Zhipeng Yu,Siji Wu,Wenzhu Zho,*,Long Ding,Dvid Shiun,Fuping Zheng,Jinrong Li,**,Jingbo Liu

a College of Food Science and Engineering,National&Local Joint Engineering Research Center of Storage,Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products,Bohai University,Jinzhou,121013,PR China

b Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Technology and Business University(BTBU),Beijing,102488,PR China

c College of Food Science and Engineering,Northwest A&F University,Yangling,712100,PR China

d Lab of Nutrition and Functional Food,Jilin University,Changchun,130062,PR China

ABSTRACT Inhibition of beta-site APP cleaving enzyme1 (BACE1) is one of the most promising therapeutic approaches for Alzheimer’s disease.To find natural products for the treatment of Alzheimer’s disease,absorption,distribution,metabolism,excretion and toxicity (ADMET) properties and in vitro BACE1 inhibitory activity of the peptides isolated from egg albumin were evaluated.Then,molecular docking and molecular dynamics simulation were used to explain the molecular mechanism of the interactions between BACE1 and peptides.The IC50 value of peptide KLPGF,with satisfactory ADMET properties,against BACE1 was (8.30 ± 0.56) mmol/L.Molecular docking revealed that KLPGF contacted with the residues of BACE1’s active sites through twelve hydrogen bonds interactions,two hydrophobic interactions,one electrostatic interaction,and two Pi-cation interactions.The 5 ns molecular dynamics simulations confirmed that the structure of KLPGF with BACE1 was stable.Peptide KLPGF contacted the residues Lys321,Asp228,and Asn233 with stable hydrogen bonds.KLPGF may be a potential anti-BACE1 candidate.

Keywords:Alzheimer’s disease BACE1 ADMET prediction Molecular docking Molecular dynamics KLPGF

1.Introduction

Alzheimer’s disease(AD)is the most common form of dementia affecting 36 million people worldwide[1].Amyloid and neurofibrillary tangles are the main characteristics of AD [2].The cause of AD remains unknown;the most popular hypothesis is aggregation and deposition of toxic amyloid fibrils(Aβ)[3,4].Aβ,a neurotoxic peptide leads to cerebral cortex neuronal apoptosis [5],thereby causing the memory deficits in AD.Beta-site APP cleaving enzyme 1 (BACE1) and γ secretase are two aspartyl proteases that cleave amyloid precursor protein (APP),and increase Aβ-40/42 peptide fragments[3,6].BACE1 catalyzes the rate-limiting step in the generation of Aβ peptide[7].Multiple factors that are associated with BACE1 increase AD incidence[2].Thus BACE1 has attracted attention as a challenging and potential therapeutic drug target for AD[7].

Since BACE1 inhibition blocks the production of Aβ and reduces Aβ levels in brain,extensive efforts are needed to explore the BACE1 inhibitors.There are mainly two types of BACE1 inhibitors,peptidics and nonpeptidics [8],and only a limited number have yet been developed.However,traditional peptidic inhibitors with a large molecular size and numerous peptide bonds possess unfavorable pharmacological properties[8].Many studies have shown that the ADMET (absorption,distribution,metabolism,excretion,and toxicity) properties are necessary and helpful in developing new natural compounds with enhanced pharmacokinetic and pharmacodynamics properties[9,10].Thus,the importance of the ADMET properties for peptidic BACE1 inhibitors is no less than their efficacy and specificity[11].Many studies have shown that the absorption,distribution,metabolism,excretion and toxicity(ADMET)properties are good predictors of new compounds with BACE1 inhibitory activity[12-14].

In our previous research,the activities of the egg albuminderived peptides(i.e.,RVPSL,KLPGF,TNGIIR,and QIGLF)against the enzyme cholinesterase were evaluated.Peptides KLPGF and TNGIIR exhibited the activity against acetylcholinesterase(AChE)[15].The deficiency in the brain levels of acetylcholine(ACh)is seen as key step in the pathogenesis of AD [2].Peptide with AChE inhibitory activity can reduce the hydrolysis rate of ACh,and play an important role in the treatment of AD.As a part of our ongoing program to identify peptides from egg albumin hydrolysates with potential anti-AD activity via bioactivity-guided fractionation,in vitroanti-BACE1 activity of the peptides (QIGLF,RVPSL,KLPGF,and TNGIIR)must be evaluated.Thus,in the present study,ADMET analyses were performed to identify the peptides with favorable pharmacological properties.Fluorescence resonance energy transfer (FRET)assay was applied to evaluate thein vitroanti-BACE1 activity of the peptides.Molecular docking and molecular dynamics simulations were carried out to evaluate the interaction between the peptide and BACE1,and to evaluate their binding stability.

