The effect of Benzothiazolone-2 on the expression of Metallothionein-3 in modulating Alzheimer's disease

Brain and Behavior. 2017;e00799.   | 1 of 9 https://doi.org/10.1002/brb3.799 wileyonlinelibrary.com/journal/brb3 Received:5January2017 | Revised:14June2017 | Accepted:2July2017 DOI: 10.1002/brb3.799 ORIGINAL RESEARCH The effect of Benzothiazolone2 on the expression of Metallothionein3 in modulating Alzheimer’s disease Sudeep Roy1 | Jaromir Gumulec2,3,6 | Akhil Kumar4 | Martina Raudenska2,3 | Mohd Hassan Baig5 | Hana Polanska2,3,6 | Jan Balvan3,6 | Mansi Gupta4 | Petr Babula3 | Jan Odstrčilík1 | Inho Choi5 | Ivo Provaznik1,3 | Michal Masarik2,3,6 ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium, provided the original work is properly cited. ©2017TheAuthors. Brain and BehaviorpublishedbyWileyPeriodicals,Inc. 1DepartmentofBiomedicalEngineering, Faculty of Electrical Engineering and Communication,BrnoUniversityof Technology,Brno,CzechRepublic 2CentralEuropeanInstituteofTechnology, BrnoUniversityofTechnology,Brno,Czech Republic 3DepartmentofPhysiology,Facultyof Medicine,MasarykUniversity,Brno,Czech Republic 4BiotechnologyDivision,CSIR–Central InstituteofMedicinalandAromaticPlants, Lucknow,India 5SchoolofBiotechnology,Yeungnam University,Gyeongsan,Korea 6Department of Pathological Physiology,FacultyofMedicine,Masaryk University,Brno,CzechRepublic Correspondence SudeepRoy,DepartmentofBiomedical Engineering,FacultyofElectricalEngineering andCommunication,BrnoUniversityof Technology,Brno,CzechRepublic. Email: r[email protected]om MichalMasarik,DepartmentofPhysiology, FacultyofMedicine,MasarykUniversity,Brno, CzechRepublic. Email:[email protected] Funding information MinistryofEducation,YouthandSports oftheCzechRepublicundertheproject CEITEC2020,Grant/AwardNumber: LQ1601;MinistryofEducation,Youthand SportsoftheCzechRepublicunderthe SpecificUniversityResearchgrants,Grant/ AwardNumbers:MUNI/A/1355/2016and MUNI/A/1401/2016 Abstract Introduction: Metallothioneins (MTs) are a class of ubiquitously occurring lowmolecularweight cysteineand metalrich proteins containing sulfurbased metal clusters. MT3 exhibits neuroinhibitory activity. The possibility to enhance the expressionofMT-3orprotectitfromdegradationisanattractivetherapeutictarget, because low levels of MT-3 were found in brains of Alzheimer’s disease (AD) patients. Objectives: The primary objective of this study was to test an enhancement of MT3 cellular concentration after MT-3 binding treatment, which could prevent MT-3 degradation. Methods: MTT assay, flow-cytometry, fluorescence microscopy, quantitative real- timepolymerasechainreaction,andimmunodetectionofMT3wereusedforanalysis ofeffectofSTOCK1N-26544,STOCK1N-26929,andSTOCK1N-72593onimmortalizedhumanmicroglia-SV40cellline. Results:AllthreetestedcompoundsenhancedconcentrationofMT-3proteinincells andsurprisinglyalsomRNAconcentration.IC50valuesoftestedmoleculesexceeded about ten times the concentration that was needed for induction of MT3 expression. ThetestedcompoundBenzothiazolone-2enhancedapoptosisandnecrosis,butitwas notofsevereeffect.About80%ofcellswerestillviable.TherewasnoseriousROS- generation and no severe decrease in mitochondria numbers or stress induced endoplasmic reticulum changes after test treatments. The selected compound showed stable hydrophobic and electrostatic interaction during MT3 ligand interaction. Conclusion:Benzothiazolone-2compoundssignificantlyenhancedMT-3proteinand mRNAlevels.Thecompoundscanbelookeduponasoneoftheprobableleadcompounds for future drug designing experiments in the treatment of Alzheimer’s disease. KEYWORDS Alzheimer’sdisease,flowcytometry,immunodetection,metallothionein-3,moleculardynamics, qRT-PCR 2 of 9 | ROY et al. 1 | INTRODUCTION Alzheimer’s is the most common form of dementia; it isa term for memory damage and other intellectual capacities that are serious enoughtoimpede dailylife.Alzheimer’sdisease accountsfor60 to 80 percent of dementia cases. The neuropathological hallmarks of Alzheimerdisease(AD)arecomprisedof“positive”lesionssuchasamyloidplaques,cerebralamyloidangiopathy,neurofibrillarykinks,glial