kinetics and molecular docking studies of the inhibitions of angiotensin converting enzyme and renin...

Kinetics and Molecular Docking Studies of the Inhibitionsof Angiotensin Converting Enzyme and Renin Activities by HempSeed (Cannabis sativa L.) PeptidesAbraham T. Girgih,† Rong He,†,‡ and Rotimi E. Aluko*,†

†Department of Human Nutritional Sciences and The Richardson Centre for Functional Foods and Nutraceuticals,University of Manitoba, Winnipeg, Manitoba R3T2N2, Canada‡College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210046,People’s Republic of China

ABSTRACT: Four novel peptide sequences (WVYY, WYT, SVYT, and IPAGV) identified from an enzymatic digest of hempseed proteins were used for enzyme inhibition kinetics and molecular docking studies. Results showed that WVYY (IC50 =0.027 mM) was a more potent (p < 0.05) ACE-inhibitory peptide than WYT (IC50 = 0.574 mM). However, WYT (IC50 =0.054 mM) and SVYT (IC50 = 0.063 mM) had similar renin-inhibitory activity, which was significantly better than that of IPAGV(IC50 = 0.093 mM). Kinetics studies showed that WVYY had a lower inhibition constant (Ki) of 0.06 mM and hence greateraffinity for ACE when compared to the 1.83 mM obtained for WYT. SVYT had lowest Ki value of 0.89 mM against renin, whencompared to the values obtained for WYT and IPAGV. Molecular docking results showed that the higher inhibitory activities ofWVYY and SVYT were due to the greater degree of noncovalent bond-based interactions with the enzyme protein, especiallyformation of higher numbers of hydrogen bonds with active site residues.

KEYWORDS: hemp seed, antihypertensive peptides, angiotensin-converting enzyme, renin, enzyme inhibition kinetics,molecular docking

■ INTRODUCTION

Food-protein-derived peptides have been shown to exert avariety of bioactivities during in vitro and in vivo testing asantioxidant,1−3 antihypertensive,4−6 and immunomodulatory7,8

agents. Other works have also produced food-protein-derivedpeptides with antibacterial,9−11 anticancer,12−14 antidia-betic,15−17 and anti-inflammatory18−20 properties. Thesebioactive peptides could be generated from animal and plantsources through enzymatic hydrolysis of the isolated proteinswith single or a combination of proteases. In this respect,several studies have produced natural antihypertensive peptidesthat may be used to prevent or manage hypertension.5,21−24

The use of natural peptide sequences as antihypertensive agentsis attractive because in some patients pharmaceutical drugs havebeen reported to cause undesirable side effects and health com-plications, such as dry cough, edema, skin rashes, and diarrheaduring prolonged administration.25−27 Hypertension in humanbeings is regulated by the renin−angiotensin systems (RAS),which also ensures fluid homeostasis; a malfunctioning of thissystem could result in the development of chronic diseases suchas hypertension, kidney failure, liver malfunction, diabetes, andcancer.28 Three enzymes, namely, renin, angiotensin I con-verting enzyme (ACE), and chymase, are the key catalystsinvolved in the operation of RAS. The RAS operates as follows:renin, an aspartyl protease produced from the kidney, convertsangiotensinogen (a product of the liver) into an inactive angio-tensin I (AT-I).29 Subsequently, ACE catalyzes the conversionof AT-I into a pro-hypertensive octapeptide and potent vaso-constrictor angiotensin II (AT-II). ACE also inactivates a vaso-dilator named bradykinin, which leads to increased vasopressive

effects and contributes to blood pressure (BP) elevation.29

Studies have shown that prolonged therapy with ACE-inhibitory antihypertensive drugs leads to AT-I accumulationand could activate an alternate pathway involving chymase,which is also capable of converting AT-I to AT-II independentof ACE; such a reaction can result in the failure of the anti-hypertensive drug therapy.30 In contrast, there is growingevidence that direct inhibition of renin could offer a bettercontrol of elevated BP than ACE because accumulation of AT-Iin substantial amounts that could be converted to AT-II insome organs via an ACE-independent pathway catalyzed bychymase is prevented. However, sole inhibition of renin maynot prevent ACE-catalyzed bradykinin degradation; therefore, adual strategy for inhibition of renin and ACE simultaneously issuggested to be the more effective therapy for BP control whencompared to single ACE therapy approach.31

