Ligand fishing using new chitosan based functionalized AndrogenReceptor magnetic particles

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ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Ligand

ﬁshing

using

new

chitosan

based

functionalized

Androgen

Receptor

magnetic

particles

Michał

Piotr

Marszałł

a,∗

_,

_Wiktor

_Dariusz

_Sroka

a

_,

_Adam

_Sikora

a

_,

_Dorota

_Chełminiak

b

_,

Marta

Ziegler-Borowska

b

_,

_Tomasz

_Siódmiak

a

_,

_Ruin

_Moaddel

c

a_Nicolaus_Copernicus_University_in_Torun,_Ludwik_Rydygier_Collegium_Medicum_in_Bydgoszcz,_Department_of_Medicinal_Chemistry,_ul._Jurasza_2,_85-089

Bydgoszcz,Poland

b_Nicolaus_Copernicus_University_in_Torun,_Faculty_of_Chemistry_Gagarina_7,_87-100_Torun,_Poland

c_Laboratory_of_Clinical_{Investigation,}_National_Institute_on_Aging_Intramural_Research_Program,_National_Institutes_of_Health,_Baltimore,_MD,_USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13November2015

Receivedinrevisedform17March2016 Accepted12April2016

Availableonline4May2016

Keywords: Androgenreceptor Antiandrogens Chitosan Ligandﬁshing Magneticbeads

a

b

s

t

r

a

c

t

Superparamagneticnanoparticleswithchemicallymodiﬁedchitosanhasbeenproposedasapotential supportfortheimmobilizationoftheandrogenreceptor(AR).Thestudyinvolvedcomparisonofdifferent ARcarrierslikecommerciallyavailablemagneticbeadscoatedwithsilica(BcMag)andchitosancoated nanoparticleswithdifferentamountofaminogroups.Theimmobilizationwascarriedoutthrough cova-lentimmobilizationoftheARthroughtheterminalaminogrouporthroughavailablecarboxylicacids. TheinitialcharacterizationoftheARcoatedmagneticbeadswascarriedoutwithdihydrotestosterone, aknownARligand.Subsequently,chitosanmodiﬁednanporticleswithlong-distancedprimaryamino groups(Fe3O4CS-(NH2)3)(upto8.34mM/g)wereusedforfurtherstudytoisolateknownARligands

(bicalutamide,flutamide,hydroxyflutamideandlevonogestrel)fromamixtureoftestedcompoundsin ammoniumacetatebuffer[10mM,pH7.4].Theresultsshowedthattheselectednanoparticlesarea promisingsemi-quantitativetoolfortheidentificationofhighaffinitycompoundstoARandmightbeof specialimportanceintheidentificationofnovelagonistsorantiandrogens.

1. Introduction

Androgenreceptor(AR)isaligand-dependenttranscription fac-torthatcontrolstheexpressionofspecificgenesandisamember ofthenuclearreceptor(NR)superfamily[1].Themechanismby whichandrogenselicittheiractionsondifferenttissueshasbecome clearer over the years. The AR protein has3 major functional domains: a variable N-terminal domain (NTD), a highly con-servedDNA-bindingdomain(DBD)andaconservedligand-binding domain(LBD). Testosteroneand dihydrotestosterone (DHT) are endogenoushormoneswhichbindtotheligand-bindingdomain andactivateARasatranscriptionfactor.Adecreasein circulat-inglevelsofthesehormonesresultsinadeclineinmusculoskeletal function,increaseinbodyfat,decreaseinmusclemassandstrength andrecentlyhasbeenidentifiedasariskfactorforAlzheimers dis-ease[2,3].Asaresultthereisapressingneedfortheidentificationof newpotentialandrogens.While,hormonereplacementtherapyis available,itisnotwidelyusedduetopotentialsideeffects.In

addi-∗ Correspondingauthor.

