DNA interactions of monofunctional organometallic osmium(II) antitumor complexes in cell-free media.

J.Med.Chem.2008,51,3635–3643 3635 DNA Interactions of Monofunctional Organometallic Osmium(II) Antitumor Complexes in Cell-Free Media Hana Kostrhunova,† Jakub Florian,† Olga Novakova,† Anna F. A. Peacock,‡ Peter J. Sadler,§ and Viktor Brabec*,† InstituteofBiophysics,AcademyofSciencesoftheCzechRepublic,V.V.i.,Kra´loVopolska´ 135,CZ-61265Brno,CzechRepublic,Schoolof Chemistry,UniVersityofEdinburgh,WestMainsRoad,EdinburghEH93JJ,U.K.,andDepartmentofChemistry,UniVersityofWarwick, GibbetHillRoad,WarwickCV47AL,U.K. ReceiVedDecember10,2007 Thisworkisthefirstin-depthstudyofosmiumbindingtoDNAandconfirmsthepharmacologicalactivity ofanewclassofanticancermetallodrugs.Weinvestigatedtheinteractionsbetweenthepotentialbiological target DNA and four osmium(II) arene complexes, of the type [(η6-arene)Os(LL)Cl]n+, where arene ) biphenyl or p-cymene and LL ) ethylenediamine, picolinate, or oxinate in an effort to understand their mechanismofaction.Most notablyweshowthatthesecomplexesbindtoDNA.DNAadductsofthe OsII complexesthatexhibitpromisingcytotoxiceffectsinovariantumorcelllineslargelydistortitsconformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involvescombinedcoordinationtoguanineresiduesandnoncovalentinteractionsbetweentheareneligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin. Introduction Nevertheless, in spite of the difference in chemical structure, DNAbindinganddownstreamintracellulareffectsofcisplatin Platinum coordination compounds are widely used as anti- and organometallic RuII arene complexes, the formation and tumor drugs. The clinical efficacy of these anticancer drugs is processing of their DNA adducts leads in both cases to cell diminishedbyintrinsicandacquiredtumorresistanceandside death. effects.Owingtotheselimitations,thereisanintenseeffortto designnewtransitionmetal-basedcompoundscontainingtransi- Recently,arenecomplexesoftheheaviercongenerOsIIhave tion-metalionsotherthanplatinumthatarecapableofovercom- been designed, and their chemical and cytotoxic activity has ing problems associated with platinum chemotherapy while been described.4,5,9,10 Interestingly, some half-sandwich OsII delivering the therapeutic effect. Possible advantages in using arenecomplexesofthetype[(η6-arene)Os(XY)Cl]wherearene transition-metalionsotherthanplatinumincludetheavailability )p-cymene(cym)orbiphenyl(bip)andXY)N,O-chelating of additional coordination sites in octahedral complexes, the ligands such as picolinate (pico) showed promising activity alteredshapeofthecomplex,alterationsinligandaffinityand toward human lung and ovarian cancer cells.5 substitution kinetics, and changes in oxidation state. In the DNA is an important potential biological target for many design of these new drugs, ruthenium complexes1–3 and quite metal-basedanticanceragents.11DistortionsofDNAstructure recentlyalsoosmiumcomplexes4,5haveattractedmuchinterest. oftencorrelatewithanticanceractivity.2,12Hence,itisofgreat CertainRuIIarenecomplexesofthetype[(η6-arene)Ru(LL)- importance to understand in detail DNA binding properties of (X)][Z](whereLLisachelatingligandsuchasethylenediamine these new osmium complexes and their possible relationship (en)a,XaleavinggroupsuchasCl-,andZacounterion)exhibit to cytotoxicity in different tumor cell lines. This may provide both in vitro and in vivo activity, in some cases with activity groundsforestablishingnewstructure-pharmacologicalactivity comparable to that of cisplatin and carboplatin.6–8 Similar to relationships for this class of metal-based complexes as new conventionalcisplatin,theseRuIIarenecomplexespreferentially antitumor drugs. No work has been reported so far on the bindtoguanineresiduesofDNAformingmonofunctionalDNA reactivity of Os arene complexes toward polymeric DNA. To adductsthatarerecognizedandrepairedinthecellinamanner addresssomefundamentalquestionsaboutDNAbindingmodes different from the bifunctional DNA adducts of cisplatin.8 ofOsIIareneantitumorcompounds,theexperimentsdescribed in the present paper were carried out. More specifically, the *Correspondingauthor.Tel.:+420-541517148.Fax:+420-541240499. interactionsofpolymericB-DNAswith[(η6-arene)Os(XY)Cl] E-mail:brabec@ibp.cz. wherearene)p-cymorbipandXY)N,O-chelatingligands †AcademyofSciencesoftheCzechRepublic. pico or 8-hydroxyquinolinate (oxinate) and [(η6-bip)Os(en)- ‡UniversityofEdinburgh. Cl]BF (en)ethylenediamine;Figure1)incell-freemediawere §UniversityofWarwick. 