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

B ain and Beha io . 2017;e00799.   | 1 o 9 h ps://doi.o g/10.1002/b b3.799 wileyonlinelib a y.com/jou nal/b b3 Recei ed:5Janua y2017 | Re ised:14June2017 | Accep ed:2July2017 DOI: 10.1002/b b3.799 ORIGINAL RESEARCH The e ec o Benzo hiazolone- 2 on he exp ession o Me allo hionein- 3 in modula ing Alzheime ’s disease Sudeep Roy1 | Ja omi Gumulec2,3,6 | Akhil Kuma 4 | Ma ina Raudenska2,3 | Mohd Hassan Baig5 | Hana Polanska2,3,6 | Jan Bal an3,6 | Mansi Gup a4 | Pe Babula3 |  Jan Ods čilík1 | Inho Choi5 | I o P o aznik1,3 | Michal Masa ik2,3,6 Thisisanopenaccessa icleunde  he e mso  heC ea i eCommonsA ibu ionLicense,whichpe mi suse,dis ibu ionand ep oduc ioninanymedium, p o ided he o iginal wo k is p ope ly ci ed. ©2017TheAu ho s. B ain and Beha io publishedbyWileyPe iodicals,Inc. 1Depa men o BiomedicalEnginee ing, Facul y o Elec ical Enginee ing and Communica ion,B noUni e si yo  Technology,B no,CzechRepublic 2Cen alEu opeanIns i u eo Technology, B noUni e si yo Technology,B no,Czech Republic 3Depa men o Physiology,Facul yo  Medicine,Masa ykUni e si y,B no,Czech Republic 4Bio echnologyDi ision,CSIR–Cen al Ins i u eo MedicinalandA oma icPlan s, Lucknow,India 5Schoolo Bio echnology,Yeungnam Uni e si y,Gyeongsan,Ko ea 6Depa men o Pa hological Physiology,Facul yo Medicine,Masa yk Uni e si y,B no,CzechRepublic Co espondence SudeepRoy,Depa men o Biomedical Enginee ing,Facul yo Elec icalEnginee ing andCommunica ion,B noUni e si yo  Technology,B no,CzechRepublic. Email: [email p o ec ed]om MichalMasa ik,Depa men o Physiology, Facul yo Medicine,Masa ykUni e si y,B no, CzechRepublic. Email:[email p o ec ed] Funding in o ma ion Minis yo Educa ion,You handSpo s o  heCzechRepublicunde  hep ojec  CEITEC2020,G an /Awa dNumbe : LQ1601;Minis yo Educa ion,You hand Spo so  heCzechRepublicunde  he Speci icUni e si yResea chg an s,G an / Awa dNumbe s:MUNI/A/1355/2016and MUNI/A/1401/2016 Abs ac In oduc ion: Me allo hioneins (MTs) a e a class o ubiqui ously occu ing low- molecula - weigh cys eine- and me al- ich p o eins con aining sul u - based me al clus e s. MT- 3 exhibi s neu o- inhibi o y ac i i y. The possibili y o enhance he ex- p essiono MT-3o p o ec i  omdeg ada ionisana ac i e he apeu ic a ge , because low le els o  MT-3 we e ound in b ains o  Alzheime ’s disease (AD) pa ien s. Objec i es: The p ima y objec i e o his s udy was o es an enhancemen o MT- 3 cellula  concen a ion a e  MT-3 binding ea men , which could p e en  MT-3 deg ada ion. Me hods: MTT assay, low-cy ome y, luo escence mic oscopy, quan i a i e eal- imepolyme asechain eac ion,andimmunode ec iono MT3we eused o analysis o e ec o STOCK1N-26544,STOCK1N-26929,andSTOCK1N-72593onimmo al- izedhumanmic oglia-SV40cellline. Resul s:All h ee es edcompoundsenhancedconcen a iono MT-3p o einincells andsu p isinglyalsomRNAconcen a ion.IC50 alueso  es edmoleculesexceeded abou en imes he concen a ion ha was needed o induc ion o MT- 3 exp ession. The es edcompoundBenzo hiazolone-2enhancedapop osisandnec osis,bu i was no o se e ee ec .Abou 80%o cellswe es ill iable.The ewasnose iousROS- gene a ion and no se e e dec ease in mi ochond ia numbe s o s ess induced endo- plasmic e iculum changes a e es ea men s. The selec ed compound showed s able hyd