Bioorganic & Medicinal Chemistry Letters 21 (2011) 38983904

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

The molecular mechanisms of interactions between bioactive peptides and angiotensin-converting enzyme
Daodong Pan a,b,, Huiqing Guo b, Bo Zhao c,, Jinxuan Cao a
a

Faculty of Life Science & Biotechnology, Ningbo University, Ningbo 315211, China Faculty of Food Science, Nanjing Normal University, Nanjing 210097, China c Faculty of Chemistry and Material Science, Nanjing Normal University, Nanjing 210097, China
b

a r t i c l e

i n f o

a b s t r a c t
The ability of milk protein derived Ile-Pro-Ala (IPA), Phe-Pro (FP) and Gly-Lys-Pro (GKP) peptides to inhibit angiotensin I-converting enzyme (ACE), a protein with an important role in blood-pressure regulation, were veried in vitro and in vivo. This work elucidates the modes and molecular mechanisms of the interaction of IPA, FP and GKP with ACE, including mechanisms that bind the peptides to the cofactor Zn2+. It was observed that the best docking poses obtained for IPA, FP and GKP were at the ACE catalytic site with very similar modes of interaction, including the interaction with Zn2+. The interactions, including H-bonds, hydrophobic, hydrophilic, and electrostatic interactions, as well as the interaction with Zn2+, were responsible for the binding between the bioactive peptides and ACE. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 24 February 2011 Revised 7 May 2011 Accepted 10 May 2011 Available online 15 May 2011 Keywords: Milk protein peptides ACE-inhibition Molecular mechanisms Elucidate

In recent years, the search for natural components in food with potential prophylactic and therapeutic effects has received great interest from researchers.1 Bioactive peptides are included in this category because they exhibit antihypertensive, antioxidative, antithrombotic, opioidic, mineral carrier, antimicrobial, and immunomodulatory properties.24 Milk protein is known to be a rich source of bioactive peptides compared to other protein sources such as animal (including sh) meat, wheat and soybean proteins. Among the bioactive peptides, angiotensin I-converting enzyme (ACE) inhibitory peptides and antihypertensive peptides have been extensively researched, because hypertension is a major risk factor in cardiovascular disease, including heart disease.5,6 Ile-Pro-Ala (IPA), Phe-Pro (FP) and Gly-Lys-Pro (GKP) are antihypertensive peptides crucial to cardiovascular homeostasis due to their inhibition of ACE, a protein that plays a fundamental role in the renin angiotensin system.4,79 ACE (EC 3.4.15.1) is a zinc metallopeptidase distributed in vascular endothelial, absorptive epithelial, neuroepithelial, and male germinal cells.1012 ACE acts on a wide range of substrates, displaying both exopeptidase and endopeptidase activity.13 It cleaves the carboxyl terminal His-Leu dipeptide from the inactive decapeptide angiotensin I to the active angiotensin II, a potent vasoconstrictor, which encourages hypertension. ACE also inuences in an indirect manner the kallikreinkinin system by promoting the degradation and inactivation of bradykinin, which is a nonapeptide involved in

Corresponding authors. Tel.: +86 0 574 87600737; fax: +86 0 574 87608347.
E-mail address: daodongpan@163.com (D. Pan). 0960-894X/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.05.033