4 investigated by various biochemical and biophysical methods aAbbreviations: bip, biphenyl; bp, base pair; cisplatin, cis-diam- minedichloridoplatinum(II);CT,calfthymus;DEPC,diethylpyrocarbonate; withthegoalofunderstandingtheirpromisingeffectsincancer dienPt, chloridodiethylenetriamineplatinum(II) chloride; EtBr, ethidium cell lines and to establish the foundations of structure- bromide; en, ethylenediamine; FAAS, flameless atomic absorption spec- pharmacologicalrelationshipsforthisclassofcytotoxicosmium t t r r a o t p io h n ot i o n m hi e b t i r t y in ; g H c P e L ll C g , ro h w ig t h h p b r y es 5 s 0 u % re ; l I i C qu P id OE ch S r , o i m nd a u to c g ti r v a e p l h y y c ; o I u C p 5 l 0 e , d c p o l n a c s e m n a - compounds. optical emission spectroscopy; oxine, 8-hydroxyquinoline; PAGE, poly- acrylamidegelelectrophoresis;p-cym,p-cymene;pico,picolinate;r b,the Results numberofmoleculesofthemetalcomplexboundpernucleotideresidue; ri,themolarratiooffreemetalcomplextonucleotide-phosphatesatthe Cytotoxicity.Thecytotoxicityofcomplexes1-4towardboth onsetofincubationwithDNA;t 50%,thetimesatwhichthebindingreached 50%;t m,DNAmeltingtemperature. cisplatin-sensitivehumanovarianA2780andresistant(A2780cisR) 10.1021/jm701538wCCC:$40.75 2008AmericanChemicalSociety PublishedonWeb05/22/2008 3636 JournalofMedicinalChemistry,2008,Vol.51,No.12 KostrhunoVaetal. Figure 1. Structures of OsII arene complexes. 1, [(η6-biphenyl)Os(ethylenediamine)Cl]+; 2, [(η6-biphenyl)Os(picolinate)Cl]; 3, [(η6-p- cymene)Os(picolinate)Cl];4,[(η6-p-cymene)Os(oxinate)Cl]. Table2. OsmiumandCisplatinUptakeinA2780Cellsa complex uptakeb cisplatin 11.4(0.2 1 13.3(1.0 2 14.0(1.2 3 34.9(2.3 4 31.6(1.8 aCellularosmiumandcisplatinaccumulationwasmeasuredbyICPOES after6hoftreatmentatequimolarconcentrationsoftheindicatedcompound. Eachpointrepresentsthemean(SEMforthreeindependentexperiments. bEachvalueshowninthistableisinpmoleOs(Pt)/106cells/µM. thehighestactivityincellssensitivetocisplatinandwith[η6- bip)Os(en)Cl]+(1)incellsresistanttocisplatin.Incontrastthe complex[η6-p-cym)Os(oxinate)Cl](4)wastheleastpotentwith IC values of 30 and 36 µM in sensitive and resistant cells, 50 respectively.Notablythesecomplexesshowsimilarpotencyin both the cisplatin-sensitive and resistant A2780 cell lines, indicating a different detoxification mechanism than cisplatin. Intriguingly, complexes 1 and 3 actually show higher activity in the cisplatin resistant A2780 cell line (5.0 and 5.6 µM, respectively)comparedtothecisplatinsensitivecells(9.0and 5.9 µM). Cellular Uptake. A factor that is usually thought to contributetometallodrugcytotoxicityiscellularuptake.To examineaccumulationofcomplexes1-4,thecellularlevels of these compounds were measured after a 6 h exposure of human ovarian A2780 cancer cells to equimolar concentra- tions of the drugs. The uptake of these compounds was comparablewiththatofcisplatinforcomplexes1and2and Figure 2. Dose response effects on the survival of A2780 (A) and approximately2-3timeshigherforcomplexes3and4(Table A2780cisR (B) cancer cell lines. The cells were exposed to the OsII arenecomplexesandcisplatinfor72hintheconcentrationrangeof0 2). to 128 µM. Cell death was determined by MTT assay. The drug KineticsofBindingtoCalfThymus(CT)DNA.Reactions concentrations causing 50% inhibition (IC ) were calculated. The of the cytotoxic complexes 1-4 with polymeric DNA were 50 resultsareexpressedasmean(standarddeviationsoffourindependent investigated, as binding to DNA is often associated with the experiments;allconcentrationsweretestedinthreereplicates. cytotoxic action of metal anticancer drugs.2,12 The rate of bindingoftheosmiumcomplexestoCTDNAwasdetermined Table1. InVitroGrowthInhibitionofHumanOvarianCisplatin at different ratios of r (molar ratio of free Os complex to i SensitiveandResistantA2780Cells,IC50(µM)a nucleotide phosphate), 0.05 and 0.1, in 10 mM NaClO 4 at 37 complex sensitive resistantb °C in the dark. The OsII complexes were incubated with the cisplatin 3.6(0.3 21.4(5.9) CTDNAandaliquotsremovedatvarioustimeintervals,rapidly 1 9.0(0.6 5.0(0.55) cooled,andprecipitatedoutbyadditionofethanolandtheOs 2 6.8(0.4 7.7(1.13) content of the supernatant determined by inductively coupled 3 5.9(0.4 5.6(0.95) plasmaopticalemissionspectroscopy(ICPOES).Thetimesat 4 30.3(0.9 36.3(1.2) whichthebindingreached50%(t )inthesebindingreactions aDrug-treatment period was 72 h. Each value represents the mean ( and total % bound after 48 h 50 c % an be found in Table 3. SEMforthreeindependentexperiments.bResistancefactor,definedasIC50 Intriguingly, complexes 2 [(η6-bip)Os(pico)Cl] and 4 [(η6-p- (resistant)/IC50(sensitive),isgiveninparentheses. cym)Os(oxinate)Cl] bind