Currently, our main research focus is to purify and identifyfood-based antihypertensive or bioactive substances that wouldpossess dual ability to inhibit renin and ACE activities. Re-cently, a number of antihypertensive peptides with multifunc-tional properties capable of inhibiting ACE and renin enzymesin vitro and in vivo conditions have been produced fromvarious plant and animal food sources. Peptides with dualability to inhibit ACE and renin have through enzymatichydrolysis been identified from seaweeds,32 yellow field pea

Received: January 15, 2014Revised: April 2, 2014Accepted: April 15, 2014Published: April 15, 2014

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seeds,33 flaxseed,34 rapeseed,21 and hemp seed.35,36 Previouswork also showed that a pea protein hydrolysate reduced renalrenin mRNA production in a chronic kidney disease rat modelafter 8 weeks of oral diet intake.37 Our previous work hasshown that dietary inclusion of a multifunctional hemp seedprotein hydrolysate was effective in the prevention and manage-ment of hypertension in spontaneously hypertensive rats.38 Wehave also determined the amino acid sequence of peptides thatmay be responsible for the antihypertensive effect of hemp seedprotein hydrolysate and confirmed BP-reducing effects of thepeptides in spontaneously hypertensive rats (SHR).36 Despitethe potential bioactive properties as well as health benefits ofhemp seed peptides, there is paucity of information in the areaof structure−function of blood-pressure-lowering peptides aswell as enzyme inhibition kinetics studies. Structure−functionstudies will provide new information on the relationships ofamino acid composition and sequence with antihypertensiveactivity, which could enhance method development to producepotent peptides from food proteins. Information fromstructure−function studies could also enhance developmentof novel and potent peptidomimetics using known peptidesequences as templates. Kinetic studies will provide usefulinformation to decide the optimal dose of the peptide neededto achieve maximal preventive effects. In addition, the mode ofinhibition of ACE and renin activities when determined bykinetics and molecular docking studies will provide additionalinformation for structure−function studies of antihypertensivepeptides. Therefore, the objective of this study was to deter-mine the kinetics of ACE and renin inhibitions as well aspotential enzyme−ligand binding configurations by four hempseed peptides that have been shown to reduce systolic BPin SHR.

■ MATERIALS AND METHODSMaterials. N-[3-(2-Furyl)acryloyl]phenylalanylglycylglycine

(FAPGG) and ACE from rabbit lung (EC 3.4.15.1) were purchasedfrom Sigma-Aldrich (St. Louis, MO). Human recombinant ReninInhibitor Screening Assay Kit was purchased from Cayman Chemicals(Ann Arbor, MI). Hemp seed protein-derived peptide sequences(WYT, WVYY, SVYT, and IPAGV) with demonstrated antihyperten-sive effects in SHR36 were synthesized (>95% purity) by GenWayBiotech (GenWay Biotech Inc., San Diego, CA).Kinetics of ACE Inhibition. The ability of WYT and WVYY to

inhibit in vitro activity of ACE was measured in triplicates aspreviously reported.34 Briefly, 1 mL of 0.5 mM FAPGG (dissolved in50 mM Tris−HCl buffer containing 0.3 M NaCl, pH 7.5) was mixedwith 20 μL of ACE (final activity of 20 mU) and 200 μL of peptide in50 mM Tris−HCl buffer. The rate of decrease in absorbance at345 nm was recorded for 2 min at room temperature. Tris−HCl bufferwas used instead of peptide solutions in the blank experiment. ACEactivity was expressed as a rate of reaction (ΔA/min) and inhibitoryactivity was calculated using the equation below

= Δ − Δ

Δ ×

A A

A

ACE inhibition (%) [[( /min) ( /min) ]

/( /min) ] 100

blank sample

blank

where (ΔA/min)sample and (ΔA/min)blank are the ACE activity in thepresence and absence of peptides, respectively. The concentration ofeach peptide that inhibited ACE activity by 50% (IC50) was calculatedusing nonlinear regression from a plot of percentage ACE inhibitionversus peptide concentration. The kinetics of ACE inhibition wasstudied with 0.0625, 0.125, 0.25, and 0.5 mM FAPGG. The mode ofACE inhibition was determined from the Lineweaver−Burk plots,while inhibition constant (Ki) was calculated as the X-axis interceptfrom a plot of the slope of the Lineweaver−Burk lines against peptideconcentration.39