blood-pressure control and inammation. There are two isoforms of ACE transcribed by the same gene; a larger one with 1277 amino acids, which is expressed in most tissues and is referred to as somatic ACE (sACE), and a smaller one which is composed of 701 amino acids and is found in adult testis. The smaller ACE is referred to as testis ACE (tACE).14,15 Somatic ACE is composed of two homologous catalytic domains (N and C), each with a functional active site and with distinct physiochemical and functional properties.16 These two ACE domains differ in both substrate and inhibitor specicity and chloride activation.17,18 Although both domains are efcient in angiotensin I cleavage, inhibition of the N domain with RXP407 (an ACE-N-selective inhibitor) has no effect on blood pressure. This suggests that the C- domain is the dominant angiotensin-converting site.14,19 Testis ACE exhibits only one domain, which is essentially identical to the C domain of sACE.15,20 Considering that ACEs are an evolutionary family of proteins,20 a structural comparison of tACE and Drosophila homolog of ACE (AnACE) has revealed a close homology between them, exhibiting 42% amino acid sequence identity.13,20 AnACE exhibits properties very similar to those of the human C domain ACE11 and AnACE activity is also inhibited by highly potent human ACE inhibitors.20 Most current ACE inhibitors, such as captopril, lisinopril and enalaprilat, are potent inhibitors of both the C and N domains of sACE and tACE.15,18 These molecules interact with the enzyme through hydrogen bonds and hydrophobic interactions at the ACE subsites S1, S2, S10 and S20 , but also directly interact with the catalytic Zn2+ ion.14,18,19,21 Nutraceuticals was used to be an auxiliary drug in hypertension treatment, but there are few antihypertensive products based on

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milk-derived peptides currently available.22 Milk-derived peptides can be liberated during food processing by chemical, physical or enzymatic methods.3 The latter can be achieved through the action of proteolytic enzymes during the fermentation of milk or upon enzymatic hydrolyzes of milk proteins in vitro.23 Hydrolysates of whole-milk protein, caseinates, whey proteins, and fractions enriched in individual milk proteins are a good source of bioactive milk peptides, and in particular, ACE-inhibitory peptides.9 Some authors have proposed that tri-peptides exhibit higher ACE-inhibitory activity.24 Peptide sequences containing hydrophobic amino acid residues or proline residues at the C terminus are potent ACE inhibitors resistant to degradation by digestive enzymes.4,12,22,24 In fact, it has been shown that binding to ACE is strongly inuenced by the C terminus of tripeptide sequence.24,25 Casein-derived FP (b-casein: f6263, f157158, f205206) and when protein derived IPA (b-lactoglobulin: f7880) and GKP (b-microglobulin: f1820) can be produced in vitro or in vivo through enzymatic hydrolysis of milk proteins (e.g., casein) by digestive enzymes (e.g., pepsin or trypsin) or by microbial fermentation of milk using a proteolytic system of lactic-acid bacteria (e.g., Lactococcus lactis, Lactobacillus helveticus).4 In vitro studies demonstrated that IPA, FP and GKP inhibit ACE activity. The concentrations of IPA, FP and GKP needed to inhibit 50% of the ACE (IC50) activity were 141, 315 and 352 lM, respectively.9 In vivo studies with milk products containing these peptides were performed using spontaneously hypertensive rats (SHR).11 Attenuation of hypertension was observed after six weeks by gastricintubation treatment of IPA, FP and GKP.8 The systolic blood pressure (SBP) of SHR decreased by 31, 27 and 26 mmHg, respectively, at dosages of 8.0 mg/kg.11 These results suggest that they may be the auxiliary drugs once they are further developed and commercialized. Despite the interest in bioactive peptides as an alternative for the control of mild hypertension, the exact mode of interaction and the molecular mechanisms between these peptides and ACE are not understood and their promise is mainly supported by data from in vitro and in vivo ACE-inhibition studies. In this work, we attempt to elucidate how IPA, FP and GKP exert their antihypertensive effects. This was accomplished by automated molecular docking of the bioactive peptides at the ACE catalytic site in the presence of cofactors (chloride and zinc ions), and by analyzing the position, type and energy of the interactions. The three-dimensional structure of native-human ACE was imported from the Protein Data Bank (PDB: 1O8A). The structures of ligands (bioactive peptides) were generated with Accelrys Discovery Studio 2.1 software and energy minimized with the CHARMm program using steepest descent and conjugate gradient techniques. Before the docking procedure, water molecules were removed from the protein-crystal structure. Automated molecular docking studies of the bioactive peptides (IPA, FP and GKP) at the ACE-binding site were performed with the Flexible Docking tool of DS2.1 software, in the presence of cofactors (zinc and chloride ions).The active site was obtained with the Binding Site tool. The docking runs were performed with a ra0 dius of 8 , with the coordinates x: 38.977; y: 38.645 and z: 50.183. A Evaluation of the molecular docking was performed according to the scores of several scoring functions, including LibDockScore, CDocker Energy, CDocker Interaction Energy, LigScore1, LigScore2, PLP1, PLP2, Jain, PMF, and PMFO4. The implicit solvent model Distance-Dependent Dielectrics was used in this work. The software returned data for the 20 best poses obtained in terms of the binding energy values. The software Accelrys Discovery Studio 2.1 was used to identify the hydrogen bonds, and the hydrophobic, hydrophilic and electrostatic interactions between residues at the ACE active site and the peptide poses.

