stable (-20.65)

8

HKEMPFPK

8

Good

Non-Toxin

unstable (141.43)

9

INKKIEK

7

Good

Non-Toxin

unstable (93.04)

10

VKEAMAPK

8

Good

Non-Toxin

unstable (44.65)

11

VLPVPQK

7

Poor

Non-Toxin

unstable (118.63)

12

FFSDKIAK

8

Good

Non-Toxin

stable (-12.48)

20


Page 21 of 27


Table 3 The -CDOCKER_Energy Scores of the Potential ACEI Peptides Obtained from Molecular Docking. -CDOCKER_ENERGY

No.

Peptides

1

EGIHAQQK

177.172

2

EKVNELSK

185.540

3

NRLNFLK

154.963

4

NRLNFLKK

159.759

5

FFSDKIAK

169.805

(kcal mol-1)

21



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Table 4 Hydrogen Bonds, Electrostatic and Hydrophobic Interactions Observed in the Best Peptide Poses Based on the ACE-peptide Complex. Interaction Residues

Distance

Types

EKVNELSK:H132 - A:ASP121:OD2

2.28054

Hydrogen Bond;Electrostatic

EKVNELSK:H133 - A:GLU123:OE2

2.353



2.16624


A:LYS118:NZ - EKVNELSK:O137

4.3831

Electrostatic

A:ARG522:NH1 - EKVNELSK:O15

5.48899

Electrostatic

A:ZN701:ZN - EKVNELSK:O15

2.30831

Electrostatic

EKVNELSK:N1 - A:GLU411:OE2

4.41662

Electrostatic

A:LYS118:HZ1 - EKVNELSK:O111

2.07162

Hydrogen Bond

A:LYS118:HZ3 - EKVNELSK:O114

2.12179

Hydrogen Bond

A:TYR360:HH - EKVNELSK:O64

2.77065

Hydrogen Bond

A:ARG522:HH12 - EKVNELSK:O17

3.02249

Hydrogen Bond

A:ARG522:HH12 - EKVNELSK:O39

2.97451

Hydrogen Bond

EKVNELSK:H66 - A:ARG402:O

2.16968

Hydrogen Bond

EKVNELSK:H67 - A:VAL399:O

3.08084

Hydrogen Bond


2.89964

Hydrogen Bond


2.05597

Hydrogen Bond

EKVNELSK:H32 - A:ASP358:O

2.658

Hydrogen Bond


2.56598

Hydrogen Bond

EKVNELSK:H130 - A:SER219:O

2.79581

Hydrogen Bond

A:ALA89 - EKVNELSK:C98

4.43141

Hydrophobic

A:ALA125 - EKVNELSK:C98

4.26213

Hydrophobic

EKVNELSK:C46 - A:PRO519

4.97062

Hydrophobic

EKVNELSK:C94 - A:ARG124

4.93511

Hydrophobic

EKVNELSK:C98 - A:ILE88

4.68749

Hydrophobic

EKVNELSK:C98 - A:ARG124

4.24887

Hydrophobic

22


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Fig. 1.

23



Fig. 2.

24


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Fig. 3.

25



Fig. 4.

A)

B)

C)

26


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Graphic for table of contents

27


Analysis and Evaluation of the Inhibitory Mechanism of a Novel

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