ophobic and elec os a ic in e ac ion du ing MT- 3 ligand in e ac ion. Conclusion:Benzo hiazolone-2compoundssigni ican lyenhancedMT-3p o einand mRNAle els.Thecompoundscanbelookeduponasoneo  hep obableleadcom- pounds o  u u e d ug designing expe imen s in he ea men  o  Alzheime ’s disease. KEYWORDS Alzheime ’sdisease, lowcy ome y,immunode ec ion,me allo hionein-3,molecula dynamics, qRT-PCR 2 o 9 | ROY e al. 1 | INTRODUCTION Alzheime ’s is he mos  common o m o  demen ia; i  isa e m o  memo y damage and o he in ellec ual capaci ies ha a e se ious enough oimpede dailyli e.Alzheime ’sdisease accoun s o 60 o 80 pe cen o demen ia cases. The neu opa hological hallma ks o Alzheime disease(AD)a ecomp isedo “posi i e”lesionssuchasam- yloidplaques,ce eb alamyloidangiopa hy,neu o ib illa ykinks,glial esponsesand“nega i e”lesionssuchasneu onalandsynap icloss (Haass&Selkoe,2007;Iqbal,Liu,Gong,AlonsoAdel,&G undke-Iqbal, 2009). The neu ochemical elemen s esponsible o his age- linked compulsi e ad ance a e s ill poo ly desc ibed. The g owing e idence suppo sasigni ican  ole o biome alssuchascoppe (Cu),i on(Fe), andzinc(Zn)inAβacc e ionandneu onal elapse(Bush&Tanzi,2008; Lo ell, Robe son, Teesdale, Campbell, & Ma kesbe y, 1998; Miu & Benga, 2006; Robe s, Ryan, Bush, Mas e s, & Duce, 2012; Za a, 2008). The plasma Cu/MT a io was ound o be symp oma ic o dis- easead ancemen inADpa ien s.Thisconno a ionhin sa ap obable connec ion be ween some pa hological ea u es o he disease and he MTs exp ession. Me allo hioneins (MTs) a e a amily o low- molecula weigh and cys eine- ich p o eins p esen in all euka yo es. The MT amily is en- compassedo  ou mainmembe s(MT-1 oMT-4)wi hmul ipleiso- o m subclasses. MT- 1 and MT- 2 a e ex ensi ely exp essed in almos all issues,MT-3mainlyin hecen alne oussys em(CNS),whe eas MT-4 is p esen  in squamous epi helial issue (Palacios, A ian, & Capde ila,2011). Many s udies show ha  MT-3 mRNA is down egula ed in AD b ains (Ba nham e al., 2004; Ca asco e al., 1999; Kim, Nam, Jeon, Han, & Suk, 2012; Na use e al., 1994; S ei , 2004; Uchida, Takio,Ti ani,Iha a,&Tomonaga,1991;Yu,Lukiw,Be ge on,Niznik, &F ase ,2001); hismigh , he e o e,con ibu e o heupsu geo  abno mal neu onal de elopmen ha is associa ed wi h he disease. Deg ada ion and de iciency in MT- 3 may lead o se e e dis up ion o lysosomal biogenesis and cy oskele on dynamics. Dys unc ion o ly- sosomes and ac in cy oskele on in MT- 3- null as ocy es con ibu ed oaccumula iono  oxicAβs p o eins and o he damaged p o eins. Apa  om he unc iono MT-3inlysosomesandcy oskele on,ho- meos asis o me al ions managed by MT- 3 may also play a key ole in AD(Bondae al.,2011). Fu he mo e,MT-3-nullas ocy eshadlowle elo zincinlyso- someandp esen edau ophagyde ec .Au ophagyseems obeabno - maldue oal e a iono  heendo-lysosomalpa hway,whichimpai s usiono au ophagosomeswi hlysosomes.Inaddi ion,a ecen  epo  de ails hecon ibu iono mi ophagy,aspecialized o mo au oph- agy ha  emo esdamagedmi ochond iainAD(Mo ei ae al.,2007). In e es ingly, heac i a iono au ophagycanp omo edeg ada iono  APP/Aβand educe aupa hology(Nixon,2007).The e o e, heau- ophagy pa hwayis conside ablymo ecomplexinAD because i is simul aneouslyinducedandimpai ed.APP/Aβ can be gene a ed in au- ophagosomesa lowle elsinaphysiologicalen i