The study of the coordination between peptides and Zn(II) was also possible. The distance between the peptides and Zn(II) was obtained from the docking results. The X-ray crystallographic structure of the ACE protein is available. The chemical structural formulas of bioactive peptides were generated using the software ChemDraw Ultra 8.0 ( Fig. 1). Based on ACEs three-dimensional structure, the possible ACE active sites were obtained via a binding site procedure. According to ACEs catalytic mechanism and relevant experimental reports, ACEs active site was identied. The site contains 19 amino acid residues: His353, Ala354, Ser355, Ala356, His383, Glu384, His387, Phe391, Pro407, His410, Glu411, Phe512, His513, Ser516, Ser517, Val518, Pro519, Arg522, and Tyr523. The zinc(II) ion is also an important component in ACE catalysis14 and is partly responsible for the binding strength between ACE and their inhibitors.26 A complete inactivation of the enzyme was observed in studies where mutations of metal-coordinating residues in human sACE were created, providing evidence for the role of zinc ions in ACE activity.20 The docking study of the tripeptide IPA at the ACE catalytic site in the presence of the cofactor Zn(II) showed a best pose (pose 14) with a binding energy value of 611.63 kJ/mol. The best pose was stabilized by hydrogen bonds, and hydrophobic, hydrophilic ( Fig. 3a) and electrostatic interactions (Table 6) with ACE residues. The values for the hydrogen bond parameters of the best pose (IPA) are shown in Table 1. The amine group of the N-terminal isoleucine residue established hydrogen bonds with the OH from Ser355 and the carbonyl group of Ala356. The carbonyl group of the proline residue also interacted with the residue Ala356 via hydrogen bonding. The amine group of the alanine residue contacted with the residues Ala354 and Glu384 via hydrogen bonds. The carboxylic group of the C-terminal alanine residue established hydrogen bonds with the OH from Tyr 523. On the basis of the hydrogen bond parameters, these H-bond interactions established with Ala354, Ala356 (N), Ser355 and Tyr523 presented more potent interactions, which greatly contributed to stabilization (Table 4). The molecular docking of IPA on the ACE binding site revealed that IPA, via hydrophobic interactions makes contact with residues including Ala354, Ala356, Phe391, Phe512, and Val518, and via hydrophilic interactions with residues including His353, 383, 387, 410, 513, Glu384, 411, and Arg522. The aromatic portion of Tyr 523 also contributed to stabilization. The research shows that, the interactions between the ACE inhibitors and the Zn2+ at the ACE active site usually play very important roles and therefore make ACE deactivation.19 In ACE, the zinc ion tetra-coordinated with the three residues (His383, Glu411 and His387) and CH3COOXT (Fig. 2). The values of bond length are shown in Table 5. After docking, we measured the distances between the zinc ion and its surrounding atoms. We found that the initial values of bond length made some changes and some atoms of the peptides were nearly closed to the zinc ion. These atoms and their distances were obtained (Table 5). The distance 0 of O15 (IPA) and Zn2+is 2.057 , which is nearly closed to the covaA 0 0 lent radius summation (2.14 ) of the oxygen (1.4 ) and the zinc A A 0 20 ion (0.74 ). This result suggests that the IPA may have the ability A to coordinate with the zinc ion through the carbonyl group of the proline residue. Together with the three residues (His383, Glu411 and His387) coordinated with zinc(II) the IPA around the zinc ion formed a distorted tetrahedral geometry (Fig. 3b). The dipeptide FP docked at the ACE catalytic site in the presence of the cofactor Zn(II). H-bonds, and hydrophobic, hydrophilic (Fig. 3c) and electrostatic interactions (Table 6) with this enzyme were suggested. The best docking pose obtained (pose 19) presented a binding energy of 713.89 kJ/mol. The values for the hydrogen bond parameters of the best pose are shown in Table 2.