E-mailaddress:mmars@cm.umk.pl(M.P.Marszałł).

tiontoandrogens,theidentificationofantiandrogenscanbeused forthetreatmentofprostatecancerinmenandhyperandrogenism inwomen[4].Forthisreason,theidentificationofantiandrogens isalsooftherapeuticinterest.Currentantiandrogenstherapiesare usedtotreatprostatecancer,includebicalutamide,flutamideand nilutamidehowever,thesedrugs haveadverseeffects and drug resistance[5].

Severalmethodshavebeendevelopedfortheidentificationof novelligandsfortheAR.Forexample,high-throughputscreening techniques,wherearapidassayiscarried outtodeterminethe biologicalorbiochemicalactivityofalargenumberofdrug-like compounds,hasbeenusedtostudytheinteractionsofendocrine disruptingchemicalswiththeAR[4].Asaresult,HTSisusefulfor thediscoveryandidentificationofnewselectiveandrogenreceptor modulators[6,7].Anothermethoddevelopedfortheidentification ofnovelARligandsistheARbinding-screeningassays.Thiswas alsodevelopedtocharacterizereceptormediatedendocrine activ-itybymeasuringtheinhibitionof ARtranscriptionalactivityby smallmoleculesormeasuringtheblockadeofligand-bindingAR [8,9].While useful,thesebiochemicalassays arelimitedbylow purityandstabilityoffunctionalARprotein.Therearealso com-merciallyavailableassays,includingtheARcompetitorassayswith http://dx.doi.org/10.1016/j.jpba.2016.04.013

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130 M.P.Marszałłetal./JournalofPharmaceuticalandBiomedicalAnalysis127(2016)129–135

ﬂuorescentorradiolabeledandrogenligand[10–12],however,due tothelownumberlabeled-ligands,wastedisposalandsafetythey havesomelimitationascommontoolsindrugdiscovery.

Anoveltechniquethathasbeensuccessfullyusedforthe iden-tificationofactivecomponentsfromacomplexmixtureisligand fishing[13–15].Inthesestudies,thetargetedproteinis immobi-lizedontomagneticparticlessurfaceandtheresultingstationary phaseisusedto“fish”outpotentialactivecompoundsfrom differ-entmixtures[11,13,16–18].Thishasbeenpreviouslydemonstrated withitsapplicationfor theisolation ofactive compoundsfrom naturalproductsfromdifferentmatrixesincludingplantandcell extracts[12,15,19–21].

Several types of magnetic beads have been employed for biomoleculeimmobilizationand drugdelivery[13,22].Recently, therehasbeenanincreasinginterestinmagnetite(triiron tetraox-ide) based micro- and nanoparticles for theirpotential use in differentbiomedicalandchemicalareas,becauseoftheir“flexible” surface.Thechemicallymodifiedsurfacewithinorganicororganic moleculesisresponsibleforoxidativestabilizationaswellas func-tionalizationofparticles.Thepolycationicpolymer,chitosan(CS), hasrecently,becomeaninterestingmaterialduetoitsnon-toxicity, goodmechanicalproperties,biocompatibility,and biodegradabil-ity[23].Asaresult,CSbasedmagneticnanoparticleshavegained increasedattentionasauniversalcarrierfordrugdeliveryandfor enzymeimmobilization[24,25].However,thispolymerdoesnot exhibitsufficientchemicalstabilityinaqueousenvironment.Asa result,anovelmagnetitenanoparticlecoatedwithamodified chi-tosanmaterialwassynthesized,specificallydesignedforenzyme (lipase)and protein (human serum albumin, HSA) immobiliza-tions[26].Theresultingcoatedsurfacewithchemicallymodified chitosanandlong-distancedprimaryaminogroupsgives riseto dispersioninorganicandwatersolventsasit isunabletoform intramolecularhydrogenbinding.Thus,thehydrophobicsurface hassignificantinfluencesoncatalyticactivityoftheimmobilized enzymese.glipases[27].Hence,thesenanoparticleswerestudied aspotentialmagneticsupportsforAR-proteinimmobilization. 2. Materialsandmethods