rapidly (t ca. 2 h) and almost 50% cancercelllineswasinvestigated.Allcomplexesshowedactivity quantitatively,whereasthecomplex[(η6-p-cym)Os(pico)Cl](3) (Figure2),andtheircorrespondingIC (concentrationinhibiting binds most slowly (t 4.9 and 8.3 h at r 0.05 and 0.1, 50 50% i cell growth by 50%) values are reported in Table 1. Similar respectively), and only ca. 75% is bound after 48 h. activitywasfoundforcomplexes1-3withIC valuesranging TranscriptionMapping.CuttingofpSP73KBDNAbyNdeI 50 from 5.0 to 9.0 µM, with [η6-p-cym)Os(pico)Cl] (3) showing andHpaIrestrictionendonucleasesyieldeda212-bpfragment OsIIAntitumorComplexDNAInteractions JournalofMedicinalChemistry,2008,Vol.51,No.12 3637 Table3. KineticsofBindingofOsmium(II)AreneComplexestoCalf ThymusDNAa t 50% b(h) 48h(%) t 50% b(h) 48h(%) atri )0.05 atri )0.05 atri )0.1 atri )0.1 1 2.1(0.2 76.0(0.7 4.6(0.2 72.1(0.8 2 1.8(0.2 98.5(0.6 2.1(0.2 94.8(0.7 3 4.9(0.2 76.8(0.8 8.3(0.2 71.8(0.8 4 0.9(0.1 87.2(0.7 1.6(0.1 84.9(0.6 aTheconcentrationofDNAwas32µg/mL.Eachvaluerepresentsthe mean(SEMforthreeindependentexperiments.bThetimesatwhichthe bindingreached50%. (asubstantialpartofitsnucleotidesequenceisshowninFigure 3B).ThisfragmentcontainedtheT7RNApolymerasepromotor. In vitro RNA synthesis by RNA polymerases on these DNA templatesmodifiedbyosmiumarenecomplexes1-4atthesame levelofmetalation(r )0.005)canbeprematurelyterminated b at the level or in the proximity of adducts (Figure 3A). Interestingly,monofunctionalDNAadductsofseveralplatinum complexes are unable to terminate RNA synthesis.13–15 The majorstopsites,primarilyguanineresidues,withsomeadenine bases,wereroughlyidenticalforallOscomplexes.Theprofiles aresimilartothatobtainedforDNAtreatedwiththeanticancer drug cisplatin (lane Cisplatin in Figure 3A) and also to those reported previously for the ruthenium arene compounds, such as [(η6-arene)Ru(en)Cl]+.16 The major stop sites for DNA modifiedby3aredemonstratedinFigure3B.Intriguinglythe distributionofthestopsitesproducedbybiphenylethylenedi- aminecomplex1isratherinfavorofshorterfragments,which is consistent with the view that the adduct of this complex presents the most difficult obstacle for RNA polymerase. ChemicalProbes.A21-basepair(bp)DNAduplex(forits sequence, see Figure 4B) was site-specifically modified with osmiumarenecomplexes1-3soastoformasinglemonofunc- tionalG-adductinthemiddleofthetop,pyrimidine-richstrand. TheduplexcontainingtheDNAadductofthep-cymeneoxinate osmiumcomplex4wasimpossibletoprepare,purifyandisolate apparently because of the instability of this adduct during the high pressure liquid chromatography (HPLC) purification process.Themetalatedduplexesweresubsequentlytreatedwith Figure 3. Inhibition of RNA synthesis by T7 RNA polymerase on the chemical agents KMnO , diethyl pyrocarbonate (DEPC), theNdeI/HpaIfragmentofpSP73KBplasmidmodifiedbyOsIIarene 4 complexesandcisplatin.(A)Autoradiogramof6%polyacrylamide/8 andbrominethatareusedastoolsformonitoringtheexistence M urea sequencing gel showing inhibition of RNA synthesis by T7 of conformations other than canonical B-DNA. These agents RNA polymerase on the NdeI/HpaI fragment containing adducts of react preferentially with base residues in single stranded and/ osmiumcomplexesandcisplatin.Lanes:control,unmodifiedtemplate; or in distorted double stranded DNA but not with the base cisplatin, 1-4, the template modified by cisplatin and OsII arene residues in intact, double-stranded DNA.17,18 The pattern and complexes 1-4 at r b ) 0.005, respectively; A, U, G, and C, chain degreeofreactivitytowardthechemicalprobeswereidentical terminatedmarkerDNAs.(B)Schematicdiagramshowingtheportion ofthesequenceusedtomonitorinhibitionofRNAsynthesisbycisplatin for the adducts formed by all three osmium arene complexes andosmiumcomplexes.ThearrowindicatesthestartoftheT7RNA 1-3 (Figure 4A), indicating a similar character of the confor- polymerase,whichwasusedasthetemplateintheupperstrandofthe mationaldistortion.TheresultsshowninFigure4alsosuggest NdeI/HpaI fragment of pSP73KB. The bullets represent major stop thattheadductsformedwiththeDNAcausedistortionswhich signals for DNA modified by 3. The numbers correspond to the extend4basepairsaroundtheadductandthatthesedistortions nucleotidenumberinginthesequencemapofthepSP73KBplasmid. aremorepronouncedinthebasepairscontainingthemetalated adduct and that containing the thymine residue flanking this site19anddienPtatthed(G)site,21keepsthehelixrodlike.We adductonits5′side(Figure4B).Alsointerestingly,theadduct have compared the electrophoretic mobility of the multimers of the complex [(η6-p-cym)Os(pico)Cl] (3) appears to distort oftheligated21-merduplex(foritssequence,seeFigure4B) DNAlessthantheadductsofthebiphenylcomplexes1and2. withandwithoutsinglemonofunctionaladductsofcomplexes Electrophoretic Mobility of Multimers of 21 bp Oligo- 1-3 formed at the central G residue in the top strand. The nucleotides.IntrinsicbendingofDNAduplexesresultsinthe correspondingmultimersexhibitvirtuallynogelmobilityshifts, abnormal electrophoretic mobility of DNA fragments. A gel migratingatalmostexactlythesamepositionsastheladderof migrationanomalyhasbeenfoundforDNAfragmentscontain- nonmodifiedmultimers(resultsnotshown).Wecan,therefore, ingbidentateadductsformedbycisplatinatthed(GG),d(AG), conclude that no bending is induced in DNA containing and d(GTG) sites.19,20 On the other hand, the monofunctional monofunctional osmium adducts of 1-3. binding of cis-[Pt(NH ) (Am)Cl]+ cations, in which Am is a UnwindingofSupercoiledDNA.Theunwindingofsuper- 3 2 derivativeofpyridine,pyrimidine,purine,oranilineatthed(G) coiled plasmid DNA induced on binding the four osmium 3638 JournalofMedicinalChemistry,2008,Vol.51,No.12 KostrhunoVaetal. Figure 5. Unwinding of supercoiled pUC19 plasmid DNA by the compounds 3 (top) and 4 (bottom). The plasmid was incubated with theOsIIarenecomplexesfor24hat37°C.Lanesinthetoppanel:1 and10,control,unmodifiedDNA;2,r )0.01;3,r )0.015;4,r ) b b b 0.021;5,r )0.028;6,r )0.032;7,r )0.036;8,r )0.04;9,r b b b b b ) 0.048. Lanes in the bottom panel: 1 and 12, control, unmodified DNA;2,r )0.02;3,r )0.03;4,r )0.05;5,r )0.08;6,r ) b b b b b 0.1; 7, r ) 0.14; 8, r ) 0.2; 9, r ) 0.25; 10, r ) 0.30; 11, r ) b b b b b 0.36. The top bands in each panel correspond to the form of nicked plasmid,andthebottombands,totheclosed,negativelysupercoiled plasmid. Table4. UnwindingofSupercoiledPlasmidDNAbyOsmium(II) AreneComplexesa Figure 4. Chemical probes of DNA conformation. (A) Piperidine- r b(c) Φ(deg)b induced specific strand cleavage at KMnO-modified, KBr/KHSO- 4 5 1 0.032(0.005 27(2 modified, and DEPC-modified bases in the 21-bp duplex (shown at 2 0.035(0.005 24(2 thebottomofthisfigure)nonmodifiedorcontainingsingle,site-specific monofunctionaladductof1-3.Lanes:ss,thenonmodifedstrand;ds, 3 0.040(0.005 21(2 4 >0.36 <2.5 the nonmodified duplex; 1, 2, 3, the duplex containing a unique monofunctionaladductof1,2,3,respectively;G,aMaxam-Gilbert aPlasmidwasincubatedwiththeosmiumcomplexfor24hin10mM specificreactionfortheunplatinatedduplex.Theoligomerswere5′- NaClO4 at 37 °C. Each value represents the mean ( SEM for three end labeled at either the top (left panel marked KMnO) or bottom independentexperiments.bTheunwindinganglewascalculatedasdescribed 4 strand (middle and right panels marked DEPC and KBr/KHSO, inthetext. 5 respectively).(B)Summaryofthereactivityofchemicalprobeswith the21-bpduplexcontainingsingle,site-specificmonofunctionaladduct of1-3.Closedandopencirclesdesignatestrongandweakreactivity, respectively. complexes1-4,respectively,wasdeterminedbyincubatingthe plasmidwiththeosmiumcomplexfor24hat37°Catvarious r (differentlanesinthegel).Theresultingelectrophoresisnative b agarosegelsofDNAmodifiedby3and4areshowninFigure 5(topandbottompanels,respectively)asexamples.Adecrease intherateofmigrationistheresultofunwindingtheDNAas this reduces the number of supercoils. The mean unwinding Figure6. Plotsshowingthedependenceof∆t valuesonr forCT m b angle is calculated from the equation Φ ) -18σ/r (c), where DNA modified by OsII arene complexes, 1 (∆), 2 (O), 3 (b), and 4 b σ is the superhelical density and r (c) is the r at which the (9).Themeltingcurvesweremeasuredin10mMNaClO 4 plus1mM b b Tris-HClwith0.1mMEDTA,pH7.4.∆t isdefinedasthedifference supercoiled and nicked forms comigrate.22 It can be seen in m betweenthevaluesofmetalatedandnonmodifiedDNAs.Datameasured Figure 5 (top) that the complex [(η6-p-cym)Os(pico)Cl] (3) intriplicatevariedonaverage(5%fromtheirmean. causes a significant unwinding of the DNA (Φ ) 21°, the comigrationpointofthemodifiedsupercoiledandnickedDNA, r (c), was reached at r ) 0.040, Table 4). In contrast, the MeltingTemperatureofModifiedCTDNA.CTDNAwas b b oxinate complex, [(η6-p-cym)Os(oxinate)Cl] (4), does not modified by the osmium arene complexes 1-4 at various r b unwind the DNA significantly, and the comigration point of values (0-0.1) in 10 mM NaClO . The effect on the DNA 4 the modified supercoiled and nicked DNA was not reached at melting temperature (t ) is dependent on the nature of the m ashighr as0.36(Figure5,bottom).Thedataaresummarized osmiumcomplexandtheamountofosmiumbound(r ),ascan b b