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O O C H3N C N NH

C
O

NH3

IPA

FP

H3N

O H3N C N H N

C
O O O

GKP
Fig. 1. The chemical structural formulas of bioactive peptides.

Table 1 Hydrogen bonds observed between ACE and the best IPA pose obtained from docking results IPA XH Y Ala354:O H42N16 Ser355:OG H23N1 Ala356:O H22N1 Ala356:O H23N1 Ala356:NHN O8 Glu384:OE2 H42N16 Tyr523:OHHH O21 d (XH) 1.00 1.04 1.04 1.04 0.99 1.00 0.95 d (H Y) 2.07 2.42 2.07 2.40 2.08 2.25 2.03 d (X Y) 3.01 3.32 2.58 2.58 3.02 2.85 2.89 d (X Y) 155.60 144.20 107.10 87.60 157.10 116.70 148.80

The N-terminal amine group of phenylalanine established two H-bond interactions with a histidine residue (His353): one with an alanine residue (Ala354) and one with Ser355. An additional H-bond interaction was observed between the carboxylic group of the C-terminal proline residue and Arg522. Arg522 greatly contributed to stabilization via the H-bond. As observed for IPA, FP also established additional hydrophobic interactions with residues including Ala354, Ala356, Phe391, Phe512, and Val518, and hydrophilic interactions with residues including His353, 383, 387, 410, 513, Glu384, 411, and Arg522. Similarly, there were some changes after FP entered the ACE active site. The CH3COOXT was replaced by the carboxylic group of the C-terminal proline of FP (Table 5). The coordination atom of Glu411 (ACE) was no longer OE1 but OE2, and this change in coordination atoms may help stabilize the complex and therefore benet the ACE-inhibition properties. Together with the three residues (His383, Glu411 and His387) coordinated with zinc ion in ACE the FP around the zinc ion formed a distorted tetrahedral geometry (Fig. 3d). The molecular docking between ACE and GKP revealed that the binding energy value of the best pose was 1335.79 kJ/mol. The best pose was stabilized by hydrogen bonds, and hydrophobic,

Fig. 2. Details of the zinc ion tetra-coordinated with the ACE residues before docking.

hydrophilic (Fig. 3e) and electrostatic interactions (Table 6) with ACE residues. The values for the hydrogen bond parameters of the best pose (GKP) are shown in Table 3. The N-terminal amine group of glycine established two H-bond interactions with glutamine residue (Glu384) and Ala354. The amine group of the rst peptide bond between the glycine and lysine residues established an H-bond interaction with histidine residue (His353). An additional H-bond interaction was observed in the

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Fig. 3. Details of the interaction between ACE and (a) IPA, (c) FP and (e) GKP after automated docking of the peptides at the ACE-active site. ACE hydrophobic residues are represented in blue, hydrophilic residues in yellow, other residues present on binding site in gray, and zinc atoms in purple. Hydrogen bonds are shown in green dashed lines and zinc coordination bonds in gray bold lines. (a) Binding of IPA peptide to ACE, (c) binding of FP peptide to ACE, (e) binding of GKP peptide to ACE. Schematic view of (b) IPA, (d) FP and (f) GKP zinc coordination at the ACE-active site (image obtained with Accelrys DS Visualizer software).