Kinetics of Renin Inhibition. In vitro inhibition of humanrecombinant renin activity was conducted according to the previouslydescribed method,33 using the Renin Inhibitor Screening Assay Kit.Prior to the assay, renin buffer was diluted with 50 mM Tris-HCl, pH8.0, containing 100 mM NaCl. The renin protein solution was diluted20 times with assay buffer before use, and the assay buffer wasprewarmed to 37 °C before the reaction was initiated in a fluorometricmicroplate reader (Spectra MAX Gemini, Molecular Devices,Sunnyvale, CA) maintained at 37 °C. Before the reaction, (1) 20 μLof substrate, 160 μL of assay buffer, and 10 μL of Milli-Q water wereadded to the background wells; (2) 20 μL of substrate, 150 μL of assaybuffer, and 10 μL of Milli-Q water were added to the control wells;and (3) 20 μL of substrate, 150 μL of assay buffer, and 10 μL ofpeptide samples (WYT, SVYT, or IPAGV) were added to the inhibitorwells. The reaction was initiated by adding 10 μL of renin to thecontrol and sample wells. The microplate was shaken for 10 s to mixfollowed by incubation at 37 °C for 15 min; fluorescence intensity (FI)was recorded using excitation and emission wavelengths of 340 and490 nm, respectively. The percentage inhibition was calculated as follows:

= −

×

inhibition% {[(FI of control well) (FI of sample well)]

/(FI of control well)} 100

The peptide concentration that inhibited 50% of renin activitywas determined and defined as the IC50 value and was calculated bynonlinear regression as performed for ACE. The renin inhibitionkinetics studies were conducted using 0.625, 1.25, 2.5, 5, and 10 μM ofsubstrate in the absence and presence of peptides. Lineweaver−Burkplots were used to calculate inhibition constant as described above forACE.

Molecular Docking Studies. Molecular docking was performedusing Accelrys Discovery Studio software 2.5 (DS 2.5) as previouslydescribed.40 The structures of WYT, WVYY, SVYT, and IPAGV weregenerated with DS 2.5, and the energy was minimized with theCHARMm program. For ACE docking, a crystal structure of humanACE bound with lisinopril (an ACE-inhibitory drug) (PDB ID 1O8A)was used as ACE molecule. A binding site was created by the digitalremoval of lisinopril with the radius of 15 Å, and coordinatesx = 41.268, y = 34.559, z = 45.393. Automated molecular docking wasperformed using the partial flexibility CDOCKER tool of the DS2.5 software in the presence of cofactors (zinc and chloride ions). Forrenin docking, a crystal structure of human renin bound with aliskiren(a renin-inhibitory drug) (PDB ID 2V0Z) was used. All the watermolecules present in the protein except two water molecules (H2O184and H2O250) and aliskiren were digitally removed and hydrogenatoms were added to the protein structure. A binding site was createdusing a radius of 10 Å around the ligand and with coordinates x =7.568, y = 46.092, z = 68.842.41 Evaluation of the molecular dockingwas performed according to the scores and binding-energy value inorder to obtain the best poses for peptides. DS 2.5 software was alsoused to identify the hydrogen bonds as well as the hydrophobic,hydrophilic, electrostatic, and coordination interactions betweenresidues located at the ACE and renin active sites.

Statistical Analysis. Enzyme inhibition assays were conducted intriplicates and analyzed by one-way analysis of variance. Data are re-ported as mean ± standard deviation. Statistical significance of dif-ferences was evaluated by Duncan’s multiple range test (p < 0.05) usingthe Statistical Analysis Systems software version 9.2 (SAS, Cary, NC).

■ RESULTS AND DISCUSSION

ACE- and Renin-Inhibitory Activities of Hemp SeedPeptides. Figure 1A shows that the ACE-inhibitory IC50 valuesfor WVYY and WYT were 0.027 and 0.574 mM, respectively(p < 0.05). These IC50 values are lower than the values previouslyreported for pea dipeptides IR (2.25 mM), KF (7.23 mM), and EF(2.98 mM).33 The IC50 value for WYT is similar to 0.3−0.7 mMbut higher than the 0.115−0.129 mM values reported for variousmushroom peptides.42 However, the IC50 value for WVYY shows