onmen ;howe e , du ing disease condi ions au ophagosomes apidly accumula e in he cell body and axonal e minals due o ei he impai ed au ophagic lux o ade ec edendosomalpa hway(Lee,2009;Lee,Pa k,Kim,&Koh, 2010). Ma in e al. (2006) showed ha in he Tg2576 ansgenic mouse modelo AD,MT-3wasless han ha  oundinwild- ypemice.I also es ablishes he oleso MT-3 andNnosinAlzheime ’sdisease.The pa hological changes ha de elop in his mode migh be esponsible o he deg ada ion o MT- 3. Roy, Kuma , Baig, Masa ik, and P o aznik (2015) epo ed ha  na u al-based compounds we e e ec i e in modula ingAlzheime ’s disease. The in silico indings (which include s udies o bo h pha ma- codynamics and pha macokine ic p ope ies) show ha MT- 3 was able obindwi hSTOCK1N-26544,26929,and72593compounds. The o me  wo compounds con ain Benzo hiazolone-2 as a ag- men  (3-(2-oxobenzo[d] hiazol-3(2H)-yl)p opanoicacid (p opanoic acido Benzo hiazolone-2)wasusedin hesyn hesis).I isob ained om Mic ococcus sp. ound in Tedania ignis,whe eas hela e com- poundisamodi iedbaseo  yp amineob ainedwidelyinplan s,such as Acacia spp.,Lens esculen a,P osopsis juli lo a,aswellas om he ungi Ponaeolus oenisicii,Cop inus micaceus and om he go gonian Pa amu icia chamaeleon. The cu en wo k alida es he in silico indings o he com- pounds. Expe imen s we e pe o med o see he e ec o hese compoundsonli eimmo alizedhumanmic ogliacells(SV40)and hei e ec on MT- 3 cellula concen a ion. The s udy also includes a binding ee- ene gy calcula ion o he es ed na u al compound complexes and he s anda d con ols h ough molecula dynamics simula ions. 2 | MATERIALS AND METHODS 2.1 | Cell cul u es and cul u ed cell condi ions Theimmo alizedhumanmic oglia-SV40(AppliedBiologicalMa e ials Inc.,Richmond,BC,Canada)we eusedin hiss udy.Thesecellsa e posi i e o NGF[ne eg ow h ac o (Be aPolypep ide)],Iba1ion- izedcalcium-bindingadap e molecule1,TREM2( igge ing ecep o  exp essedonmyeloidcells2),CD11b,andCD68.Theimmo alized humanmic ogliawascul u edinP ig owIIIMediumwi h10% e al bo ine se um in collagen- coa ed lasks. The medium was supple- men edwi h penicillin(100U/ml) and hecells we emain ained a  37°Cinahumidi iedincuba o wi h5%CO2. 2.2 | RNA isola ion and e e se ansc ip ion T iPu e Isola ion Reagen  (Roche, Basel, Swi ze land) was used o  RNAisola ion.RNAsampleswi hou  e e se ansc ip ionwe eused as henega i econ ol o qRT-PCR,ino de  oexcludeDNAcon- amina ion.Theisola edRNAwasused o  hecDNAsyn hesis.RNA (1000ng)was ansc ibedusing heT ansc ip o Fi s S andcDNA Syn hesisKi (Roche),whichwasappliedacco ding o hemanu ac- u e ’sins uc ions. ThecDNA (20μl) p epa ed om he o al RNA wasdilu edwi hRNase- eewa e  o100μl; 5 μl was hen di ec ly analyzedusing heLigh Cycle ®480IISys em(Roche). | 3 o 9 ROY e al. 2.3 | Quan i a i e eal- ime polyme ase chain eac ion qRT- PCR was pe o med using TaqMan gene exp ession as- says and he Ligh Cycle ®480 II Sys em (Roche). The ampli ied DNAwas analyzed by he compa a i e C me hod using β- ac in asa e e encegene.Thep ime andp obese s o ACTB(assay ID: Hs99999903_m1) and MT- 3 (assay ID: Hs01921768_s1) we e selec ed om he TaqMan gene exp ession assays (Li e Technologies,USA).TheqRT-PCRwaspe o medunde  he ol- lowing ampli ica ion condi ions: o al olume o 20 μl,ini ialincu- ba ion a 50°C/2 min ollowed