2.1. Materials

Androstanedione,bicalutamide,dexamethasone, dibutylphtha-late, dihydrotestosterone (DHT), ﬂutamide, hydroxyﬂutamide, levonorgestrel, lidocaine hydrochloride, 1-ethyl-3-(3-methylaminopropyl)carbodiimide (EDC), gluteraldehyde, hydroxylaminehydrochloride,N-hydroxysulfosuccinimide (Sulfo-NHS), potassium phosphate dibasic, pyridine (99.8%), sodium azide, sodiumcyanoborohydride, sodiumchloride, trizmabase, calciumchloride,glycerolandsodiumphosphatemonobasicwere purchasedfromSigma–Aldrich(Stainhaim,Germany).HPLCgrade Acetonitrile(ACN)and methanol(MeOH)werefromPOCh (Gli-wice,Poland).Humanandrogenreceptor(AR)fulllengthprotein wasfromAbcam(Cambridge,UK).

Commercially available amine terminated magnetic beads (BcMag)(50mg/mL,1␮mdiameter)of“sophisticallycoatediron oxideparticlestoprovideprimaryaminogroups”,werepurchased fromBioclonInc.(SanDiego,CA,USA).Amanualmagnetic sepa-ratorDynalMPC-SwaspurchasedfromInvitrogen(Carlsbad,CA, USA).Solutionswerepreparedusingpuriﬁed waterusedinthe studyusingaMilli-QWaterPuriﬁcationSystem(Millipore,Bedford, MA,USA).

2.2. Synthesisofchitosanmagneticnanoparticles

The3groupsofmagneticnanoparticleswerepreparedbased ontheFe3O4,coatedwithchemicallymodiﬁedchitosanand

char-acterized by ATR-FTIR, 13C NMR, TGA/DTG/DSC, as previously described [21]. All of chitosan coated nanoparticles were pre-paredviastandardco-precipitationprocedure.Toobtainonelong aminesubstituentinchain,thecoatedchitosanwasreactedwith gluteraldehydeandaqueoussolutionofethylenediamine. Materi-alscontainingtwoandthreeaminesubstituentswerepreparedby thereactionofchitosanwithepichlorohydrininalkalisolutionto formcarbonylgroupswhichweretreatedwithglutaraldehydeand ﬁnallywithethylenediamine[28].

Theresultingmagneticmaterialswithsurfacemodiﬁed with long-distanced amino groups were used as a support for AR-bioligands binding. In Fig. 1 the magnetic nanoparticles (Fe3O4 CS-(NH2),Fe3O4 CS-(NH2)2andFe3O4 CS-(NH2)3)with

a1,2or3aminogroupsdistancedfromthepolymerchainwere presented.

2.3. ARimmobilizationontothesurfaceofmagneticbeads

Thefollowingmagneticbeads(MB)wereusedinAR-bioligands bindingstudy:(a)commerciallyavailableamine-terminated mag-neticbeads(BcMag)withfunctionalgroupdensityof∼250␮mol/g ofMBand(b)chitosancoatedMBwithsurfacemodiﬁedwith long-distancedaminogroups−Fe3O4 CS-(NH2),(c)Fe3O4 CS-(NH2)2

andd)Fe3O4 CS-(NH2)3 withtheamountoffreeaminegroups

3.15, 5.93and 8.34mM/g,respectively. TheAR-fullprotein was immobilizedusingpreviouslyprotocolwithmodiﬁcation[16]. 2.3.1. ImmobilizationviaN-terminal