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that this peptide is a better ACE inhibitor than the mushroompeptides. The strong ACE-inhibitory activities exhibited by WVYYare consistent with earlier reported results, which showed that thepresence of hydrophobic or aromatic amino acid residues such asphenylalanine, tyrosine, and proline at the C-terminal of peptidespositively contributed toward the enhancement of ACE-inhibitoryactivities.43,44 Hayes et al.45 reported the identification of a rangeof novel propeptide angiotensin converting enzyme inhibitors in acasein fermentate produced by the action Lactobacillus animalisDPC6134. The casein fermentate peptides showed showed a

correlation between the C-terminal tripeptide sequences anddegree of ACE inhibition. In particular, binding of caseinfermentate peptides to ACE active sites was shown to beinfluenced by the hydrophobicity of the three C-terminal aminoacid residues, with aromatic or branched side chain amino acidresidues being preferred. Thus, the presence of valine (V) inthe tripeptide C-terminal sequence of WVYY may have con-tributed to the strong ACE-inhibitory properties of this peptide.The availability of two bulky tyrosine (Y) groups at theC-terminal may also have potentiated stronger inhibition ofACE activities when compared to WYT that has a hydrophilicamino acid threonine (T) at the C-terminal. Moreover, on thebasis of other studies,46−48 the presence of a branched-chainaliphatic amino acid such as valine at the N-terminal as well asthe availability of an ionizable functional group (e.g, intryptophan, W) may have also contributed to making WVYYa better ACE inhibitor than WYT.Renin catalyzes the first and rate-limiting step that initiates

production of vasoconstricting peptides responsible for BPelevation; therefore, down-regulation of renin’s activity mayenhance vasodilation. To the best of our knowledge, there isscanty information about the properties and occurrence ofrenin-inhibitory peptides from natural sources. Currently, thereis only one known commercially available potent nonpeptidicrenin inhibitor (aliskerin, IC50 = 0.6 nM), thus justifying theongoing research for renin inhibitors from naturally occurringfood sources. The IC50 values of the synthesized hemp seedpeptides identified in this study with potent renin inhibitoryproperties are shown in Figure 1B. The tripeptide WYT ex-hibited the strongest inhibitory capacity with the lowestIC50 value of 0.054 mM when compared to the tetrapeptide(SVYT = 0.063 mM) and the pentapeptide (IPAGV = 0.093 mM).Renin-inhibitory IC50 values of the hemp seed peptides are verylow when compared to the 9.20, 17.78, and 22.66 mM values forpea peptides IR, KF, and EF, respectively. Similarly, higher renin-inhibitory IC50 values were reported for rapeseed peptidesRALP (0.968 mM), LY (1.868 mM), and TF (3.061 mM).21

The potent renin-inhibitory data obtained in this study is inagreement with results from previous works that have suggestedthe presence of all or at least some of the following char-acteristics for good renin inhibition: one hydrophobic sidechain at the N-terminal, bulky chains at the C-terminal, one

Table 1. Kinetics Constants of Enzyme-Catalyzed Reactions at Different Peptide Concentrationsa

(A) Angiotensin-Converting Enzyme

WVYY WYT

catalytic parameter control 0.04 mM 0.08 mM 0.21 mM 0.85 mM

Vmax (ΔA/min) 0.014 ± 0.000 0.002 ± 0.000 0.006 ± 0.000 0.012 ± 0.000 0.008 ± 0.000Km (mM) 0.543 ± 0.004 0.841 ± 0.003 0.744 ± 0.003 0.870 ± 0.003 0.767 ± 0.001CE 0.026 ± 0.000 0.002 ± 0.000 0.008 ± 0.000 0.014 ± 0.000 0.011 ± 0.000Ki (mM) 0.06 ± 0.00 b 1.83 ± 0.03 a

(B) Renin

WY SVYT IPAGV

catalytic parameter control 0.43 mM 1.07 mM 0.43 mM 1.07 mM 0.43 mM 1.07 mM

Vmax (FI/min) 87.56 ± 0.97 36.87 ± 0.14 16.42 ± 0.05 25.73 ± 0.26 16.57 ± 0.07 29.94 ± 0.03 17.84 ± 0.01Km (μM) 5.62 ± 0.28 4.63 ± 0.01 2.62 ± 0.01 4.90 ± 0.07 3.90 ± 0.04 4.86 ± 0.02 3.42 ± 0.04CE 15.58 ± 0.82 7.96 ± 0.01 6.26 ± 0.01 5.25 ± 0.03 4.25 ± 0.02 6.15 ± 0.02 5.22 ± 0.06Ki (mM) 1.93 ± 0.02 b 0.89 ± 0.01 c 6.17 ± 0.02 a

aKm is the Michaelis constant, Vmax is maximum reaction velocity, CE is the catalytic efficiency (Vmax/Km), and Ki is the enzyme−inhibitordissociation constant.