by dena u a ion a 95°C/10 min; hen45cyclesa 95°C/15sanda 60°C/1min.Allsampleswe e measu ed in duplica es. 2.4 | Immunode ec ion o MT- 3 Ex ac ion o o al p o ein om he cells was p ocessed using he Pie ce®RIPABu e (The moScien i ic,Rock o d,IL,USA)inacco d- ance wi h he p o ocol p o ided. All p o ein concen a ions we e de e mined wi h he Pie ce® BCA P o ein Assay Ki . P o ein sam- ples we e dilu ed o 10 μg/μl, hen 1μl was slowly spo ed on o a 0.2μmImmun- Blo ®PVDFmemb ane(BioRadLabo a o ies,He cules, CA,USA)andai -d ied.Toblocknonspeci icbindingsi es, hemem- b anes we e incuba ed using 5% Blo ing-G ade Blocke  (BioRad Labo a o ies) o  one hou  a  oom empe a u e. They we e hen incuba edusingAn i-MT-3an ibody(p oduc no.ab76618;Abcam, Camb idge,UK),andwi hAn i-β-ac in(p oduc no.ab8227;Abcam). The an ibody was dilu ed 1:1000 using 5% Blo ing-G ade Blocke  (BioRadLabo a o ies). The memb anes we e hen washed ou  imes in PBS (Sigma- Ald ich,S .Louis,MO,USA) o  i eminu eseach,be o ebeingin- cuba edwi ha seconda yan ibody (Pe oxidase-LabeledAn i-Rabbi  IgG(p oduc no.PI-1000;Vec o ,Bu lingame,CA,USA)).Thiswasdi- lu ed1:2000using5%Blo ing-G adeBlocke .A e washinginPBS, allblo swe e isualizedonpho osensi i e ilm.Densi ome icanal- yses o  he memb aneswe e pe o med using GeneSnap (Syngene, Camb idge,UK). 2.5 | Cy o oxici y es ing – MTT es The suspension o 5000 cells was added o each well o s anda d mi- c o i e pla es.A olumeo 200μlwas ans e ed owells2–11.The medium (200 μl) was added o he i s and he las column (1 and 12(con ol)). The pla es we e incuba ed o 2 days a 37°C o ensu e cellg ow h.Themediumwas emo ed omcolumns2–11.Columns 3–10we e illedwi h200μlo  hemedium,whichcon ainedanin- c eased concen a ion o es ed compound dilu ed in dime hylsul ox- ide(DMSO)(0–1μmol/l).Asacon ol,columns2and11we e illed wi h he medium wi hou es ed compound. Thepla eswe eincuba ed o  12and 24h , hen he medium was emo edand hecellswe ewashedinPBS.Columns1–11we e illed wi h 200 μlo medium,whichcon ained50μl o MTT (5 mg/ mlinPBS)incuba edinahumidi ieda mosphe e o  ou hou sa  37°Cand henw appedinaluminum oil.A e  heincuba ion, he MTT- con aining medium was eplaced wi h 200 μlo 99.9%DMSO in o de  o dissol e he MTT- o mazan c ys als. Subsequen ly, 25 μl o glycine bu e was added o all wells; abso bance was im- media ely de e mined o be 570nm (Ve saMax mic opla e eade , Molecula De ices,Sunny ale,CA,USA).Allsampleswe emeasu ed in duplica es. 2.6 | Con ocal mic oscopy and cell s aining Fo  hepu poseo  luo escencemic oscopy, hecellswe ecul i a ed di ec ly on mic oscope glass slides (75×25mm, hickness 1mm, MenzelGlässe ,B aunschweig,Ge many)inPe idishesin heabo e- desc ibedcul i a ionmedia(see“Cul u edcellcondi ions”).Thecells we e ans e eddi ec lyon o heslides,whichwe e hensubme ged in he cul i a ion media. A e  he ea men , hemic oscopeglassslideswi hamono- laye  o  cells we e emo ed om he Pe i dishes, insed in he cul i a ionmediumwi hou  supplemen a ionandPBS bu e and di ec ly used o s aining and luo escence mic oscopy. The cells we e incuba ed using he ollowing highly speci ic luo escen p obes: • Reac i eoxygenspecieswe e isualizedusingCellROXDeepRed eagen (Li eTechnologies, 5μmol/L, cell-pe mean ,li e-cells ain wi habso p ion/emissionmaximao 644/665nm) • Mi ochond ia