carbonyl group of the second peptide bond between the lysine and proline residues and the OH from Tyr523. The carboxylic group of the C-terminal proline residue also established a hydrogen bond with the amide group of Arg522. Based on the hydrogen bond parameters, the H-bond interactions established with His353,

Ala354, Glu384 (OE1), Arg522, and Tyr523 presented potent interactions that greatly contributed to stabilization. These three poses were very similar in terms of their hydrophobic and hydrophilic interactions with ACE. In GKP it was the carbonyl group of the rst peptide bond that replaced the CH3COOXT (Table 5).The three

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Table 2 Hydrogen bonds observed between ACE and the best FP pose obtained from docking results FP XH Y His353:NE2 H20N1 His353:NE2 H21N1 Ala354:O H20N1 Ser355:OG H22N1 Arg522:NH2HH22 O19 d (XH) 1.04 1.04 1.04 1.04 1.00 d (H Y) 2.21 2.49 2.03 2.29 2.22 d (X Y) 2.70 2.70 2.77 3.05 2.99 (XHY) 106.80 89.80 125.90 129.50 132.60

Table 3 Hydrogen bonds observed between ACE and the best GKP pose obtained from docking results GKP XH Y His353:NE2 H27N5 Ala354:O H24N1 Glu384:OE1 H22N1 Glu384:OE2 H22N1 Arg522:NH2HH22 O21 Tyr523:OHHH O13 d (XH) 1.00 1.04 1.04 1.04 1.00 0.95 d (H Y) 2.46 1.82 2.13 1.92 2.11 2.19 d (X Y) 3.46 2.76 3.14 2.69 3.02 3.07 (XHY) 177.40 148.90 162.60 128.00 150.70 154.40

residues (His383, Glu411 and His387) and their atoms coordinated with the zinc ion made no changes after the GKP entering (Fig. 3f). As shown in Table 6, electrostatic energy played a more important role in the interactions between the bioactive peptides and ACE than did van der Waals energy. In the interaction of ACE with IPA, the six residues from the ACE active site, Ala354 (23.14 kJ/ mol), Ser355 (32.55 kJ/mol), Ala356 (74.31 kJ/mol), His383 (36.65 kJ/mol), His513 (33.64 kJ/mol), and Tyr523 (55.52 kJ/ mol) greatly contributed to stabilization. Ala356 and Tyr523 were especially important components in the ACE active site and were

partly responsible for the binding strength. In FP, it is the residues, Ala354 (71.55 kJ/mol), Glu384 (65.61 kJ/mol), His410 (38.03 kJ/mol), Arg522 (142.67 kJ/mol), and Tyr (35.61 kJ/mol) that stabilized the interaction between FP and ACE. Of the above residents, Ala354, Glu384 and Arg522 made the greatest contribution to stabilization. For GKP, the eight residues from the ACE active site, His353 (31.46 kJ/mol), Ala354 (78.87 kJ/mol), Ala356 (21.63 kJ/mol), His410 (66.78 kJ/mol), His513 (31.38 kJ/mol), Pro519 (45.81 kJ/mol), Arg522 (200.46 kJ/mol) and Tyr523 (58.32 kJ/mol) greatly contributed to stabilization. Ala354, His410, Arg522 and Tyr523 were especially important components in the ACE active site. In fact, these three poses are very similar in terms of the residues from the ACE active site established via Hbonds and electrostatic interactions, specically, Ala354, Ala356, Arg522, and Tyr523. As exhibited in Figure 4(ac), the best peptide docking site was located in the deep narrow channel of the ACE active site. Binding energy was believed to be the best choice as a parameter for identifying the best binder to a given target between a set of different ligands.11,27 The peptide IPA presented the lowest IC50 value (IC50 = 141 lM) but the highest binding energy (611.63 kJ/mol). The peptide GKP presented the highest IC50 value (IC50 = 352 lM) but the lowest binding energy (1335.79 kJ/mol). The IC50 value and binding energy of FP were 315 lM and 713.89 kJ/mol, respectively. Both FP values were between those for IPA and GKP. We found no correlation between the lowest binding energies obtained and the lowest known IC50 values. The IPA, FP and GKP have the potential to inhibit ACE via very similar modes of inhibition. Their promise rests, in part, dependent on their small size and generally high hydrophobic, hydrophilic and electrostatic character. The three peptides poses reveal interactions of peptides with ACE via H-bonding and electrostatic inter-