TheaminegroupsontheBcMagbeadsandtheARwerelinked byapreviouslydescribedmethod[18],withslightmodiﬁcations. Brieﬂy,5mgofMBwerewashedwith1mLpyridinebuffer[10mM, pH6.0]inmicrocentrifugetube.Thesupernatantwasdiscardedand MBweresuspendedin1mLof5%gluteralaldehydeandshakenfor 3h.TheMBwerethenwashed3timeswith1mLpyridinebuffer [10mM,pH6.0]toremovetheunreactedgluteraldehyde.200␮Lof bufferwastransferredwith200␮goffulllengthARproteintothe MBandleftundergentlerotationat4◦Cfor24h.Next,the super-natantwasdiscardedand0.5mLofglycine(1M,pH8)wasadded. Themixturewasshakenfor30minandsupernatantdiscarded.The resultingMBwasrinsedandstoredinphosphatebuffer[10mM, pH7.4]with0.02%sodiumazide.Thecontrolglycine-coatedMB weremadeforeach groupofparticlesinthesamemannerbut withoutARprotein.Finally,the4groupsofMBwerepreparedby immobilizationofARproteinontothesurfaceofMBthroughthe aminogroup:(a)BcMag(NT)-AR,(b)Fe3O4 CS-(NH2)(NT)-AR(c)

Fe3O4 CS-(NH2)2(NT)-ARand(d)Fe3O4 CS-(NH2)3(NT)-AR.

2.3.2. ImmobilizationviaCOOH group

Fortheimmobilizationofthe COOHgroup,wefolloweda pre-viouslypublishedmethodwithslightmodifications[16].Briefly, 5mgofMBaftertherinsingwith1mLofMES[100mM,pH5.5]in amicrocentrifugetubeweresuspendedin300␮Lofrinsingbuffer, 200␮goffulllengthARproteinand50␮Lofamixtureof10mg ofEDCand15mgofsulfo-NHSin1mLofwater.Themixturewas vortex-mixedandleftfor3hat4◦Cwithgentlerotation.Next,the 20␮Lof1Mhydroxylaminewasaddedtothefinalreactionandleft for3hat4◦Cwithgentlerotation.Thesupernatantwasdiscarded andtheMBwithimmobilizedARproteinwererinsed3timeswith 1mLofphosphatebuffer[10mM,pH7.4]containing0.02%sodium azide.Thecontrolhydroxylamine-coatedMBweremadeforeach groupofparticles inthesame mannerbutwithoutARprotein. Finally,the4groupsofMBwerepreparedbyimmobilizationof ARprotein ontothe surfaceof MBthrough thecarboxy group: BcMag(C)-AR,Fe3O4 CS-(NH2)(C)-AR(c)Fe3O4 CS-(NH2)2(C)-AR

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Fig.1.StructureofpreparedFe3O4CSnanoparticles(a)anddifferentmodiﬁcationsofchitosan:witha1(b),2(c)or3(d)aminogroupsdistancedfromthepolymerchain.

2.4. Ligandﬁshing

5mg ofthe ARcoatedbeads or controlwassuspended in a microcentrifugetubewith10nMoftheselecteddrugs(Table1) andincubatedin500␮Lofammoniumacetatebuffer[10mM,pH 7.4]individually.Thetubewasmixedfor2minandthenplaced formagneticseparatorfor30s.Theﬁrstsupernatant(S1) includ-ingnon-bindingcompoundswascollected.Thebeadswerethen washed with 500␮L of ammonium acetate buffer[10mM, pH 7.4]for 2minandthesupernatant (S2)wasseparated and col-lected.Theboundligandswerethenelutedwith500␮Lofelution buffer(ammoniumacetatebuffer[10mM,pH7.4])containing20% methanol(v/v twicefor 2min(S3 andS4). Themagneticbeads washingprocedurebetweenextractionsconsistedofadoublewash with500␮Lofammoniumacetatebuffer[10mM,pH7.4]for2min. Thebindingtestswereperformedindividuallyforeachcompound andtoanequalmixtureofDHT,bicalutamide,dexamethasoneand lidocaine.