inTable4,anditcanbeseenthattheunwindingisgreatestfor beseeninFigure6.IngeneralDNAmodifiedbyosmiumwas biphenyl ethylenediamine complex 1 (27°), followed by the destabilized and to a greater extent with increasing r . The b biphenyl picolinate complex 2 (24°) and p-cymene picolinate destabilizingeffectoftheOsIIarenecomplexesonDNAismore complex 3 (21°). The high level of unwinding induced by pronounced in the case of the p-cymene complexes (3 and 4) osmium arene complexes 1-3 is notable. than in the case of the biphenyl complexes (1 and 2). On the OsIIAntitumorComplexDNAInteractions JournalofMedicinalChemistry,2008,Vol.51,No.12 3639 Figure7. LineardichroismspectraofCTDNAmodifiedbyOsIIarenecomplexes.LDspectrawererecordedforDNAin10mMNaClO,20mM 4 NaCl,and10mMsodiumcacodylate,pH7.0.TheconcentrationofDNAwas0.1mg/mL.(A-D)LDspectraofCTDNAmodifiedby1(A)(thick solidline,control,nonmodifiedDNA;dashedline,r )0.008;dash-dottedline,r )0.035;solidline,r )0.07);2(B)(thicksolidline,control, b b b nonmodifiedDNA;dashedline,r )0.01;dottedline,r )0.05;dash-dottedline,r )0.1);3(C)(thicksolidline,control,nonmodifiedDNA; b b b dashedline,r )0.008;dottedline,r )0.037;dash-dottedline,r )0.075);and4(D)(thicksolidline,control,nonmodifiedDNA;dashedline, b b b r )0.01;dottedline,r )0.045;dash-dottedline,r )0.085).(E)PlotsoftheintensityofthebandinLDspectraat258nmofDNAmodified b b b bycomplexes1(∆),2(O),3(b),and4(9)versusr. b contrary, there is little difference between the complexes carrying the same axial ligand, either p-cymene or biphenyl. LinearDichroism.Bindingofallthreeosmiumcomplexes to CT DNA was also monitored by linear dichroism spectros- copy(Figure7).Itiswellestablishedthatthemagnitudeofthe LD signal measured within the DNA absorption band (e.g., at the258nmmaximum)isafunctionofitspersistencelength.It isknownthatchangesinflexibilities,ortheformationofrigid bends or kinks induced by strongly bound compounds, can manifestthemselvesasdecreasesintheabilitiesofthemodified DNAmoleculestoalignthemselvesinthehydrodynamicflow gradient of the LD cell. The magnitudes of the LD signals at 258nmdecreaseasafunctionofr forallOsIIarenecomplexes Figure8. PlotsoftheEtBrfluorescenceversusr b forDNAmodified 1-4 (Figure 7E). These results s b uggest that the formation of by cisplatin, dienPt, and OsII arene complexes in 10 mM NaClO 4 at 37°Cfor24h:(()cisplatin,(*)[PtCl(dien)]Cl,1(∆),2(O),3(b), stronglyboundadductsderivedfromOsIIarenecomplexesis and4(9).Datapointsmeasuredintriplicatevariedonaverage(3% accompanied by the appearance of flexible hinge joints at fromtheirmean. thesiteofthelesion.Anothereventuality,suchasappearance of rigid bends or kinks, is unlikely based on the results of gelelectrophoresisanalysisofmultimersofsite-specifically amineplatinum(II)chloride).Theadductsofallfourmonofunc- modifiedoligonucleotides(videsupra).Inaddition,treatment tionalOsIIarenecomplexescompetitivelyreplacedintercalated of the DNA with complexes 2 and 3 produces a new and EtBrmarkedlymoreeffectivelythantheadductsofmonofunc- weak positive band at 330 nm, which increases more tional dienPt. The adducts of biphenyl complexes 1 and 2 are significantlyforthebiphenylpicolinatecomplex2compared most potent. The adducts of the other two OsII p-cymene to the p-cymene picolinate complex 3. complexes reduced EtBr fluorescence less effectively but still slightly more than the adducts of bifunctional cisplatin. Ethidium Bromide (EtBr) Fluorescence. The ability of a complextodisplacetheDNAintercalatorEtBrfromCTDNA Discussion was probed by monitoring the relative fluorescence of the EtBr-DNA complex after treating the DNA with varying The four complexes investigated differ from each other in concentrationsoftheOsIIarenecomplexes1-4.Figure8shows thefollowingways.Firstofallcomplex1,[(η6-bip)Os(en)Cl]+, a plot of relative fluorescence versus r for complexes 1-4, is the only positively charged complex and after hydrolysis b cisplatin, and monofunctional dienPt (chloridodiethylenetri- (believed to activate the complex) would possess an overall 3640 JournalofMedicinalChemistry,2008,Vol.51,No.12 KostrhunoVaetal. positivechargeof+2.Clearlyitselectrostaticinteractionswith analogueof1,[(η6-bip)Ru(en)Cl]+,andinbothcasesisthought negativelychargedDNAwillbedifferentthanfortheremaining tobesignificantintheircytotoxicmechanismofaction(Figure threecomplexes.SecondthechelatedNH groupsincomplex 3). The major stopsites were guanine residues, which agrees 2 1 are capable of hydrogen bonding.23 In contrast, in the wellwiththesmallmoleculebindingstudiesperformedprevi- remaining complexes the N-donor group is a pyridine, which ously