B ain and Beha io . 2017;e00799.  
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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
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Re ised:14June2017
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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
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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).
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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),
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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
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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

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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
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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.
COMPETING FINANCIAL INTERESTS
We ce i y ha we ha e no a ilia ion wi h o inancial in ol e-
men wi hanyo ganiza iono en i ywi hadi ec  inancialo any
o he in e es in he subjec ma e o ma e ials discussed in he
manusc ip .
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SUPPORTING INFORMATION
Addi ional Suppo ing In o ma ion may be ound online in he sup-
po ing in o ma ion ab o his a icle.
How o ci e his a icle:RoyS,GumulecJ,Kuma A,e al.
Thee ec o Benzo hiazolone-2on heexp essiono 
Me allo hionein-3inmodula ingAlzheime ’sdisease.B ain
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