Figure 1. Inhibitory concentration of synthesized hemp seed peptidesthat reduced 50% (IC50) in vitro activity of (A) ACE and (B) renin.Bars with different letters have mean values that are significantlydifferent at p < 0.05. Values are means ± SD (n = 3).



hydrogen bond donor, and two hydrogen bond acceptors.41,49,50

This is because the hemp seed peptides had most of thesequalities as a result of the presence of P, L, Y, W, I, and Vamino acid residues.Kinetics of ACE Inhibition. A Lineweaver−Burk plot was

used for the estimation of the modes of ACE inhibition bythe hemp seed peptides, and results are shown in Figure 2 for

WVYY (A) and WYT (B). From the linear regression fit of thekinetics data, the Km value of ACE activity in the absence of theinhibiting peptide was estimated to be 0.543 mM (Table 1A).The Km value was higher in the presence of peptides, which isan indication that more substrate was required for enzymecatalysis, and hence, the enzyme was less efficient. However, theKm value reduced when peptide concentration was increased,which suggests that, at higher peptide levels, more peptidesbecome bound to the enzyme active site to prevent formationof an enzyme−substrate complex. The Vmax for uninhibitedACE reaction was 0.014 A min−1, and this velocity decreaseddose-dependently in the presence of peptides (WVYY andWYT), though WVYY appeared to be more effective (Table 1A).The lower Vmax in the presence of WVYY is consistent with thehigher potency (lower IC50 value) when compared to WYT as

ACE inhibitors. Thus, the results indicate that the activationenergy of the catalytic reaction was increased in the presence ofWVYY and WYT because the rate of enzyme reaction wasslowed down. The Km and Vmax values obtained in this study areslightly higher than those previously reported for hemp seedprotein hydrolysate and its ultrafiltration membrane fractions.35

The catalytic efficiency (CE) of the uninhibited ACE-catalyzedreaction was 0.026, which decreased in a dose-dependent man-ner in the presence of WVYY and WYT. The reduced CE isdirectly related to lower Vmax and IC50 values, suggesting thatthe peptides had some binding affinity to the enzyme, whichled to reduced catalytic ability. WVYY and WYT exhibited a

Figure 3. Lineweaver−Burk plots of the inhibition of humanrecombinant renin by different concentrations of synthesized hempseed peptides at varying substrate concentrations (0.625−10 μM); V =initial rate of reaction (change in fluorescence intensity/min).

Figure 2. Lineweaver−Burk plots of angiotensin I-converting enzyme(ACE) inhibition by different concentrations of synthesized hempseed peptides at varying substrate concentrations (0.0625−0.5 mM).V = the initial rate of reaction [ΔA345 (nm)/min].



mixed-type mode of enzyme inhibition, implying that thepeptides bind to the ACE enzyme at positions that include boththe active and nonactive sites in order to reduce catalyticactivity. Binding to either the active or nonactive site wouldhave, respectively, blocked substrate binding or produce

enzyme conformations that reduced substrate affinity for theactive site. The Ki of WVYY and WYT for ACE inhibition werecalculated to be 0.039 mg/mL (0.06 mM) and 0.858 mg/mL(1.83 mM), respectively, which are lower than the2.55−4.74 mg/mL (3.92−7.31 mM) values reported forthe hemp seed protein hydrolysate and its membrane frac-tions,35 indicating a stronger inhibitory capacity of the purepeptides in comparison to the peptide mixture. The lower Kivalue for WVYY is consistent with the observed greater reductionsin Vmax and CE when compared to the reductions produced byWYT.

Renin-Inhibitory Kinetics. Parts A, B, and C of Figure 3illustrate the double-reciprocal plots of the renin-catalyzedreaction in the absence or presence of WYT, SVYT, andIPAGV, respectively. These three peptide sequences were

Figure 4. Automated molecular docking of WVYY (A) and WYT (B) at the angiotensin-converting enzyme (ACE) active site. ACE hydrophobicresidues are represented in red, positively charged residues in blue, and negatively charged residues and hydrogen bonds in green, while otherresidues and the zinc atom are represented automatically. Image obtained with Accelrys DS Visualizer software.