responsesand“negative”lesionssuchasneuronalandsynapticloss (Haass&Selkoe,2007;Iqbal,Liu,Gong,AlonsoAdel,&Grundke-Iqbal, 2009). The neurochemical elements responsible for this agelinked compulsive advance are still poorly described. The growing evidence supportsasignificantroleforbiometalssuchascopper(Cu),iron(Fe), andzinc(Zn)inAβaccretionandneuronalrelapse(Bush&Tanzi,2008; Lovell, Robertson, Teesdale, Campbell, & Markesbery, 1998; Miu & Benga, 2006; Roberts, Ryan, Bush, Masters, & Duce, 2012; Zatta, 2008). The plasma Cu/MT ratio was found to be symptomatic of diseaseadvancementinADpatients.Thisconnotationhintsataprobable connection between some pathological features of the disease and the MTs expression. Metallothioneins (MTs) are a family of lowmolecular weight and cysteinerich proteins present in all eukaryotes. The MT family is encompassedoffourmainmembers(MT-1toMT-4)withmultipleisoform subclasses. MT1 and MT2 are extensively expressed in almost alltissues,MT-3mainlyinthecentralnervoussystem(CNS),whereas MT-4 is present in squamous epithelial tissue (Palacios, Atrian, & Capdevila,2011). Many studies show that MT-3 mRNA is downregulated in AD brains (Barnham etal., 2004; Carrasco etal., 1999; Kim, Nam, Jeon, Han, & Suk, 2012; Naruse etal., 1994; Streit, 2004; Uchida, Takio,Titani,Ihara,&Tomonaga,1991;Yu,Lukiw,Bergeron,Niznik, &Fraser,2001);thismight,therefore,contributetotheupsurgeof abnormal neuronal development that is associated with the disease. Degradation and deficiency in MT3 may lead to severe disruption of lysosomal biogenesis and cytoskeleton dynamics. Dysfunction of lysosomes and actin cytoskeleton in MT3null astrocytes contributed toaccumulationoftoxicAβs proteins and other damaged proteins. ApartfromthefunctionofMT-3inlysosomesandcytoskeleton,homeostasis of metal ions managed by MT3 may also play a key role in AD(Bondaetal.,2011). Furthermore,MT-3-nullastrocyteshadlowlevelofzincinlysosomeandpresentedautophagydefect.Autophagyseemstobeabnormalduetoalterationoftheendo-lysosomalpathway,whichimpairs fusionofautophagosomeswithlysosomes.Inaddition,arecentreport detailsthecontributionofmitophagy,aspecializedformofautophagythatremovesdamagedmitochondriainAD(Moreiraetal.,2007). Interestingly,theactivationofautophagycanpromotedegradationof APP/Aβandreducetaupathology(Nixon,2007).Therefore,theautophagy pathwayis considerablymorecomplexinAD because itis simultaneouslyinducedandimpaired.APP/Aβ can be generated in autophagosomesatlowlevelsinaphysiologicalenvironment;however, during disease conditions autophagosomes rapidly accumulate in the cell body and axonal terminals due to either impaired autophagic flux oradefectedendosomalpathway(Lee,2009;Lee,Park,Kim,&Koh, 2010). Martin et al. (2006) showed that in the Tg2576 transgenic mouse modelofAD,MT-3waslessthanthatfoundinwild-typemice.Italso establishesthe rolesofMT-3 andNnosinAlzheimer’sdisease.The pathological changes that develop in this mode might be responsible for the degradation of MT3. Roy, Kumar, Baig, Masarik, and Provaznik (2015) reported that natural-based compounds were effective in modulatingAlzheimer’s disease. The in silico findings (which include studies of both pharmacodynamics and pharmacokinetic properties) show that MT3 was abletobindwithSTOCK1N-26544,26929,and72593compounds. The former two compounds contain Benzothiazolone-2 as a fragment (3-(2-oxobenzo[d]thiazol-3(2H)-yl)propanoicacid (propanoic acidofBenzothiazolone-2)wasusedinthesynthesis).Itisobtained from Micrococcus sp. found in Tedania ignis,whereasthelattercompoundisamodifiedbaseoftryptamineobtainedwidelyinplants,such as Acacia spp.,Lens esculenta,Prosopsis juliflora,aswellas fromthe fungi Ponaeolus foenisicii,Coprinus micaceus and from the gorgonian Paramuricia chamaeleon. The current work validates the in silico findings for the compounds. Experiments were performed to see the effect of these compoundsonliveimmortalizedhumanmicrogliacells(SV40)and their effect on MT3 cellular concentration. The study also includes a binding freeenergy calculation of the tested natural compound complexes and the standard controls through molecular dynamics simulations. 