ThecollectedsupernatantswereanalyzedbyShimadzuUPLC Nexera X2 system consisting of: LC-30AD pomp with Kinetex C18, 150×4.6mm, 2.6u, 100A column, SIL-30AC autosampler, CTO-20AC column oven, FCV-20-AH2 valve unit and DGU-20A5RdegassercoupledwithShimadzu8030ESI–MS/MStriple quadrupolemassspectrometer.Mobilephaseconsistedof(A)0.1% formicacidinmethanol(v/v)and(B)0.1%formicacidinwater (v/v).ProportionsbetweensolventAandsolventBwere60–40. Flowratewassetto0.8mL.min−1,chromatographictemperatureat 35◦Cwithmonitoredpressurebelow500bar.Sourceandcollision

energiesaswellasfragmentorvoltageswereoptimizedforeach analytebyinfusingapurestandardofeachcompounddissolved in 50%methanol.Sourceconditionsweresetto:nebulizing gas flow1.5L/min,dryinggasflow15.0L/min,DLtemperature250◦C, heat blocktemperature400◦C.Injectionvolume,precursor and productsions,ionizationmodeofeachcompoundarepresented inTable1.Allanalysesofsupernatantweredoneafterincubation processintriplicate.Thesemi-quantificationwascarriedoutby meansofsignalratioofincubatedligandsinsupernatantto sig-nalofpurestandard.Thecalculatedstandardcurvesforalltested compoundswerelinearwithcorrelationcoefficientsgreaterthan 0.9932withalinearrangefrom0.1nMto10nM.

3. Resultsanddiscussion

CovalentimmobilizationoftheAR-proteinwasperformedon thesurfaceofamineterminatedmagneticbeadsbytheformation oftheamidebound((C)-AR)orreductiveamination((NT)-AR)ofa carboxylgrouportheaminoterminal,respectively.TheAR-protein wasimmobilizedonto4differenttypesofiron-oxide multifunc-tionalmagneticnanomaterialby2linkers,resultingin8typesof AR-proteinmagneticbeads(4(NT)-ARand4(C)-AR)(section2.3). InordertodeterminewhichARcoatedmagneticparticleresultedin moreefficientimmobilization,10nMofdihydrotesterone(DHT),a knownARligand,wasincubatedwitheachofthe8magnetic parti-cles(4Cand4NT)versuscontrolfor2mininbufferaccordancewith protocolofligandfishing(section2.4)andanalyzedbyESI–MS/MS massspectrometer.Forthecontrolbeads,thenon-specific

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bind-132 M.P.Marszałłetal./JournalofPharmaceuticalandBiomedicalAnalysis127(2016)129–135

Table1

Structuralformulas,activityandanalyticalparametersofstudiedcompounds.

Compound Activity/function Injectionvolume(␮L) Ionizationmode Precursor[m/z] Products[m/z]

androgen 10 (+) 291.3 255.3 273.3 androgen 1 (+) 287.2 109.1123.0263.3 anti-androgen 1 (−) 429.0 255.0185.0184.0 anti-androgen 3 (+) 393.2 373.3147.0237.1 anti-androgen 10 (+) 279.2 149.0205.1121.0 anti-androgen 5 (−) 275.1 201.9205.0186.0 anti-androgen 1 (−) 291.0 205.0175.0155.0 androgen 10 (+) 313.3 109.1245.3135.0 non-binder 5 (+) 234.6 58.1 86.1

ingfora2minincubation withDHTresultedina similarrange

ofunboundDHTinthesupernatant(85.9–90.2%)andboundDHT

(S3+S4)<3%foralltypesofstudiedmagneticbeads.Forthe(C)-AR

magneticparticles,nodifferencewasobservedbetweenthe

(C)-ARandcontrolmagneticbeads(datanotshown),withthelevels

ofunboundligandsimilarforallthe(C)-ARandcontrolparticles

studied.Similarlyfor3ofthe4NT-AR(Fig.2a)andforall4

con-trols(Fig.2b)thelevelsof unboundligand(DHT)in S1ranged from80to90%,withtheexceptionoftheFe3O4 CS-(NH2)3