Table 2. Residues of tACE Having at Least One Atom at aDistance of 3.5 Å around the Docked Peptidea

tACE residues WVYY WYT lisinopril

Glu162 √ √ √Gln281 √ √ √Thr282 √ √ −Cys352 √ − −His353 √ √ √Ala354 √ √ √Ser355 √ √ √Ala356 √ √ √Gln369 √ − √Thr371 √ − −Thr372 √ − −Glu376 √ √ −Asp377 √ − −Val379 √ √ −Val380 √ √ √His383 √ √ √Glu384 √ √ √His387 − √ √Glu411 − √ √Asp415 − √ −Asp453 √ − −Phe457 √ √ √Lys511 √ − √Phe512 √ √ √His513 √ √ √Val518 √ √ √Tyr520 √ √ √Tyr523 √ √ √Phe527 √ √ √total 26 22 20

aThe residues around lisinopril in the tACE (PDB ID 1O86) are alsoshown.

Table 3. Hydrogen Bonds Observed between the DockedTop Ranked Pose of Peptides and tACEa

no. of H-bonds and corresponding distance (Å)

tACE residues involved inH-bondingb WVYY WYT lisinopril

Glu162:OE1 − − 1 (2.23)Glu162:OE2 − 1 (2.18) −Gln281:HE22 2 (2.12, 2.21) − 2 (2.18, 2.41)His353:NE22 1 (2.21) − −Ala354:O − 1 (2.35) 1 (2.04)Ala354:HN − − 1 (2.01)Asp377:OD1 1 (2.09) − −Asp377:OD2 1 (2.22) − −Glu384:OE1 − 1 (2.31) −Glu384:OE2 − 1 (2.11) 1 (2.48)Lys511:HZ1 − − 1 (1.93)His513:NE2 1 (2.192) − −Tyr520:HH 1 (1.93) 1 (1.97) 1 (2.06)Tyr522:OH − − 1 (2.42)total 7 5 9

aThe residue numbers belong to tACE (PDB ID 1O86). bH-bondposition:donor or acceptor atoms. Repeated residues indicateformation of two separate H-bonds by the same amino acid. “E”,“D”, and “Z” represent the presence of other residues near the donoror acceptor atom.



found to display mostly uncompetitive mode of renininhibition. For example, the Km of WYT showed a greaterreduction from 4.631 to 2.623 μM when compared to thedecrease in Km observed for SVYT and IPAGV (Table 1B).SVYT, however, exhibited the lowest ranges of Vmax (25.73−16.57 FI/min) and CE (5.249−4.247) values, which is anindication of reduced enzyme velocity in the presence of thepeptide. The uncompetitive mode results suggest that thepeptide bind mostly to the renin−substrate complex to causereductions in the reaction velocity as well as the catalyticefficiency. It is presumed that the binding of the peptide to therenin−substrate complex is responsible for the simultaneousdecreases in Vmax and Km values (Table 1). This is because thereduced effectiveness of the renin−substrate complex (reducedVmax) will lead to increased affinity of renin for the substrateand hence reduction in the Km value. Renin inhibition by thepeptides, which was reflected in the reduced CE as peptide levelwas increased, indicated the lower ability of the enzyme toconvert the substrate into products. The lower Ki of SVYTindicated that this peptide may be able to more strongly bind tothe renin−substrate complex (resistant to substrate-dependentdislodgement) when compared to WYT and IPAGV. The

Km for renin substrate in the absence of the inhibitor wasdetermined to be 5.621 μM, which is higher than those fromother studies, such as 1.3,51 4.416,34 and 4.01 μM,52 but is,however, lower than the 6.4 μM value reported by Yuan et al.53

The difference in Km values could be attributed to the differentsources from which renin enzyme was procured for thesestudies and/or the variations in experimental conditions/approaches that may have been used.

Molecular Docking Studies. Figure 4 shows the bestposes for the WVYY and WYT when present within the ACEcatalytic site and in the presence of Zn2+. The best pose foreach peptide was stabilized mainly by electrostatic interactionsand hydrophobic interactions that exist between amino acidresidues of ACE and those of each peptide within a distance of3.5 Å (Table 2). The poses were also stabilized by hydrogenbond formation with ACE amino acid residues (Table 3).WVYY had a higher number of total interactions (26) withACE residues, which could have contributed to the higherinhibitory activity (lower IC50 value) when compared to WYT,which showed 22 interactions. This was confirmed by thelowest binding electrostatic energy value of −114.06 kJ/mol forWVYY when compared to −92.91 kJ/mol for WYT. Similarly,

Figure 5. Automated molecular docking of IPAGV (A), SVYT (B), and WYT (C) at the renin active site. Renin hydrophobic residues arerepresented in red, hydrophilic residues are represented in black, and negatively charged residues and hydrogen bonds are represented in green, whileother residues are represented automatically. Image obtained with Accelrys DS Visualizer software.