2 | MATERIALS AND METHODS 2.1 | Cell cultures and cultured cell conditions Theimmortalizedhumanmicroglia-SV40(AppliedBiologicalMaterials Inc.,Richmond,BC,Canada)wereusedinthisstudy.Thesecellsare positiveforNGF[nervegrowthfactor(BetaPolypeptide)],Iba1ionizedcalcium-bindingadaptermolecule1,TREM2(triggeringreceptor expressedonmyeloidcells2),CD11b,andCD68.Theimmortalized humanmicrogliawasculturedinPrigrowIIIMediumwith10%fetal bovine serum in collagencoated flasks. The medium was supplementedwith penicillin(100U/ml) and thecells weremaintained at 37°Cinahumidifiedincubatorwith5%CO2. 2.2 | RNA isolation and reverse transcription TriPure Isolation Reagent (Roche, Basel, Switzerland) was used for RNAisolation.RNAsampleswithoutreversetranscriptionwereused asthenegativecontrolforqRT-PCR,inordertoexcludeDNAcontamination.TheisolatedRNAwasusedforthecDNAsynthesis.RNA (1000ng)wastranscribedusingtheTranscriptorFirstStrandcDNA SynthesisKit(Roche),whichwasappliedaccordingtothemanufacturer’sinstructions. ThecDNA (20μl) preparedfrom thetotal RNA wasdilutedwithRNase-freewaterto100μl; 5 μl was then directly analyzedusingtheLightCycler®480IISystem(Roche). | 3 of 9 ROY et al. 2.3 | Quantitative realtime polymerase chain reaction qRTPCR was performed using TaqMan gene expression assays and the LightCycler®480 II System (Roche). The amplified DNAwas analyzed bythe comparative Ctmethod using βactin asareferencegene.TheprimerandprobesetsforACTB(assay ID: Hs99999903_m1) and MT3 (assay ID: Hs01921768_s1) were selected from the TaqMan gene expression assays (Life Technologies,USA).TheqRT-PCRwasperformedunderthefollowing amplification conditions: total volume of 20 μl,initialincubation at 50°C/2 min followed by denaturation at 95°C/10 min; then45cyclesat95°C/15sandat60°C/1min.Allsampleswere measured in duplicates. 2.4 | Immunodetection of MT3 Extraction of total protein from the cells was processed using the Pierce®RIPABuffer(ThermoScientific,Rockford,IL,USA)inaccordance with the protocol provided. All protein concentrations were determined with the Pierce® BCA Protein Assay Kit. Protein samples were diluted to 10 μg/μl, then 1μl was slowly spotted onto a 0.2μmImmunBlot®PVDFmembrane(BioRadLaboratories,Hercules, CA,USA)andair-dried.Toblocknonspecificbindingsites,themembranes were incubated using 5% Blotting-Grade Blocker (BioRad Laboratories) for one hour at room temperature. They were then incubatedusingAnti-MT-3antibody(productno.ab76618;Abcam, Cambridge,UK),andwithAnti-β-actin(productno.ab8227;Abcam). The antibody was diluted 1:1000 using 5% Blotting-Grade Blocker (BioRadLaboratories). The membranes were then washed four times in PBS (Sigma- Aldrich,St.Louis,MO,USA)forfiveminuteseach,beforebeingincubatedwitha secondaryantibody (Peroxidase-LabeledAnti-Rabbit IgG(productno.PI-1000;Vector,Burlingame,CA,USA)).Thiswasdiluted1:2000using5%Blotting-GradeBlocker.AfterwashinginPBS, allblotswerevisualizedonphotosensitivefilm.Densitometricanalyses of the membraneswere performed using GeneSnap (Syngene, Cambridge,UK). 2.5 | Cytotoxicity testing – MTT test The suspension of 5000 cells was added to each well of standard microtiterplates.Avolumeof200μlwastransferredtowells2–11.The medium (200 μl) was added to the first and the last column (1 and 12(control)). The plates were incubated for 2 days at 37°C to ensure cellgrowth.Themediumwasremovedfromcolumns2–11.Columns 3–10werefilledwith200μlofthemedium,whichcontainedanincreased concentration of tested compound diluted in dimethylsulfoxide(DMSO)(0–1μmol/l).Asacontrol,columns2and11werefilled with the medium without tested compound. Theplateswereincubatedfor 12and 24hr,then the medium wasremovedandthecellswerewashedinPBS.Columns1–11were filled with 200 μlofmedium,whichcontained50μl of MTT (5 mg/ mlinPBS)incubatedinahumidifiedatmosphereforfourhoursat 37°Candthenwrappedinaluminumfoil.Aftertheincubation,the MTTcontaining medium was replaced with 200 μlof99.9%DMSO in order to dissolve the MTT-formazan crystals. Subsequently, 25 μl of glycine buffer was added to all wells; absorbance was immediately determined to be 570nm (VersaMax microplate reader, MolecularDevices,Sunnyvale,CA,USA).Allsamplesweremeasured in duplicates. 