(NT)-ARmagneticparticles,wherethelevelsofsupernatants(unbound ligand)decreased to62.3% (Fig.2a).Of interest, when compar-ingthe chitosan magnetic particlesfor the 4-NT-ARs,was that withincreasingconcentrationsoffreeaminogroup,anincreasein retainedDHTwasobserved.Forexample,increasingtheamountof freeaminogroupsfrom3.15mM/g(Fe3O4 CS-(NH2))to5.93mM/g

(Fe3O4 CS-(NH2)2),resultedinasigniﬁcantincreaseofretained

DHTfrom5%to10%(S3+S4)(p<0.05)andadecreaseinunbound ligand(S1)from∼90%to80%,respectively.Similarly,increasing thefreeaminogroupsto8.34mM/g(Fe3O4 CS-(NH2)3),resulted

inasigniﬁcantdropinunboundligand(S1)to∼60%and a cor-respondingincreaseinretainedDHTto∼20%(p<0.0001).These resultsleadtotheassumptionthatthehigheramountoffreeamino

groupsresultsinahigheryieldofARimmobilizationviathe N-terminus.Similarobservationsweredescribedpreviouslywiththe useofnanoparticlesasasupportforthecovalentimmobilization oflipaseandhumanserumalbumin[21].Theresultsindicatethat magneticnanoparticlescoatedwithchemicallymodifiedchitosan withanincreaseinfreelong-distancedaminogroupscanimprove theimmobilizationprocessofbiomolecules.Theresultofinactivity ofthe(C)-ARisnotsurprising,astheARcontainsmultiple sur-facecarboxylicacidsthataresolventaccessibleforEDC-mediated crosslinking, whilethe (NT)-AR is specifictothe N-terminal of theprotein.Thisnon-specificityinthe(C)-AR,mayresultinthe immobilizationoftheproteinthatmayblockaccesstotheligand bindingsite,orcausestericeffectsthatmaypreventthebinding ofknownligands.Thedeterminationoftheoptimalcrosslinkerfor newproteinsanditsmolarratiosforreactionsisoftenempirically determined.

As a result, the Fe3O4CS-(NH2)3(NT)-AR nanoparticles were

usedforalltheligandfishingstudies.Theselectedmagneticbeads wereincubatedwitha seriesofknowncompoundswith differ-ent affinities to AR (Fig. 3). The results demonstrate that the highaffinitybinders,bicalutamide,flutamide,hydroxyflutamide and levonogestrel, were selectively retained on the Fe3O4

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CS-Fig.2.Comparisonofdihydrotestosterone(DHT)bindingtodifferentmagneticnanoparticleswitha)immobilizedAR-proteinvieaminegroupandb)controlnanoparticles; S1-S4—supernatantsdescribedinligandﬁshingprocedure.

Fig.3.Comparisonofbindingofandrostenedione,bicalutamide,dexamethasone,dibutylphthalate,flutamide,hydroxyflutamide,levonorgestrelandlidocaineHClto a)Fe3O4CS-(NH2)3(NT)-ARmagneticbeadsandb)controlnanoparticles;S1-S4—supernatantsdescribedinligandfishingprocedure.

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134 M.P.Marszałłetal./JournalofPharmaceuticalandBiomedicalAnalysis127(2016)129–135

Fig.4.ThemixtureligandﬁshingofDHT,bicalutamide,dexamethasoneandlidocaineHClwiththeuseofFe3O4CS-(NH2)3(NT)-ARmagneticbeads;S1-S4—supernatants

describedinligandﬁshingprocedure.