2.Materials and methods

2.1.Chemical reagents

The β-secretase (BACE1) activity detection kit was purchased from Sigma Co(St.Louis,Mo,USA).All the other reagents and chemicals used were analytical grade.The peptide KLPGF identified in our previous study[15],was synthesized by Shanghai Top-peptide Co.,Ltd(Shanghai,China).

2.2.Bioavailability and ADMET parameters screening of peptides

The SMILLES input files of the peptides were generated using ChemSketch12.0.The Bank-Formatter service (http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py?form=FAF

Drugs4#forms::Bank-Formatter) was used to convert SMILES input file to a suitable SDF file for FAFDrugs4 (http://fafdrugs4.mti.univ-paris-diderot.fr/).Parameters such as molecular weight(MW),percentage of human oral absorption(%ABS),octanol-water partition coefficient (log Po/w),topological polar surface area(TPSA),and the numbers of hydrogen bond acceptors (HBA),hydrogen bonds donors(HBD),and rotatable bonds of the peptides were assessed using FAF Drug4 server [16].Blood-brain barrier(BBB) permeability,solubility,human intestinal absorption (HIA),and Caco-2 permeability for each peptide were estimated using admetSAR (http://lmmd.ecust.edu.cn/admetsar1/predict/) server based on 50 high quality QSAR models for chemical ADMET profiling[17].

2.3.In vitro BACE1 activity assay BACE1 activity assay

The assay is based on FRET,and enhanced fluorescence signals will be observed after the cleavage of substrate by BACE1.Mixtures of 28 μL fluorescent assay buffer(pH 4.5),20 μL BACE1 Substrate Solution (50 μmol/L),2 μL BACE1 Enzyme (0.3 U/μL),and 50 μL test samples were incubated for 2 h at 37°C.For each reaction,the wavelength of excitation light is 320 nm,and the wavelength of emission light is 405 nm.The percentage of inhibition was obtained by using the following equation[18]:

WhereCis fluorescence of the mixture of enzyme,buffer,and substrate,Sis the fluorescence of the mixture of enzyme,egg albumin-derived peptide solution,and substrate.

2.4.Molecular docking simulation of the BACE1 and peptides

To demonstrate the possible binding modes for peptides inside the binding site of BACE1,CHARMM-based software (CDOCKER)was used to carry out the molecular docking.The crystal structure(1TQF) of human BACE1 complexed with inhibitor was download from the Protein Data Bank[19].The structure of BACE1 was prepared by removing water and adding up the hydrogen atoms.The docking was carried out with coordinatesx:31.1709,y:43.8270,andz:1.8431 with a radius of 9 ?.The structure of peptides was generated by Discovery Studio 2017 Client software.

2.5.Molecular dynamics simulation

Molecular dynamics (MD) simulations were c arried out to investigate the binding stability and solvent effect of the top hit compounds screened from docking.Before MD,the BACE1-KLPGF complexes were immersed in an orthorhombic box of explicit periodic boundary solvent model with 8634 waters,31 sodium,and 23 chlorides.MD simulation of the best docked BACE1-KLPGF complexes was carried out by using the simulation package of DS 2017 R2 client under force field with a time of 2 fs.Steepest Descent and Adopted Basis NR was selected for the minimization of the energy,and the maximum cycles were set to 2000,respectively.Then,the system was gradually heated from 50 K to 300 K during 200 ps and equilibrated time was set to 200 ps.The production ran for 5 ns by using NPT type[20,21].Finally,MD trajectories and the BACE1-KLPGF complexes root mean square deviation (RMSD)and root mean square fluctuation (RMSF) were analyzed by using analyze trajectory module in DS.