which show that complex 1 binds selectively to mono- isunabletotakepartinhydrogenbonding.Complexes2and3 meric guanine9 and complex 3 binds more strongly and differ from one another in that the arene varies, extended selectivelytomonomericguanineincompetitionexperiments.5 biphenyl arene in 2 (capable of intercalation in the adducts of The distortions induced on binding to DNA extend 4 base analogousRuIIarenecomplexescontainingasymmetricalN,N- pairs around the adduct for the three OsII arene complexes chelating ligand16) and a single ring arene with bulky substit- studied(1-3)(Figure4B),whichissimilarcomparedtoDNA uents (p-cymene) in 3. Complex 4 differs from 3 primarily in bindingofrutheniumareneanaloguescontainingmultiringarene thenatureoftheO-donorgroup,whichinthelattercaseisan ligands.16 aryloxide donor as opposed to the carboxylate group in 3. A Thesignificantresultobtainedfromtheligationexperiment majordifferenceisthepK a ofthischelatedoxygen(itsacidity) wasthatformationoftheDNAadductsofOsIIarenecompounds andthehigherpartialnegativechargedonatedtotheosmium. doesnotresultinDNAbending.Asthisbendingandsubsequent Clearly the chemistry of these four complexes is different,5,9 binding of HMG (high mobility group) proteins to damaged/ andconsequentlywewouldanticipatethattheireffectsonDNA bentDNAisthoughttoberesponsibleforthecytotoxicaction would be different as well. of cisplatin in tumor cells,12,26 we can conclude that the WereportinthepresentworkthefirstdetailedDNAbinding cytotoxic mechanism of action of OsII arene complexes is study of OsII arene complexes, which have been shown5,24 to different from that of cisplatin. be a potential new class of anticancer agents (Table 1). In The binding of OsII complexes 1-3 to DNA results in a addition, we investigated the effects and extent of changes significantlylargedegreeofunwinding(21-27°;Table4),much inducedintheDNAonbindingofosmiumandcomparedthese larger than that observed for the RuII complexes [(η6-ar- observations with other metal-based anticancer agents. ene)Ru(en)Cl]+ (7-14°)16 or cisplatin (6° and 13° for mono- Though we have previously reported the binding of these orbifunctionaladducts,respectively).22Similarlargeunwinding complexestonucleobases,4,5,9itisnotablethatthesecomplexes angles in the range of 17-30° have been observed for the all bind polymeric DNA. Binding to DNA has often been adducts of several antitumor platinum compounds containing associated with the cytotoxic action of metal-based anticancer heterocyclic planar or nonplanar ligands.27,28 Thus, the large agents,2,11,12 and therefore DNA may be a possible biological unwinding angles produced by the adducts of OsII arene target for this class of OsII arene complexes. The cell uptake compounds1-3canbeexplainedbytheadditionalcontribution studies (Table 2) also suggest that the type of DNA lesion is tounwindingassociatedwiththeinteractionoftheareneligand importantforactivitysince,despiteitslowactivity,thecellular with the duplex upon strong binding of osmium. Complex 4 levelsofOsfrom4arehigherthanthoseofcomplexes1or2. consistently behaves differently to the other OsII arene com- CT DNA was treated with osmium solutions, yet their plexes, which is similar to the reports of its aqueous solution subsequentratesofreactionwithDNA(Table3)donotcorrelate behavior (rapid hydrolysis and high acidity of coordinated with their rates of hydrolysis. Rates of irreversible binding to water). Most notably it was reported that the chelated oxygen DNAincreaseintheorder3(bindsmostslowly)<1∼2<4 atom is readily protonated about physiological pH, and a (rapid binding), whereas their rates of hydrolysis, previously dynamic pH-dependent ring-opening process at the osmium determinedat25°CandpH2,aresuchthat1hydrolyzesmost centerwasobserved.5Thissuggeststhatadductsof4onDNA slowly,followedby2andthen3,with4hydrolyzingtoorapidly would be less stable and the chelate ring opening would not tobemeasuredby1HNMR.4,5,9Thereforealthoughhydrolysis allowtheosmiumtoenforceanysignificantconstraintsonthe may be rate-determining for some complexes (e.g., 4) other DNA.Inotherwords,thearenemoietyinDNAadductsofOsII factors such as electrostatic interactions may also play a role. arenecompounds1-3couldbegeometricallywell-positioned The rate of binding to DNA compares well with that to interact with the double helix. In contrast, the oxinate determinedfortheanticancerdrugcisplatin(t ca.2hunder complex, [(η6-p-cym)Os(oxinate)Cl] (4), does not unwind the 1/2 similarconditions),25forwhichDNAbindingisthoughttobe DNA significantly (<2.5°). The explanation behind