2.6 | Confocal microscopy and cell staining Forthepurposeoffluorescencemicroscopy,thecellswerecultivated directly on microscope glass slides (75×25mm, thickness 1mm, MenzelGlässer,Braunschweig,Germany)inPetridishesintheabove- describedcultivationmedia(see“Culturedcellconditions”).Thecells weretransferreddirectlyontotheslides,whichwerethensubmerged in the cultivation media. Afterthetreatment,themicroscopeglassslideswithamonolayer of cells were removed from the Petri dishes, rinsed in the cultivationmediumwithout supplementationandPBS bufferand directly used for staining and fluorescence microscopy. The cells were incubated using the following highly specific fluorescent probes: • ReactiveoxygenspecieswerevisualizedusingCellROXDeepRed reagent(LifeTechnologies, 5μmol/L, cell-permeant,life-cellstain withabsorption/emissionmaximaof644/665nm) • Mitochondria were visualized using MitoTracker Green FM (Life Technologies,300nmol/L,cell-permeantlife-cellstainwithabsorption/emissionmaximaof490/516nm) • The endoplasmic reticulum was visualized using ER-Tracker Red (LifeTechnologies,1μmol/L,cell-permeant,life-cellstainwithabsorption/emission maxima of 587/615 nm). After incubation (45min, 37°C, dark), the cells were washed three times with PBS buffer (0.05mol/L, pH 7.0) and observed under the confocalmicroscope(LeicaTCSSP8 X, Germany) using appropriateexcitationandemissionwavelengths.Quantitativeanalysis was performed typically from 10 fields of view. For image analysis, NIS elements BRAnalysis (Nikon instruments, Tokyo, Japan) softwarewasused.Averagefluorescenceintensitywasmeasuredin equally confluent fields of view. Results were compared statistically using ttests. 2.7 | Flow cytometry analysis of cell death Doublestaining with fluorescein isothiocyanate (FITC)/propidium iodide(PI)wasundertakenusing theAnnexinV-FLUOS-stainingkit (RocheAppliedScience)accordingtothemanufacturer’sprotocols,in ordertodeterminethepercentagesofviable,apoptotic,andnecrotic cells following the exposure to Plumbagin. Briefly,thecellswereharvestedbyrepetitivepipettingandwere washed two times with PBS (centrifuged at 2000rpm for 5min), 4 of 9 | ROY et al. resuspended in 100 μlofAnnexin-V-FLUOSlabelingsolutionandincubatedfor15mininthedarkat15–25°C.AnnexinV-FITCbinding wasdetectedbyflowcytometry(PartecGmbH,Münster,Germany) (Ex=488nm,Em=533nm,FL1filterforAnnexin-V-FLUOSandFL3 filter for PI). 2.8 | Thermodynamic studies - MD simulation The MM-PBSA (Molecular Mechanics energies combined with Poisson–BoltzmannSurfaceArea)approach,combinedwithmoleculardynamics(MD)simulations, iswidelyusedin biomolecularcomplexes as a way to estimate the protein ligand interaction energies and predict bindingfree energies. It also evaluates the relative stabilities of different biomolecular structures. Additionally, it can be used to rescoreasetofdockedcomplexes,therebyimprovingtheabilityto distinguish between active and inactive lead molecules. The molecular dynamics simulation of MT3 docked complexes was performed using the GROMACS 4.5.52 (Berendsen etal., 1995; Lindahl etal., 2001)package,withastandardGROMOS96forcefieldfor10,000ps. Binding freeenergy calculations were performed in this work for the ligandbound complexes in order to provide further insight into the study. 3 | RESULTS 3.1 | The cytotoxic evaluation of benzothiazolone2containing substances in microglia cells The IC50 value for STOCK1N-26544 is 162.4nmol/L, for STOCK1N-26929 it is 100nmol/L and for STOCK1N-72593 it is 233.4nmol/L. Thus, STOCK1N-26929 shows the highest effect on cell metabolic activity. Nevertheless, the IC50 values for all tested molecules exceed the required concentration for the induction of MT3 expression by about 10 times (see Figure 1). TheAnnexinV+/PI+(Q2quadrant)depictslatenecrosisanddying cells. The rate of the necrosis triggered by tested compounds did not exceed4%(onlyshowinga2.12%increase,comparedtothestained control). The Annexin V+/PI- (Q4) population represents apoptotic cells or early oncotic cells (see Figure 2). The largest amount of apoptoticorearlyoncoticcellswasfoundin10nmol/LSTOCK1N-72593 after treatment (19.93%; 12.31% increase compared