the van der Waals energy of −5.23 kJ/mol was lower for WVYYwhen compared to −0.67 kJ/mol for WYT. The binding energydata agree with the fact that WVYY had higher ACE-bindingaffinity (lower Ki value) when compared to WYT, as shownin Table 1. The higher activity of WVYY may also be due to ahigher degree of interactions with S1 (Ala354, Glu384, Tyr523residues) and S2′ (Gln281, Tyr520, Lys511, His513, His353)subsites of the active site of ACE (Table 2). WVYY interactedwith these S1 and S2′ subsites residues in a similar manner tothe observed interactions between lisinopril and ACE.54 Incontrast, the weaker WYT interacted with all the ACE S1 andS2′ subsites residues with the exception of Lys511 (Table 2).As shown in Table 3, the ability of peptides to form multipleH-bond interactions with ACE could have contributed topeptide-induced inhibition of enzyme activity by stabilizing thenoncatalytic enzyme−peptide complex structure. Similar resultsshowing multiple H-bond formation between ACE-inhibitorypeptides VPP or IPP and ACE residues have also been re-ported.55 Also important is that the inhibitory capacity seem tobe directly correlated to the number of H-bonds formed;lisinopril has the highest ACE-inhibitory activity and formednine H-bonds, followed by WVYY (seven bonds) and WYT(five bonds). It is not only the number of H-bonds that seemsto contribute to ACE-inhibitory potency of the peptides butalso the ACE residues involved. This is because the morepotent WVYY formed two H-bonds with Gln281 as well oneH-bond each with His353 and His513 (Table 3), all amino acidresidues that are critical components of the ACE active site.The weaker-acting WYT does not form any H-bond withGln281, His353, and His513. The docking results also showedthat the interactions between ACE-inhibitory pepitdes andZn2+ at the enzyme active site also play a significant role inmodulating catalysis, as previously suggested.56 Figure 4 showsthat WVYY and WYT were well-positioned to interact with theactive site Zn2+ atom. Since the distance between the peptideC-terminal residue carbonyl oxygen and Zn2+ has been shownto account for the varied degrees of inhibition by peptides, thenthe shorter the Zn2+ distance to the carbonyl oxygen of thepeptide, the greater the degree of inhibition.57 The averagedistance between the WVYY peptide bond carbonyl oxygen andZn2+ was 2.28 Å in comparison to 2.38 Å for WYT, hence thehigher ACE-inhibitory activity (lower IC50 value, Table 1)observed for WVYY. A previous work has also shown thatarachin-derived peptides IKP and IEP with 2.73 and 3.036 ÅZn2+ coordination distances had 7 and 18 μM IC50 values,respectively, for ACE inhibition.57

Figure 5A−C shows the docking of peptides with the activesite of renin. SVYT had the lowest electrostatic binding energyvalue of −113.08 kJ/mol when compared to −83.96 kJ/mol forIPAGV and −92.91 kJ/mol for WYT. The results are consistentwith the kinetics data (Table 1), which showed that SVYT hadthe highest binding affinity (lowest Ki value) followed by WYTand then IPAGV. Potential binding energy was also lowestfor SVYT (−106.16 kJ/mol) when compared to IPAGV(−70.37 kJ/mol) and WYT (−80.74 kJ/mol). In contrast, the vander Waals energy values were −13.51, −8.36, and −0.67 kJ/molfor IPAGV, SVYT, and WYT, respectively. The results suggest thatSVYT had better affinity toward renin followed by WYT andIPAGV in that order. The total number of interactions betweeneach peptide and renin active site residues is very similar; incontrast, aliskiren had a greater number of interactions(Table 4). A previous report had shown that binding to theunique S3 subpocket (S3sp) (Gln13, Tyr14, Val30, Tyr155,

Ala218, Ser219, Ala303) of renin is necessary for high-affinityinhibition of enzyme activity.58 In this respect, SVYT showedinteractions with five of these residues (Gln13, Tyr14, Val30,Ala218, and Ser219), while WYT (Val30 and Ala218) andIPAGV (Ala218 and Ser219) interacted with two residues each(Table 4). In contrast, the powerful drug aliskiren formedinteractions with all the S3sp residues. The lower peptidebinding capacity to S3sp residues may be responsible for lowerrenin-inhibitory activity when compared to aliskiren. Theresults are in agreement with a previous work that had sug-gested peptide modification to improve lipophilic character as ameans of enhancing binding to S3sp residues and increase renin-inhibitory capacity.58 However, the fact that the three peptidesstill showed high renin-inhibitory capacity (low IC50 values)may be due to their ability to form electrostatic and hydro-phobic interactions with the aspartate residues Asp32 andAsp215 (Table 4); these two residues are critical participantsduring renin catalysis.59 The inhibitory activity of the threepeptides may also be correlated to their ability to form multipleH-bonds with renin residues, as shown in Table 5. While thehigh renin-inhibitory activity of SVYT may be due to the higher