we e isualized using Mi oT acke  G een FM (Li e Technologies,300nmol/L,cell-pe mean li e-cells ainwi habso p- ion/emissionmaximao 490/516nm) • The endoplasmic e iculum was isualized using ER-T acke  Red (Li eTechnologies,1μmol/L,cell-pe mean ,li e-cells ainwi hab- so p ion/emission maxima o 587/615 nm). A e  incuba ion (45min, 37°C, da k), he cells we e washed h ee imes wi h PBS bu e  (0.05mol/L, pH 7.0) and obse ed unde  he con ocalmic oscope(LeicaTCSSP8 X, Ge many) using app op ia eexci a ionandemissionwa eleng hs.Quan i a i eanal- ysis was pe o med ypically om 10 ields o iew. Fo image anal- ysis, NIS elemen s BRAnalysis (Nikon ins umen s, Tokyo, Japan) so wa ewasused.A e age luo escencein ensi ywasmeasu edin equally con luen ields o iew. Resul s we e compa ed s a is ically using - es s. 2.7 | Flow cy ome y analysis o cell dea h Double- s aining wi h luo escein iso hiocyana e (FITC)/p opidium iodide(PI)wasunde akenusing heAnnexinV-FLUOS-s ainingki  (RocheAppliedScience)acco ding o hemanu ac u e ’sp o ocols,in o de  ode e mine hepe cen ageso  iable,apop o ic,andnec o ic cells ollowing he exposu e o Plumbagin. B ie ly, hecellswe eha es edby epe i i epipe ingandwe e washed wo imes wi h PBS (cen i uged a  2000 pm o  5min), 4 o 9 | ROY e al. esuspended in 100 μlo Annexin-V-FLUOSlabelingsolu ionandin- cuba ed o 15minin heda ka 15–25°C.AnnexinV-FITCbinding wasde ec edby lowcy ome y(Pa ecGmbH,Müns e ,Ge many) (Ex=488nm,Em=533nm,FL1 il e  o Annexin-V-FLUOSandFL3 il e o PI). 2.8 | The modynamic s udies - MD simula ion The MM-PBSA (Molecula  Mechanics ene gies combined wi h Poisson–Bol zmannSu aceA ea)app oach,combinedwi hmolecu- la dynamics(MD)simula ions, iswidelyusedin biomolecula com- plexes as a way o es ima e he p o ein ligand in e ac ion ene gies and p edic binding- ee ene gies. I also e alua es he ela i e s abili ies o  di e en  biomolecula  s uc u es. Addi ionally, i  can be used o esco ease o dockedcomplexes, he ebyimp o ing heabili y o dis inguish be ween ac i e and inac i e lead molecules. The molecu- la dynamics simula ion o MT- 3 docked complexes was pe o med using he GROMACS 4.5.52 (Be endsen e al., 1995; Lindahl e al., 2001)package,wi has anda dGROMOS96 o ce ield o 10,000ps. Binding ee- ene gy calcula ions we e pe o med in his wo k o he ligand- bound complexes in o de o p o ide u he insigh in o he s udy. 3 | RESULTS 3.1 | The cy o oxic e alua ion o benzo hiazolone- 2- con aining subs ances in mic oglia cells The IC50 alue o  STOCK1N-26544 is 162.4nmol/L, o  STOCK1N-26929 i  is 100nmol/L and o  STOCK1N-72593 i  is 233.4nmol/L. Thus, STOCK1N-26929 shows he highes  e ec  on cell me abolic ac i i y. Ne e heless, he IC50 alues o  all es ed molecules exceed he equi ed concen a ion o he induc ion o MT- 3 exp ession by abou 10 imes (see Figu e 1). TheAnnexinV+/PI+(Q2quad an )depic sla enec osisanddying cells. The a e o he nec osis igge ed by es ed compounds did no exceed4%(onlyshowinga2.12%inc ease,compa ed o hes ained con ol). The Annexin V+/PI- (Q4) popula ion ep esen s apop o ic cells o ea ly onco ic cells (see Figu e 2). The la ges amoun o apop- o ico ea lyonco iccellswas oundin10nmol/LSTOCK1N-72593 a e  ea men  (19.93%; 12.31% inc ease compa ed o he s ained con ol). Ne e heless, some po ion o  onco ic cells is able o e- e