to the stained control). Nevertheless, some portion of oncotic cells is able to reverseoncosisandcouldtherebysurvive;thus,nosevereincreasein the total number of dying cells was found after treatment with tested compounds (see Figure 2). Confocal microscopy revealed no significant morphological changes that had been induced by studied compounds. On the other hand,weusedtwoprobestolocalizemitochondriaandtheendoplasmicreticulum.WhereasMitoTrackerGreenFMlocalizesmitochondria regardless of mitochondrial membrane potential, ER-Tracker Red, a conjugateofgreen-fluorescentBODIPY®TRdyeandglibenclamide, alsoreflectsexpressionofsulphonylureareceptorsofATP-sensitive K+ channels and thereby reflects the ER function. The doublestaining revealed no significant decrease in fluorescence corresponded to both mitochondria and ER for the tested compounds. Compound 26544showedsomeabilitytogenerateROS,whichwerecolocalized withmitochondria(seeFigure3).Nevertheless,ROSproductionwas rather weak. 3.2 | Benzothiazolone2 enhances expression of MT3 in microglia cells mRNAexpressionofMT-3wasrelativelylowincontrolcellswithout treatment.Allthethreetestedcompounds enhancedexpressionof MT-3mRNAinbothtestedconcentrations(5nmol/Land10nmol/L, respectively)(seeFigure4a). Expression of MT3 protein was undetectable in control cells withouttreatment.Allthethreetestedcompoundsenhancedexpression of MT-3 protein (see Figure4b). Nevertheless, compound 72593 caused visible enhancement only in 5nmol/L concentration. Other compounds enhanced expression of MT3 protein in tested concentrations(seeFigure4c). FIGURE1 MTT tests for selected compounds on cell lines. The halfmaximal inhibition concentration (IC50) is indicated in the graph. Error bars indicate standard deviations | 5 of 9 ROY et al. 3.3 | Thermodynamic studies on MT3benzothiazolone complexes Binding freeenergy studies that validate the above findings were also performed.The analysis was carried out for the Benzothiazolone-2 docked complexes only, since the above results were encouraging.The molecular docking representation is provided in Figure S1. It clearly states that the tested molecules (STOCK1N-26929 and STOCK1N-26544) both have stable binding free-energies values in theformofpotential,polar,andnonpolarsolvationenergiesthatare comparable to the standard controls (Table 1). It also states that both hydrophobic and electrostatic interactions are important for protein ligandinteractionincaseofMT-3forAlzheimer’sdisease. 4 | DISCUSSION The possibility to enhance the expression of MT3 or protect it from degradation is an attractive therapeutic target, because low levelsofMT-3werefoundinADbrains.Inthisstudy,wetestedan FIGURE2 Flowcytometricanalysisofapoptosis/necrosisbyAnnexinV/Propidiumiodide(PI)staining.(a)Stainingofcontrols(notstained andnottreated,andstainednontreated).(b)Flow-cytometricanalysisofcelllinestreatedwith5and10nmol/Loftestedagents.Notethe differencesinAnnexinV+/PI+(Q2)andAnnexinV+/PI-(Q4)populations 6 of 9 | ROY et al. FIGURE3 (Fluorescencemicroscopyanalysisofmitochondriaandendoplasmicreticulum(ER).ERisstainedwithredERtracker, mitochondria)[MR1]withgreenMitoTracker.ReactiveoxygenspecieswerevisualizedusingCellROXDeepRedreagent | 7 of 9 ROY et al. enhancement of MT3 cellular concentration after MT3 binding treatment,whichcouldpreventMT-3degradation. ThethreecompoundsunderinvestigationwereSTOCK1N-26929, STOCK1N-26544 (Benzothiazolone-2 as fragment), and STOCK1N- 72593 (modified tryptaminebased). The expression of MT3 protein was undetectable in control cells withouttreatment.AllthreetestedcompoundsenhancedconcentrationofMT-3proteinincellsandsurprisinglyalsomRNAconcentration.Nevertheless,STOCK1N-72593wasrelativelylesseffective. It is to be noted here that different concentrations of tested compounds can influence expression of MT3 differently. Epigenetic mechanisms and different types of activation of signaling pathways are suspected inthiscase.Furthermore,theexpressionofMT-3couldberegulated at the posttranscriptional level, which was also observed in earlier studies(Garrettetal.,2005).Theexperimentscarriedoutdidnotassume that triggering of MT3 expression