Table2

Thecomparisonvaluesofbindingparametersreportedinliteratureandmeasured inpresentedstudy. Compound Relative binding afﬁnitya_(nM) IC50b(uM) %ofligand bindingc Androstenedione 640 – 0.93 Bicalutamide – 12.0 14.76 Dexamethasone – 188.5 0.53 DHT 10 0.057 17.60 Dibutylphthalate 0.0017 – 1.75 Flutamide 0.0082 73.4 8.37 Hydroxyﬂutamide – 33.0 11.02 Levonogestrel 11.87 – 8.05 Lidocaine – – 0.65

a_Relative_binding_afﬁnities_(recombinant_rat_LBD)_[7]_.

b _IC

50measuredwithScintillationProximityAssay(recombinantratLBD)[8]. c _Percent_of_ligand_{binding–signal}_ratio_of_incubated_ligands_in_supernatant_to

signalofpurestandard.

(NH2)3(NT)-AR magnetic beads versus control (Fig. 3).Further,

incubationofFe3O4CS-(NH2)3(NT)-ARmagneticbeadswithother

compounds(androstenedione,dexamethasone,dibutylphthalate, lidocaine),resultedinnoretentioninS3andS4,indicating that thesecompounds were eitherweak binders or non-binders. In Table 2, a clear difference is seen by the% of ligand retained (S3+S4)betweenweakbinders/non-bindersandstrongbinders (0.96±0.54)%vs(11.97±4.14)%,respectively(p=0.0012).Further, alineartrend wasfoundbetweenreportedIC50 values andthe

amountof retained ligand (y=0.838+15.601, r2₌_0.9477),

indi-catingthatFe3O4CS-(NH2)3(NT)-ARmagneticbeadsarea viable

optionforscreeningofcomplexmixturestoisolateandidentify novelandrogensoranti-androgens.Inordertodeterminewhether the beads were able to sort binders from weak/non-binders simultaneously,amixtureofDHT,bicalutamide,dexamethasone and lidocaine wasincubated with theFe3O4CS-(NH2)3(NT)-AR nanoparticles(Fig.4).Aswasclearlydemonstrated,the Fe3O4CS-(NH2)3(NT)-AR nanoparticles had correctly retained DHT and bicalutamidewith12%and9%beingbound(S3+S4),respectively, and with less than 2% of dexamethasone and lidocaine, weak binders,beingretained,similartocontrol.While,someofthe com-poundsthatwerenotretainedbytheAR-coatedmagneticbeads, werenotreportedtobenon-binders,theliteraturedataonwhether acompoundisaweakAR-binderornon-binderisconﬂicting. Cur-rently,therecombinantARbindingassayisthemostcommonly usedmethodfordeterminingtheactivityofendocrineactive com-pounds,inthis methodthedatareportedismostoftenrelative totheappliedconditionsandasaresultisdifﬁculttocompareto classicbindingassay.Asaresult,basedonthereportedARbinding

propertiesofthetestedcompounds,intermsofrelativerankingof bindingaffinitiesofstrong,weakandno-marginalaffinity[8],our methodisonlycapableofdistinguishinghighaffinitybindersfrom weakbindersornon-binders(boundlessthan2%).

Thereportedresultsconﬁrmthatmagneticnanoparticlesmight beof specialimportanceinscreeningfornewpotentialligands. ThetypeofcoatingofFe3O4corehasthesigniﬁcantimpactonthe

effectiveimmobilizationofbiomolecules.ForAR-proteinthenew multifunctionalmagneticnanomaterialwithchemicallymodiﬁed chitosan(Fe3O4 CS-(NH2)3)wasdemonstratedtobeeffectivein

fishingoutligandswithhighaffinityfortheARfromamixtureof highaffinityandweak/non-binders.Thesubsequentdevelopment ofligandfishingtechniquebasedonthemagneticbeadsshould befocusedonthechemicalandphysicalmodificationofsurfaceof nanoparticles.Thiscanleadtomoreproductiveandeffectivetoolin newpotentialdrugscreening,evenwithweakbindingtotargeted protein.

Acknowledgments

Theprojectwassupportedbyresearchgrant:NationalScience Centre:DEC-2011/03/D/NZ7/02296.Thisworkwasalsosupported inpartbyfundsfromtheNIAIntramuralResearchProgram(RM).

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