seoncosisandcould he ebysu i e; hus,nose e einc easein he o al numbe o dying cells was ound a e ea men wi h es ed compounds (see Figu e 2). Con ocal mic oscopy e ealed no signi ican mo phological changes ha had been induced by s udied compounds. On he o he hand,weused wop obes olocalizemi ochond iaand heendoplas- mic e iculum.Whe easMi oT acke G eenFMlocalizesmi ochond ia ega dless o  mi ochond ial memb ane po en ial, ER-T acke  Red, a conjuga eo g een- luo escen BODIPY®TRdyeandglibenclamide, also e lec sexp essiono sulphonylu ea ecep o so ATP-sensi i e K+ channels and he eby e lec s he ER unc ion. The double- s aining e ealed no signi ican dec ease in luo escence co esponded o bo h mi ochond ia and ER o he es ed compounds. Compound 26544showedsomeabili y ogene a eROS,whichwe ecolocalized wi hmi ochond ia(seeFigu e3).Ne e heless,ROSp oduc ionwas a he weak. 3.2 | Benzo hiazolone- 2 enhances exp ession o MT- 3 in mic oglia cells mRNAexp essiono MT-3was ela i elylowincon olcellswi hou  ea men .All he h ee es edcompounds enhancedexp essiono  MT-3mRNAinbo h es edconcen a ions(5nmol/Land10nmol/L, espec i ely)(seeFigu e4a). Exp ession o MT- 3 p o ein was unde ec able in con ol cells wi h- ou  ea men .All he h ee es edcompoundsenhancedexp ession o  MT-3 p o ein (see Figu e4b). Ne e heless, compound 72593 caused isible enhancemen  only in 5nmol/L concen a ion. O he  compounds enhanced exp ession o MT- 3 p o ein in es ed concen- a ions(seeFigu e4c). FIGURE1 MTT es s o selec ed compounds on cell lines. The hal - maximal inhibi ion concen a ion (IC50) is indica ed in he g aph. E o ba s indica e s anda d de ia ions | 5 o 9 ROY e al. 3.3 | The modynamic s udies on MT- 3- benzo hiazolone complexes Binding ee- ene gy s udies ha alida e he abo e indings we e also pe o med.The analysis was ca ied ou  o  he Benzo hiazolone-2 docked complexes only, since he abo e esul s we e encou ag- ing.The molecula  docking ep esen a ion is p o ided in Figu e S1. I  clea ly s a es ha  he es ed molecules (STOCK1N-26929 and STOCK1N-26544) bo h ha e s able binding ee-ene gies alues in he o mo po en ial,pola ,andnonpola sol a ionene gies ha a e compa able o he s anda d con ols (Table 1). I also s a es ha bo h hyd ophobic and elec os a ic in e ac ions a e impo an o p o ein ligandin e ac ionincaseo MT-3 o Alzheime ’sdisease. 4 | DISCUSSION The possibili y o enhance he exp ession o MT- 3 o p o ec i om deg ada ion is an a ac i e he apeu ic a ge , because low le elso MT-3we e oundinADb ains.In hiss udy,we es edan FIGURE2 Flowcy ome icanalysiso apop osis/nec osisbyAnnexinV/P opidiumiodide(PI)s aining.(a)S ainingo con ols(no s ained andno  ea ed,ands ainednon ea ed).(b)Flow-cy ome icanalysiso celllines ea edwi h5and10nmol/Lo  es edagen s.No e he di e encesinAnnexinV+/PI+(Q2)andAnnexinV+/PI-(Q4)popula ions 6 o 9 | ROY e al. FIGURE3 (Fluo escencemic oscopyanalysiso mi ochond iaandendoplasmic e iculum(ER).ERiss ainedwi h edER acke , mi ochond ia)[MR1]wi hg eenMi oT acke .Reac i eoxygenspecieswe e isualizedusingCellROXDeepRed eagen | 7 o 9 ROY e al. enhancemen o MT- 3 cellula concen a ion a e MT- 3 binding ea men ,whichcouldp e en MT-3deg ada ion. The h eecompoundsunde in es iga ionwe eSTOCK1N-26929, STOCK1N-26544 (Benzo hiazolone-2 as agmen ), and STOCK1N- 72593 (modi ied