3.Results and discussions

3.1.ADMET properties prediction and analysis of peptides

The pharmacological parameters are good predictors of the viability of new compounds with BACE1 inhibitory activity.The ADMET properties of peptides KLPGF,TNGIIR,QIGLF,and RVPSL were performed by FAF Drug4 server (Table1).Compared to TNGIIR,peptides KLPGF,QIGLF,and RVPSL were more in line with Lipinski’s rule of five(RO5)and showed enhanced pharmacokinetic and pharmacodynamic properties.RO5 requires the following criteria:MW ≤500,log Po/w≤5,number of HBD ≤5,number of HBA≤10,and number of rotatable bonds ≤10.The smaller MW,the better absorption,bioavailability,and solubility of a molecular will be observed[22].

Compared with traditional peptidic OM99-2,peptides KLPGF,QIGLF,and RVPSL are smaller in size,which give them obvious advantages for penetration into the central nervous system to reduce Aβ levels [8],as compound 1 isolated fromArtocarpus anisophyllusMiq [23].TPSA is a descriptor associated with passive molecular transport through a membrane.The percentage of absorption (%ABS) was calculated as followed:%ABS = 109/(0.345×TPSA)[24].The peptide KLPGF had the highest absorption among four peptides,with the percentage of absorption value of 38.96%.The peptide QIGLF exhibited moderate absorption with the value of 30.59%.The peptides TNGIIR and RVPSL had lower percentage of absorption than peptides QIGLF and KLPGF,with the values of 8.38%and 19.65%,respectively.In addition,the toxicity of these peptides was negative.

Using the admetSAR server,BBB,HIA,Caco-2 permeability,and cytochrome P450 were collected.The Caco-2 and HIA indicate intestinal absorption,which help to predict the absorption of the peptides through the small intestine.The movement across the epithelial barrier affects the bioavailability of oral food-derivedpeptides[25].The HIA value identifies potent BACE1 inhibitor candidates.Peptides KLPGF,TNGIIR,and QIGLF with HIA probability of 80.90%,54.96%,and 89.94%,respectively,indicated that these three peptides have good intestinal absorption(HIA+),whereas the peptide RVPSL has poor intestinal absorption(HIA-)[17](Table2).

Table1 In silico peptide(physicochemical)properties.

Table2 Key ADMET parameters for four peptides purified from egg white protein.Classification.

BBB is also a crucial factor for CNS active compounds[26].None of the peptides could penetrate the BBB(BBB-).Cytochrome P450(CYP450)is a main route of drug metabolism.CYP2D6 and CYP3A4 are the most important members involved in metabolism.Thus,prediction of peptides with CYP450 enzymes reveals that both peptides are either substrate or inhibitor,which means CYP450 cannot recognize the peptide.The peptides did not interact with CYP450 indicating that the peptides could not affect the metabolism of compound which is the substrates for CYP450 enzymes.There are less drug-drug interactions in the course of metabolism[27].

Taking together the results got from FAF Drug4 server and admetSAR server,peptides QIGLF and KLPGF,with enhanced pharmacokinetic and pharmacodynamics properties were selected for further evaluation.

3.2.BACE1 inhibitory activities of egg albumin-derived peptides

The peptide KLPGF evaluated in our study inhibited BACE1 activity with an IC50value of(8.30±0.56)mmol/L,whereas the peptide QIGLF had no significant BACE1 inhibitory activity,which was consistent with our previous research.Peptide KLPGF could effectively inhibit acetylcholinesterase and butyrylcholinesterase,with the percentage of inhibition was 61.23%and 3.29%at a concentration of 50 μg/mL,respectively[15].Peptide KLPGF inhibited the activity of three key enzymes for causing AD simultaneously.Baicalein exhibited stronger inhibitory activities against BACE1 and AChE with IC50value of (23.71 ± 1.91) μmo/L and (45.95 ± 3.44) μmo/L,respectively[28].KLPGF could be considered a lead compound of peptide drugs from ovalbumin hydrolysate.In the ongoing study,the chemical modification of the candidate peptide KLPGF should be done to enhance the activity.