Table 4 Hydrogen bonds observed between the best bioactive peptide poses obtained from docking results with ACE Hydrogen bonds caused by molecular docking IPA(14) pose His353 NE2 Ala354 O Ala356 O Ala356 N Ser355 OG Glu384 OE1 Glu384 OE2 Tyr523 OH Arg522 NH2
p *

Amount 1 2 1 1 1 1

p p p p p p

FP(19) pose p p p p

Amount 2 1 1 1

GKP(03) pose p p p p p p

Amount 1 1 1 1 1 1

Existence of hydrogen bond interactions. Nonexistence of hydrogen bond interactions. Strong hydrogen bond interactions.

Table 5 Atoms from ACE residues and bioactive peptides (poses obtained from docking results) involved in interaction with Zn2+ before and after docking Residues involved in interaction with Zn2+ Initial ACE His 383 NE2 His 387 NE2 Glu 411 OE1 Glu 411 OE2 CH3COOXT IPA O15 FP O18 GKP O4
p
0

Distance () A 2.02 2.06 1.97 2.06

Pose 14 IPA p p p p

Distance () A 2.26 2.15 2.12 2.06

Pose 19 FP p p p p

Distance () A 2.33 2.17 2.11 2.28

Pose 03 GKP p p p p

Distance () A 2.19 2.11 2.14 2.15

p p p p

ACE residues involved in interaction with Zn2+. ACE residues not involved in interaction with Zn2+.

D. Pan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 38983904 Table 6 Electrostatic energy, van der Waals energy and total potential energy (kJ/mol) between the best bioactive peptides poses obtained from docking results with ACE Residue Eele Total His353 Ala354 Ser355 Ala356 His383 Glu384 His387 Phe391 Pro407 His410 Glu411 Phe512 His513 Ser516 Ser517 Val518 Pro519 Arg522 Tyr523 90.63 6.19 12.51 14.18 67.99 26.36 3.72 19.41 30.71 15.65 0.33 136.19 12.47 27.36 8.20 9.16 24.43 9.29 49.45 42.93 IPA Evdw 134.77 9.12 10.63 18.37 6.32 10.29 11.72 13.14 3.64 0.67 7.28 9.08 4.48 6.28 0.42 0.17 5.06 0.67 4.85 12.59 Etotal 44.14 2.93 23.14 32.55 74.31 36.65 7.99 6.28 27.07 14.98 6.95 127.11 16.95 33.64 7.78 9.33 19.37 8.62 44.60 55.52 Eele 73.14 55.77 73.89 6.65 4.18 15.06 57.03 24.98 10.29 7.07 32.97 269.28 2.85 4.10 12.76 6.36 1.30 5.23 135.31 24.27 FP Evdw 1738.28 84.81 11.00 10.88 10.25 1442.64 8.58 47.57 3.05 0.71 5.06 243.09 2.47 4.44 0.17 0.08 3.93 0.50 7.36 11.34 Etotal 1811.42 140.58 84.89 4.23 14.43 1457.71 65.61 72.55 7.24 6.36 38.03 512.37 0.38 8.54 12.59 6.44 2.64 4.73 142.67 35.61 Eele 464.72 20.38 71.55 3.60 16.28 13.14 422.67 42.17 19.08 5.73 57.03 324.13 20.13 25.10 14.85 8.45 2.22 44.64 209.95 46.19 GKP Evdw 1559.04 11.09 7.32 15.65 5.36 5.36 1102.27 9.29 1.13 1.00 9.75 520.82 21.80 6.28 3.39 0.46 5.98 1.17 9.50 12.13 Etotal