yp amine- based). The exp ession o MT- 3 p o ein was unde ec able in con ol cells wi hou  ea men .All h ee es edcompoundsenhancedconcen a- iono MT-3p o einincellsandsu p isinglyalsomRNAconcen a- ion.Ne e heless,STOCK1N-72593was ela i elylesse ec i e. I is o be no ed he e ha di e en concen a ions o es ed compounds can in luence exp ession o MT- 3 di e en ly. Epigene ic mechanisms and di e en ypes o ac i a ion o signaling pa hways a e suspec ed in hiscase.Fu he mo e, heexp essiono MT-3couldbe egula ed a  he pos ansc ip ional le el, which was also obse ed in ea lie  s udies(Ga e e al.,2005).Theexpe imen sca iedou didno as- sume ha igge ing o MT- 3 exp ession should be in posi i e co - ela ion wi h es ed compound concen a ion. The esul s showed ha ou compounds a e able o in luence MT- 3 exp ession. I is o be men ion ha ou s udy is no o ien ed owa d exac mechanism o  oshowexac quan i ieso MT-3p o ein.Inou opinion, woo  ou compoundssigni ican lyenhancedMT-3p o einandmRNAle els. The h ee es edmolecules(STOCK1N-26544,STOCK1N-26929, and STOCK1N-72593) ha e no se e e cy o oxic e ec s on neu al issue(Figu e4).IC50 alueso  es edmoleculesexceededabou 10 imes he concen a ion ha was needed o induc ion o MT- 3 ex- p ession (Figu e 1). Ou goal o he p ecise expe imen is he assess- men o IC50 alue. The indings also demons a e ha concen a ions o  es edcompounds,whicha eable o igge MT-3syn hesis,a eno  oxic. The expe imen s ca ied a e no ocused owa d inhibi ion o mi- c oglia.Ou compoundsenhancedapop osisandnec osis,bu i was no o se e ee ec .Abou 80%o cellswe es ill iable.The a eo  henec osis igge edby es edcompoundsdidno exceed4%(only showinga2.12%inc ease,compa ed o hes ainedcon ol).Thela g- es amoun o apop o ico ea lyonco iccellswas oundin10nmol/L STOCK1N-72593 a e  ea men  (19.93%; 12.31% inc ease com- pa ed o he s ained con ol). Flow cy ome ic analysis on apop osis/nec osis showed ha a lowe dose(5nmol/L)inducedahighe  oxice ec whencompa ed wi hhighe doses 10nmol/L.Thisinc easewasno  he se e eone (onlyabou 4%).Epigene icmechanismsanddi e en  ypeso ac i- a iono signalingpa hwaysa esuspec edin hiscase.Aswasshown in o he  s udies, mechanisms o  d ug-induced cell dea h a e o en concen a ion dependen . C oss alk be ween apop osis, nec osis, and au ophagy could be di e en in di e en d ug concen a ions and numbe s o apop o ic cells could be a enua ed because o some p o ec i eau ophagye ec s(Kemp,2017;Miyoshie al.,2008;Teng e al.,2016;To es&Ho wi z,1998). Acco ding o luo escences aining,nose iousROSgene a ion and no se e e dec ease in mi ochond ia numbe s o endoplasmic e iculum changes a e es - ea men we e ound. This is e y impo an ,(becauseoxida i es ess,mi ochond ialdys unc ion,and endoplasmic e iculum s ess ha e been implica ed in be a- amyloid neu o oxici y). The abo e indings we e u he alida ed by calcula ing he bind- ing ee- ene gy o he docked complexes h ough molecula dynamics s udies.TheMM-PBSAme hoduses h eeene ge ic e msino de  o FIGURE4 Exp essionanalysiso MT-3gene.