3.3.Molecular interaction mechanism of peptides and BACE1

Peptide KLPGF was subjected for molecular docking analysis by using Discovery Studio 2017 software.The X-ray crystallographic structure of BACE1 was used as receptor.A higher ‘-CDOCKERINTERACTION-Energy’ denotes a more favorable binding.Peptide KLPGF represented higher‘-CDOCKER-INTERACTION-Energy’with the value of 104.015 kcal/mol.The complex of peptide KLPGF and BACE1 with least energy are prioritized.The interaction of the peptide KLPGF with BACE1 enzyme is shown in Fig.1.Peptide KLPGF contacts the residues Asp32,Gln73,Phe108,Thr232,and Asp228 with conventional hydrogen bonds,contacts the residues Gly11,Asp228,Gly230,and Thr231 with carbon hydrogen bonds,and contacts the residues Asp32,Asp228,and Lys321 with salt bridges.One electrostatic interaction was found with Arg307 and two Pi-cation interactions were found with Phe108 and Lys321.Moreover,alkyl hydrophobic interactions involved residues LEU30 and ILE110.Other bonds were mainly van der Waals interactions.

The structure of BACE1 is composed of eleven pockets [8].Pockets are an essential feature of the protein surface and influence the interactions with small molecular [29].The cavity of the peptides occupied is mainly composed of three pockets:S1(Asp32,Asp228,Tyr71,Phe108,Trp115,and Ile118),S2(Thr232 and Asn233),and S3(Tyr14 and Ile110).Residues 69-75 are outstanding active sites of BACE1 [30].Most BACE1 inhibitors usually possess hydrogen bonds with Asp32 and Asp228 contained in the catalytic domain of BACE1[31,32],which may account for the high inhibitory activity.The interaction analysis revealed that peptide KLPGF occupies similar binding sites as the normal substrate in the catalytic domain.

3.4.Molecular dynamics simulation

A 5-ns molecular dynamics simulation was performed for further analysis of the dynamic properties related to the conformation of BACE1-ACE complexes.The stability of BACE1-KLPGF complexes were observed by comparing RMSD and RMSF values [33].The RMSD of the trajectory for BACE1-KLPGF complexes with respect to initial protein structure is displayed in Fig.2.BACE1-KLPGF complexes reached equilibrium after 4.2 ns,and their average RMSD value was 1.84213.The results indicated that the systems were stable and well equilibrated during the simulation.The binding site of the peptide KLPGF bind with BACE1 is correct.

Fig.1.Docking for the interaction of peptide KLPGF with BACE1.(a)Details of peptide KLPGF at catalytic site.(b)A two-dimensional plan of the interaction of KLPGF with BACE1.

From the RMSF plots(Fig.3),all residues fluctuate below 4 nm.The slightly higher fluctuations were observed in the 68-75,93-97,108-111,131-136,300-305,350-351,and 370-371 amino acids region.In the active site pocket areas (residues 36,219,112,119,122,223,224,18,114),the RMSF fluctuated at about 1 nm.The fluctuations were relatively small,which described the stability of the BACE1-KLPGF interactions[34].

The frequency of hydrogen bonds was calculated.The peptide KLPGF maintained favorable residual(i.e.,Lys321,Asp228,Asn233,Gly230,Thr232,Phe108,Asp32,and Lys107 stable hydrogen bonds with the BACE1 protein for whole simulation)(Fig.4).Among these,Lys321,Asp228,and Asn233 always retained close interactions with KLPGF in the whole simulation with the frequency values of 92.32%,78.32%,and 60.64%respectively,which is indicative of the importance of Lys321,Asp228,and Asn233 in the BACE1 inhibitory peptide design.

Fig.2.The RMSD plot for BACE1-KLPGF during the simulation.

Fig.3.The RMSF values for each residue of BACE1 during simulation.

Fig.4.The heat map of the hydrogen bonds between BACE1 and KLPGF during the simulation.

4.Conclusions

Peptide KLPGF from egg albumin protein hydrolysate underwent ADMET properties analyses and significantly inhibited BACE1 activity with the IC50value of 8.3 mmol/L.In addition,the peptide KLPGF formed twelve strong hydrogen bonds,two hydrophobic interactions,and three electrostatic interactions with the residues of BACE1,which is indicative of its significance as novel BACE1 inhibitor.Furthermore,the MD simulations reveal that BACE1-KLPGF complexes were stable,and the residues (Lys321,Asp228,and Asn233) played important roles in the BACE1 inhibitory peptide design.In the ongoing study,furtherin vivostudies of the peptide KLPGF should be evaluated.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Funds of China(No.31901635)and the National Key Research and Development Program of China(2018YFD0400301).