Table 4. Residues of Renin Having at Least One Atom at aDistance of 3.5 Å around the Docked Peptidea

tACE residues IPAGV SVYT WYT aliskiren

Thr12 − √ − √Gln13 − √ − √Tyr14 − √ − √Val30 − √ √ √Asp32 √ √ √ √Gly34 √ √ √ √Ser35 √ √ √ √Ser36 √ − − −Trp39 − − − √Arg74 √ − √ √Tyr75 √ √ √ √Ser76 √ √ √ √Thr77 √ √ √ √Pro111 √ − √ √Phe112 − √ √ √Leu114 − − − √Ala115 − − − √Phe117 √ √ √ √Val120 − √ √ √Gln128 √ − − √Ile130 √ − − √Tyr155 − − − √Leu213 √ − − √Asp215 √ √ √ √Thr216 − − − √Gly217 √ √ √ √Ala218 √ √ √ √Ser219 √ √ − √Tyr220 − √ − −Met289 √ √ √ −Ile291 √ √ √ √Pro292 − − √ −Thr295 √ − − √Ala303 − − − √total 20 20 18 30

aThe residues around aliskiren in the renin (PDB ID 2V0Z) are alsoshown.



number (nine) of H-bond formation, only WYT formedH-bonds with the critical catalytic residues Asp32 and Asp215(Table 5). Aliskiren also formed H-bonds with Asp32 andAsp215. In contrast, IPAGV formed the lowest number ofH-bonds (four) and none of the bonds involved Asp32 andAsp215; the lower number and absence of interaction withactive site residues may be responsible for this peptide havingthe lowest renin-inhibitory activity (higher IC50 value). It isimportant to note that though both WYT and aliskiren formedH-bonds with Asp32 and Asp215 residues, the bond length wasshorter for the drug (2.16 and 2.11 Å) when compared tothe peptide (2.47 and 2.33 Å), hence the higher drugrenin-inhibitory activity. These results suggest that the reninor ACE-inhibitory properties of peptides depend not only onthe number of interactions with active site residues but thequality of such interactions such as type of residue (catalyticversus noncatalytic) and length of the bond.

■ AUTHOR INFORMATION

Corresponding Author*Telephone +1(204) 474-9555. Fax +1(204) 474-7593. E-mail:[email protected].

FundingThis work was funded through an operating grant from theManitoba Agri-Food Research and Development Initiative(ARDI) and a Discovery grant from the Natural Sciences andEngineering Research Council of Canada (NSERC) to R.E.A.

NotesThe authors declare no competing financial interest.

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Table 5. Hydrogen Bonds Observed between the Docked Top Ranked Pose of Peptides and Renina

no. of H-bonds and corresponding distance (Å)

renin residues involved in H-bondingb IPAGV SVYT WYT aliskiren

Tyr14:O − − − 1 (2.25)Asp32:OD1 − − − 1 (2.04)Asp32:OD2 − − 1 (2.47) 1 (2.16)Gly34:O − 1 (1.97) − 1 (1.99)Arg74:O − − − 1 (1.99)Ser76:HN 1 (2.11) − 1 (2.23) 1 (2.11)Ser76:HG 1 (1.88) − 1 (1.97) −Thr77:OG1 − 2 (2.07, 2.34) − −Thr77:HG1 1 (1.92) 1 (2.30) 1 (2.10) −Asp215:OD1 − − 1 (2.33) 1 (2.11)Gly217:O − 3 (2.07, 2.07, 2.15) − −Ser219:HN 1 (2.66) 1 (1.92) − −Ser219:OG − 1 (2.35) − −water:HOH184:H2 − − − 1 (2.02)water:HOH250:H2 − − 1 (2.20) −total 4 9 6 8

aThe residue numbers belong to renin (PDB ID 2V0Z). bH-bond position:donor or acceptor atoms. Repeated residues indicate formation of twoseparate H-bonds by the same amino acid. “D”, “G”, and “N” represent the presence of other residues near the donor or acceptor atom.



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kinetics and molecular docking studies of the inhibitions of angiotensin converting enzyme and renin...

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