(a)mRNA exp ession oldchangeincelllines ea edwi h0,5,and10nmol/L o selec ed compounds. (b) Do blo o MT- 3. (c) (P o ein le el o MT- 3 based on do blo de ec ion; displayed as mean and s anda d de ia ion) 26544 26929 72593 5 nM conc. MT3 b-ac in subs ance 10 nM 5 nM 10 nM 5 nM 10 nM con ol MT3 p o ein AUC 5 nM 10 nM 2654426929 72593SV40 0 5000 10000 15000 20000 Gene exp ession old 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 ee- ene gy calcula ion o he es ed compounds and he s anda d con ols Ligand Van de Waal ene gy (kJ/mol) Elec os a ic ene gy (kJ/mol) Pola sol a ion ene gy (kJ/mol) SASA ene gy (kJ/mol) Binding ene gy (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 o 9 | ROY e al. calcula e changes in he ee- ene gy on binding. The g_mmpbsa ool de elopedbyKuma ie al.(2014),Bake e al.(2001)(h p:// ashmi- kuma i.gi hub.io/g_mmpbsa/)isused ocalcula e heMM-PBSA om eachcomplexescombinewi hGROMACS.Theexpe imen was un o  1000ps; se e al he modynamic pa ame e s (including RMSD, RMSF, SASA, and H-bond) ha e al eady been epo ed (Roy e al., 2015).g_mmpbs e chesin o ma ion om heGROMACS ajec o y ileandcalcula es he o al ee-ene gyo  hep o ein-ligandcomplex, aswellas he ee-ene gyo p o einandligand,indi idually.I alsocal- cula es he po en ial ene gy o molecula mechanics and ee- ene gy o sol a ion excep en opy con ibu ion. In e ac ion ene gy o  h ee con ol molecules wi h STOCK1N compounds was calcula ed wi h he MT-3 ecep o , in o de  o compa e i  wi h he ac i e binde . BDBM50342769, BDBM9019, BDBM50260394 binding ene gies we e app oxima ely −96, −59, and −55kJ/mol. I  we look in o he indi idual componen  o  p e- dic ed ee-ene gy, an de  Waals ene gies we e dominan  in he BDBM50342769, BDBM50260394 binding wi h MT-3. Simila ly STOCK1N-26929 showed highe  an de  Waals ene gy as well as goodbinding ee-ene gycompa ed oSTOCK1N-26544(lowcon i- bu ion o an de Waals ene gy). This sugges s one o he majo MT- 3 binding in e ac ions is go e ned by he hyd ophobic o ces. The sec- ondmajo componen waselec os a icene gy,whichiscompa ably highe inSTOCK1Nmolecules hanincon ols. The o e all indings sugges ha hyd ophobic and elec os a ic in e ac ion is impe a i e o cu en MT- 3 ligand in e ac ion. Pola sol- a ion and nonpola sol a ion ene gies a e compa able o he s anda d con ols (Table 1). 5 | CONCLUSIONS The cu en wo k has made an e o o see he e ec o Benzo hiazolone-2on hecellula concen a iono MT-3.Manys ud- iesshow ha MT-3mRNAisdown egula edinADb ainsand ha  hismigh , he e o e,con ibu e o heupsu geo abno malneu onal de elopmen associa ed wi h he disease. Ou  p e ious indings sugges s ha  Benzo hiazolone-2 pos- sesses be e pha macokine ic and pha macodynamics p ope ies. I also o ms s able complex wi h MT- 3 acco ding o molecula dy- namics indings. The abo e indings we e alida ed by sc eening he compounds agains  li e immo alized human mic oglia cells (SV40). This es ed compound showed nei he se e ed cy o oxici y no did i  igge nec osis.Inaddi ion, he ewasnose iousROS-gene a ion, dec easeinmi ochond ialnumbe s,s ess-inducedendoplasmic e ic- ulum changes a e es - ea men s.All h ee es edcompoundsen- hanced cellula concen a ion o MT- 3 p o ein in cells and su p isingly alsomRNAexp ession. ACKNOWLEDGMENTS This wo k was suppo ed by he Minis y o  Educa ion, You h and Spo so  heCzechRepublicunde  hep ojec CEITEC2020(LQ1601) andbySpeci icUni e si yResea chg an s–MUNI/A/1355/2016and MUNI/A/1401/2016–p o idedby heMinis yo Educa ion,You h andSpo so  heCzechRepublicin heyea 2017. 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