should be in positive correlation with tested compound concentration. The results showed that our compounds are able to influence MT3 expression. It is to be mention that our study is not oriented toward exact mechanism ortoshowexactquantitiesofMT-3protein.Inouropinion,twoof ourcompoundssignificantlyenhancedMT-3proteinandmRNAlevels. Thethreetestedmolecules(STOCK1N-26544,STOCK1N-26929, and STOCK1N-72593) have no severe cytotoxic effects on neural tissue(Figure4).IC50valuesoftestedmoleculesexceededabout10 times the concentration that was needed for induction of MT3 expression (Figure 1). Our goal of the precise experiment is the assessment of IC50 value. The findings also demonstrate that concentrations oftestedcompounds,whichareabletotriggerMT-3synthesis,arenot toxic. The experiments carried are not focused toward inhibition of microglia.Ourcompoundsenhancedapoptosisandnecrosis,butitwas notofsevereeffect.About80%ofcellswerestillviable.Therateof thenecrosistriggeredbytestedcompoundsdidnotexceed4%(only showinga2.12%increase,comparedtothestainedcontrol).Thelargestamountofapoptoticorearlyoncoticcellswasfoundin10nmol/L STOCK1N-72593 after treatment (19.93%; 12.31% increase compared to the stained control). Flow cytometric analysis on apoptosis/necrosis showed that a lowerdose(5nmol/L)inducedahighertoxiceffectwhencompared withhigherdoses 10nmol/L.Thisincreasewasnotthe severeone (onlyabout4%).Epigeneticmechanismsanddifferenttypesofactivationofsignalingpathwaysaresuspectedinthiscase.Aswasshown in other studies, mechanisms of drug-induced cell death are often concentration dependent. Crosstalk between apoptosis, necrosis, and autophagy could be different in different drug concentrations and numbers of apoptotic cells could be attenuated because of some protectiveautophagyeffects(Kemp,2017;Miyoshietal.,2008;Teng etal.,2016;Torres&Horwitz,1998). Accordingtofluorescencestaining,noseriousROSgeneration and no severe decrease in mitochondria numbers or endoplasmic reticulum changes after testtreatment were found. This is very important,(becauseoxidativestress,mitochondrialdysfunction,and endoplasmic reticulum stress have been implicated in betaamyloid neurotoxicity). The above findings were further validated by calculating the binding freeenergy of the docked complexes through molecular dynamics studies.TheMM-PBSAmethodusesthreeenergetictermsinorderto FIGURE4 ExpressionanalysisofMT-3gene.(a)mRNA expressionfoldchangeincelllinestreatedwith0,5,and10nmol/L of selected compounds. (b) Dot blot for MT3. (c) (Protein level of MT3 based on dot blot detection; displayed as mean and standard deviation) 26544 26929 72593 5 nM conc. MT3 b-actin substance 10 nM 5 nM 10 nM 5 nM 10 nM control MT3 protein AUC 5 nM 10 nM 2654426929 72593SV40 0 5000 10000 15000 20000 Gene expression fold change 0 nM 5 nM 10 nM 2654426929 72593 0.75 2.50 5.00 7.50 25.00 50.00 75.00 (a) (b) (c) TABLE1 Binding freeenergy calculation of the tested compounds and the standard controls Ligand Van der Waal energy (kJ/mol) Electrostatic energy (kJ/mol) Polar solvation energy (kJ/mol) SASA energy (kJ/mol) Binding energy (kJ/mol) BDBM50342769 −129.891±5.824 −25.876±5.056 71.608±5.845 −11.920±0.763 −96.078±11.107 BDBM9019 −83.033±9.812 −13.835±4.606 46.691±13.885 −8.855±1.547 −59.033±11.597 BDBM50260394 −101.236±10.190 −42.368±20.609 98.840±35.764 10.603±0.848 −55.367±20.694 STOCK1N-26544 −60.097±41.578 −92.875±62.292 97.052±69.708 −7.644±5.449 −63.564±40.740 STOCK1N-26929 −107.860±7.382 −71.870±14.138 118.127±28.044 −9.691±0.886 −71.294±21.520 8 of 9 | ROY et al. calculate changes in the freeenergy on binding. The g_mmpbsa tool developedbyKumarietal.(2014),Bakeretal.