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1094.33 31.46 78.87 12.05 21.63 7.78 679.61 32.89 17.95 4.73 66.78 844.96 41.92 31.38 11.46 7.99 8.20 45.81 200.46 58.32

Fig. 4. General overview of best docking poses (gray) at the ACE catalytic site: (a) IPA, (b) FP and (c) GKP poses (images obtained with Accelrys DS Visualizer software).

actions with four residues: Ala354, Ala356, Arg522, and Tyr523. Especially the charged amine groups of the N-terminal were the steering groups in amino acid residues. The carbonyl groups of the IPA, FP and IPP were closed to the zinc ion and may be coordinating with zinc ion; it indicated that the atoms coordinating with zinc are equivalent and the tetrahedral coordination geometry resembles a captopril interaction. Binding energy does not directly correlate with the experimental IC50 values in some cases, since estimating binding energy is a challenging task. The performance

of site mutational changes in ACE able to suppress both drug and peptide inhibition may also provide more information about the interaction modes of antihypertensive products and ACE. Acknowledgment This work was supported by the Natural Science Funding of China (project 30972130), the NSF projects of Jiangsu (BK 2009403) and Ningbo (2009A610180) of China, Science and Tech-

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D. Pan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 38983904 15. Tzakos, A. G.; Galanis, A. S.; Spyroulias, G. A.; Cordopatis, P.; Manessi-Zoupa, E.; Gerothanassis, I. P. Protein Eng. 2003, 16, 993. 16. Corradi, H. R.; Schwager, S. L. U.; Nchinda, A. T.; Sturrock, D.; Acharya, K. R. J. Mol. Biol. 2006, 357, 964. 17. Georgiadis, D.; Beau, F.; Czarny, J.; Cotton, J.; Yiotakis, A.; Dive, V. Circ. Res. 2003, 93, 148. 18. Natesh, R.; Schwager, S. L. U.; Evans, H. R.; Sturrock, E. D.; Acharya, K. R. Biochemistry 2004, 43, 8718. 19. Zhao, Y. H.; Li, B. F.; Liu, Z. Y. Process Biochem. 2007, 42, 1586. 20. Oxtoby, D. W.; Gillis, H. P. Nachr. Princ. Mod. Chem. 1986. 21. Andjar-Snchez, M.; Cmara-Artigas, A.; Jara-Prez, V. Biophys. Chem. 2004, 111, 183. 22. Lopez-Fandio, R.; Otte, J.; Van Camp, J. Int. Dairy J. 2006, 16, 1277. 23. Otte, J.; Shalaby, S. M.; Zakora, M.; Pripp, A. H.; El-Shabrawy, S. A. Int. Dairy J. 2007, 17, 488. 24. Wu, J.; Aliko, R. E.; Nakai, S. J. Agric. Food. Chem. 2006, 54, 732. 25. Jkl, P.; Vapaatalo, H. Pharmaceuticals 2010, 3, 251. 26. Corradi, H. R.; Chitapi, I.; Sewell, B. T.; Georgiadis, D.; Dive, V.; Sturrock, E. D.; Acharya, K. R. Biochemistry 2007, 46, 5473. 27. Thomsen, R.; Christensen, M. H. J. Med. Chem. 2006, 49, 3315.

nology Department projects of Zhejiang (2010C12015) of China, and the K.C. Wong Magna Fund at Ningbo University. References and notes
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