this phe- responsible for its cytotoxic properties. In contrast, the RuII nomenon is unclear, nevertheless it may be hypothesized that analogueof1,[(η6-bip)Ru(en)Cl]+,whichhasalsobeenshown the presence of the oxinate chelating ligand in OsII arene tobecytotoxictocancercells,6–8reactsmuchmorerapidlywith complexesisnotfavorablefortheinteractionofthearenerings DNA under similar conditions (t ca. 10 min).16 The slower inthesecomplexeswiththedoublehelix.Insummary,itseems 50 kineticsofosmiumbinding(28timesslowercomparing1with reasonabletosuggestthattheligandsin4donotinteractwith itslighterRuIIanalogue)mayallowmoreofittoreachitstarget thedoublehelixinawaysimilartootherOsIIcomplexes1-3, in vivo than the ruthenium analogue, which is more reactive thus also supporting a different DNA binding mode for this and likely to be deactivated by reacting with other biological compound in comparison with the other three OsII arene molecules before reaching the target DNA within the cell complexesstudiedinthepresentwork.Inparticular,complexes nucleus.TheOsIIbiphenylpicolinatecomplex,[(η6-bip)Os(pi- 1 and 2 containing the extended biphenyl arene capable of co)Cl] (2), reacts almost quantitatively with the DNA and is intercalating were potent at replacing the EtBr intercalator theonlycomplextodoso.Inaddition,replacingtheextended comparedtocomplexes3and4containingthesinglearenering, biphenyl arene by the single ring arene, p-cymene, as in 3, p-cymene. resultsinamarkeddecreaseinDNAbinding(toca.76%).For EtBr as a fluorescent probe can be used to distinguish alltheosmiumcomplexes,>90%oftheequilibriumhadbeen intercalatingandnonintercalatingligands.16,22,28BindingofEtBr reached within the first 24 h. toDNAbyintercalationisblockedinastoichiometricmanner OsmiumbindingtoDNAinhibitsRNAsynthesisinasimilar by formation of a wide spectrum of DNA-binding ligands fashionandwithsimilarstopsitestocisplatinandtheruthenium includingintercalators.Ontheotherhand,modificationofDNA OsIIAntitumorComplexDNAInteractions JournalofMedicinalChemistry,2008,Vol.51,No.12 3641 by monofunctional nonintercalative ligands, such as dienPt, TheresultsofDNAunwindingexperiments(Figure5,Table resultsinonlyaslightdecreaseofEtBrfluorescenceintensity 4) suggest that the arene ligand (p-cymene) in3 also interacts as compared with that for the complex of nonmodified DNA withthedoublehelixuponcoordinationoftheosmiumcomplex. with EtBr. Competitive binding of other intercalators leads to However,theadductsofthisOsIIcomplexthermallydestabilize alossoffluorescencebecauseofdepletionoftheDNA-EtBr DNA similarly as those of 4 [whose arene ligand (p-cymene) complex (free EtBr is poorly fluorescent). apparentlydoesnotinteractnoncovalentlywithDNAtoinduce TheadductsofallOsIIcomplexesreplacetheEtBrintercalator distinctunwindingofitsdoublehelicalstructure].Theexplana- tion of this is unclear but may be associated with a different slightly or markedly more efficiently than those of cisplatin (Figure 8). The adducts of compounds 1-3 unwind DNA by DNAnoncovalentbindingmodeofthep-cymeneligandinOsII 21-27° (Table 4); that is, the values of the unwinding angles arenecomplexescomparedtothatofthebiphenylligand.This hypothesis is corroborated by the observation that the single- areconsiderablyhigherthanthoseproducedbythemonofunc- tionaladductsofdienPt(unwindingangle6°16,22,28).Thus,the ringp-cymeneareneligand(incontrasttodouble-ringbiphenyl arene ligand) in analogous monofunctional RuII arene ethyl- resultsofunwindingexperimentsareconsistentwiththeview that the arene ligands in 1-3 interact substantially with the enediamine complexes does not intercalate in the DNA base- pair stack.16,31 doublehelixuponcoordinationoftheosmiumcomplex.16,22,28 Theresultsofthepresentworkdemonstratecytotoxicityfor Hence, these results strengthen the case for combined nonco- these complexes in ovarian cell lines, and importantly, the valent, perhaps intercalative and monofunctional coordination binding modes of 1-3. activityinthecellssensitiveandresistanttocisplatinwasalso determined(Figure2andTable1).Thattheosmium(II)arene Incontrast,theoxinatecomplex,[(η6-p-cym)Os(oxinate)Cl] complexesshowverysimilaractivityinbothcelllinesishighly (4), does not unwind the DNA significantly (<2.5°, Table 4) significant and indicates a different detoxification mechanism but replaces the EtBr intercalator as efficiently as the OsII for this class of complexes. Intriguingly, complex 1, [(η6- complex 3. In aggregate, it seems reasonable to suggest that bip)Os(en)Cl]+, shows even greater activity in the cisplatin- theligandsin4donotinteractwiththedoublehelixinaway