(2001)(http://rashmikumari.github.io/g_mmpbsa/)isusedtocalculatetheMM-PBSAfrom eachcomplexescombinewithGROMACS.Theexperimentwasrun for 1000ps; several thermodynamic parameters (including RMSD, RMSF, SASA, and H-bond) have already been reported (Roy etal., 2015).g_mmpbsfetchesinformationfromtheGROMACStrajectory fileandcalculatesthetotalfree-energyoftheprotein-ligandcomplex, aswellasthefree-energyofproteinandligand,individually.Italsocalculates the potential energy of molecular mechanics and freeenergy of solvation except entropy contribution. Interaction energy for three control molecules with STOCK1N compounds was calculated with the MT-3 receptor, in order to compare it with the active binder. BDBM50342769, BDBM9019, BDBM50260394 binding energies were approximately −96, −59, and −55kJ/mol. If we look into the individual component of predicted free-energy, van der Waals energies were dominant in the BDBM50342769, BDBM50260394 binding with MT-3. Similarly STOCK1N-26929 showed higher van der Waals energy as well as goodbindingfree-energycomparedtoSTOCK1N-26544(lowcontribution of van der Waals energy). This suggests one of the major MT3 binding interactions is governed by the hydrophobic forces. The secondmajorcomponentwaselectrostaticenergy,whichiscomparably higherinSTOCK1Nmoleculesthanincontrols. The overall findings suggest that hydrophobic and electrostatic interaction is imperative for current MT3 ligand interaction. Polar solvation and nonpolar solvation energies are comparable to the standard controls (Table 1). 5 | CONCLUSIONS The current work has made an effort to see the effect of Benzothiazolone-2onthecellularconcentrationofMT-3.ManystudiesshowthatMT-3mRNAisdownregulatedinADbrainsandthat thismight,therefore,contributetotheupsurgeofabnormalneuronal development associated with the disease. Our previous findings suggests that Benzothiazolone-2 possesses better pharmacokinetic and pharmacodynamics properties. It also forms stable complex with MT3 according to molecular dynamics findings. The above findings were validated by screening the compounds against live immortalized human microglia cells (SV40). This tested compound showed neither severed cytotoxicity nor did ittriggernecrosis.Inaddition,therewasnoseriousROS-generation, decreaseinmitochondrialnumbers,stress-inducedendoplasmicreticulum changes after testtreatments.Allthreetestedcompoundsenhanced cellular concentration of MT3 protein in cells and surprisingly alsomRNAexpression. ACKNOWLEDGMENTS This work was supported by the Ministry of Education, Youth and SportsoftheCzechRepublicundertheprojectCEITEC2020(LQ1601) andbySpecificUniversityResearchgrants–MUNI/A/1355/2016and MUNI/A/1401/2016–providedbytheMinistryofEducation,Youth andSportsoftheCzechRepublicintheyear2017. COMPETING FINANCIAL INTERESTS We certify that we have no affiliation with or financial involvementwithanyorganizationorentitywithadirectfinancialorany other interest in the subject matter or materials discussed in the manuscript. REFERENCES Baker,N.A.,Sept,D.,Joseph,S.,Holst,M.J.,&McCammon,J.A.(2001). Electrostaticsofnanosystems:Applicationtomicrotubulesandtheribosome. Proceedings of the National Academy of Sciences of the United States of America,98,10037–10041. Barnham, K. J., Haeffner, F., Ciccotosto, G. D., Curtain, C. C., Tew, D., Mavros,C.,…Bush,A.I.(2004).Tyrosinegatedelectrontransferiskey to the toxic mechanism ofAlzheimer’s disease beta-amyloid. FASEB Journal,18,1427–1429. Berendsen,H.J.C.,vanderSpoel,D.,&vanDrunen,R.(1995).GROMACS: A message-passing parallel molecular dynamics implementation. Computer Physics Communications,91,43–56. Bonda,D.J.,Lee,H.G.,Blair,J.A.,Zhu,X.,Perry,G.,&Smith,M.A.(2011). Role of metal dyshomeostasis in alzheimer disease. Metallomics, 3, 267–270. Bush,A.I.,&Tanzi,R.E.(2008).TherapeuticsforAlzheimer’sdiseasebased on the metal hypothesis. Neurotherapeutics: the Journal of the American Society for Experimental NeuroTherapeutics,5,421–432. Carrasco,J.,Giralt,M.,Molinero,A.,Penkowa,M.,Moos,T.,&Hidalgo,J. (1999).Metallothionein(MT)-III:Generationofpolyclonalantibodies, comparisonwithMT-I+IIinthefreezelesionedratbrainandinabioassaywithastrocytes,andanalysisofAlzheimer’sdiseasebrains.Journal of Neurotrauma,16,1115–1129. Garret,S.H.,Park,S.,Sens,M.A.,Somji,S.,Singh,R.K.,Namburi,V.B., & Sens, D.A. (2005). Expression of metallothionein isoform 3 is restricted at the post transcriptional level in human bladder epithelial cells. Toxicological Sciences,87,66–74. Haass, C., & Selkoe, D.J. (2007). Soluble protein oligomers in neurodegeneration:LessonsfromtheAlzheimer’samyloidbeta-peptide.Nature Reviews Molecular Cell Biology,8,101–112. Iqbal,K.,Liu,F.,Gong,C.X.,AlonsoAdel,C.,&Grundke-Iqbal,I.(2009). Mechanisms of tauinduced neurodegeneration. Acta Neuropathologica, 118,53–69. Kemp, M. G. (2017). Crosstalk between apoptosis and autophagy: environmentalGenotoxins,infection,andinnateimmunity.Journal of Cell Death,9,1–6. Kim,J.H.,Nam,Y.P.,Jeon,S.M.,Han,H.S.,&Suk,K.(2012).Amyloidneurotoxicity is attenuated by metallothionein: Dual mechanisms at work. Journal of Neurochemistry,121,751–762. Kumari,R.,Kumar,R.,OpenSourceDrugDiscoveryConsortium,&Lynn, A. (2014). g_mmpbsa A GROMACS tool for high throughput MM PBSA calculations. Journal of Chemical Information and Modeling, 54, 1951–1962. Lee,J.A.(2009).Autophagyinneurodegeneration:Twosidesofthesame coin. BMB Reports,42,324–330. Lee,S.J.,Park,M.H.,Kim,H.J.,&Koh,J.Y.(2010).Metallothionein-3regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions. Glia,58,1186–1196. Lindahl, E., Berk, H., & David, V. S. (2001). GROMACS 3.0: A package for molecular simulation and trajectory analysis. Journal of Molecular Modeling,7,306–317. | 9 of 9 ROY et al. Lovell,M.A.,Robertson,J.D.,Teesdale,W.J.,Campbell,J.L.,&Markesbery, W. R. (1998). Copper, iron and zinc in Alzheimer’s disease senile plaques. Journal of the Neurological Sciences,158,47–52. Martin,B.L.,Tokheim,A.M.,McCarthy,P.T.,Doms,B.S.,Davis,A.A.,& Armitage, I. M. (2006). Metallothionein-3 and neuronal nitric oxide synthaselevelsinbrainsfromtheTg2576mousemodelofAlzheimer’s disease. Molecular and Cellular Biochemistry,283,129–137. Miu,A.C.,&Benga,O.(2006).AluminumandAlzheimer’sdisease:Anew look. Journal of Alzheimer’s Disease,10,179–201. Miyoshi,N.,Watanabe,E.,Osawa,T.,Okuhira,M.,Murata,Y.,Ohshima,H.,& Nakamura,Y.(2008).ATPdepletionaltersthemodeofcelldeathinduced bybenzylisothiocyanate.Biochimica et Biophysica Acta,10,566–573. Moreira,P.I.,Siedlak,S.L.,Wang,X.,Santos,M.S.,Oliveira,C.R.,Tabaton, M.,…Perry,G.(2007).IncreasedautophagicdegradationofmitochondriainAlzheimerdisease.Autophagy,3,614–615. Naruse,S.,Igarashi,S.,Furuya,T.,Kobayashi,H.,Miyatake,T.,&Tsuji,S. (1994).Structuresofthehumanandmousegrowthinhibitoryfactor- encoding genes. Gene,144,283–287. Nixon, RA. (2007). Autophagy, amyloidogenesis and Alzheimer disease. Journal of Cell Science,120,4081–4091. Palacios, O.,Atrian, S., & Capdevila, M. (2011). Zn- and Cu-thioneins:A functional classification for metallothioneins? JBIC Journal of Biological Inorganic Chemistry,16,991–1009. Roberts,B.R.,Ryan,T.M.,Bush,A.I.,Masters,C.L.,&Duce,J.A.(2012). Theroleofmetallobiologyandamyloid-betapeptidesinAlzheimer’s disease. Journal of Neurochemistry,120(Suppl1),149–166. Roy,S.,Kumar,A.,Baig,M.H.,Masarik,M.,&Provaznik,I.(2015).Virtual screening, ADMET profiling, molecular docking and dynamics approaches to search for potent selective natural molecule based inhibitorsagainstmetallothionein-IIItostudyAlzheimer’sdisease.Methods, 83,105–110. Streit,W.J.(2004).MicrogliaandAlzheimer’sdiseasepathogenesis.Journal of Neuroscience Research,77,1–8. Teng,Y.H.,Li,J.P.,Liu,S.L.,Zou,X.,Fang,L.H.,Zhou,J.Y.,…Wang,R. P.(2016).Autophagyprotectsfromraddeanina-inducedapoptosisin SGC-7901humangastriccancercells.EvidenceBased Complementary and Alternative Medicine: eCAM,2016,1–8. Torres,K.,&Horwitz,S.B.(1998).Mechanismsoftaxol-inducedcelldeath are concentration dependent. Cancer Research,58,3620–3626. Uchida,Y.,Takio,K.,Titani,K.,Ihara,Y.,&Tomonaga,M.(1991).Thegrowth inhibitoryfactorthatisdeficientintheAlzheimer’sdiseasebrainisa68 amino acid metallothioneinlike protein. Neuron,7,337–347. Yu,W.H.,Lukiw,W.J.,Bergeron,C.,Niznik,H.B.,&Fraser,P.E.(2001). Metallothionein III is reduced inAlzheimer’s disease. Brain Research, 894,37–45. Zatta, P. (2008). Metallothioneins in biochemistry and pathology (336 pp). Singapore: World Scientific Publishing Company. ISBN: 978-981-277-893-2. SUPPORTING INFORMATION Additional Supporting Information may be found online in the supporting information tab for this article. How to cite this article:RoyS,GumulecJ,KumarA,etal. TheeffectofBenzothiazolone-2ontheexpressionof Metallothionein-3inmodulatingAlzheimer’sdisease.Brain Behav. 2017;e00799. https://doi.org/10.1002/brb3.799