resistantcellline(resistancefactorof0.55).Suchresultsindicate similar to other OsII complexes 1-3, thus also supporting a promisingcompoundswithwhichtotacklethecommonproblem differentDNAbindingmodeforthiscompoundincomparison of developed cisplatin resistance which can occur during withtheotherthreeOsarenecomplexesstudiedinthepresent chemotherapytreatment.Ontheotherhand,themarkedlylower work. activityassociatedwithcomplex4,[(η6-p-cym)Os(oxinate)Cl], The noncovalent interactions of arenes, which may be correlateswithitsdifferentbindingtoDNAandwithitsdifferent involvedinthebindingoftheOsIIarenecompounds1and2to aqueous solution chemistry compared with the picolinate double-helical DNA (vide supra), may also affect its melting complexes (2 and 3). behavior (Figure 6). Previously,16 two important factors have been invoked to account for the thermal stability of DNA Experimental Section modified by monofunctional RuII complexes in media of relatively low ionic strength (0.01 M Na+): (i) a destabilizing StartingMaterials.Theosmiumcomplexeswerepreparedand characterized as described previously.5,9 Cisplatin was obtained effectofconformationaldistortionsand(ii)astabilizingeffect from Sigma-Aldrich sro (Prague, Czech Republic). dienPt was a of the positive charge on the ruthenium moieties and of generousgiftofProfessorG.NatilefromUniversityofBari.Stock noncovalent binding, such as changes in solvent structure and solutionsofmetalcomplexesforthebiophysicalandbiochemical thecounteriondistributionaroundthephosphategroupsofDNA studies were prepared at the concentration of 2 × 10-4 M in 10 whichmayhelptoovercomeelectrostaticsunfavorableforthe mM NaClO 4 and stored at 4 °C in the dark. Stock solutions of hybridization of the strands of the duplex.29,30 Under the metal complexes for the cytotoxicity studies were prepared in conditions of our experiments, we expect all OsII arene DMSOandusedimmediatelyafterdissolution.Theconcentrations ofosmiumorplatinuminthestocksolutionsweredeterminedby complexestohaveproducedmonofunctionaladducts.Inherently, ICP OES. CT DNA (42% G + C, mean molecular mass ca. 2 × wepredictthatconformationaldistortionsduetotheformation 107)wasalsopreparedandcharacterizedasdescribedpreviously.32,33 oftheadductswilldestabilizethehelix,ashasbeenconsistently pSP73KB(2455bp)andpUC19(2686bp)plasmids(superhelical observedinearlierstudieswithvariousrutheniumandplatinum density σ ) -0.063 and -0.055, respectively) were isolated compounds. Hence, it is possible that the less pronounced according to standard procedures. The synthetic oligodeoxyribo- decrease in t due to the modification by the OsII compounds nucleotides (21-mers) were purchased from VBC-Genomics (Vi- m 1 and 2 (Figure 6) is a consequence of compensation of enna,Austria)andpurifiedasdescribedpreviously.21,34Restriction endonucleasesEcoRIandNdeIandT4polynucleotidekinasewere destabilizingeffectsofconformationalchanges.Thisstabilizing purchased from New England Biolabs. Dimethyl sulfate (DMS), compensationmightbeassociatedwithnoncovalentinteraction DMSO, KMnO, DEPC, KBr, and KHSO were from Sigma of the arene ligand with the duplex inferred from DNA 4 5 (Prague,CzechRepublic).Acrylamide,bis(acrylamide),andEtBr unwinding(Figure5,Table4)andquenchingEtBrfluorescence werefromMerckKgaA(Darmstadt,Germany).Agarosewasfrom (Figure 8) and with the overall positive charge on these OsII FMCBioProducts(Rockland,ME).Radioactiveproductswerefrom compounds. In addition, the stabilizing effects of the positive MPBiomedicals,LLC(Irvine,CA). charge on the osmium atom of the compounds 3 and 4 might Metalation Reactions. CT DNA and plasmid DNAs were beconsiderablyreducedduetoasubstantiallydifferentlocation incubated with osmium or platinum complex in 10 mM NaClO 4 (pH∼6)at37°Cfor48hinthedark,ifnotstatedotherwise.The oftheosmiumatomintheadductsofthesecompoundsrelative to the DNA sugar-phosphate backbone. This location might number of atoms of the metal bound per nucleotide residue (r b values)wasdeterminedbyICPOES(osmium)orFAAS(platinum). be unfavorable from the viewpoint of the efficiency of the The single-stranded oligonucleotide (the top, pyrimidine rich, positive charge on the osmium atom to neutralize negative strandcontainingasinglecentralGoftheTGT(21)duplex,Figure chargesofDNAphosphategroups.Thus,thesolutionbehavior 4B)(5×10-4M)wasreactedinstoichiometricamountswith1, oftheDNAadductsofOsIIarenecomplexesappearsinteresting 2, and 3. The metalated oligonucleotides were purified by ion- and merits further study. exchange HPLC. It was verified by ICP OES and by absorbance 3642 JournalofMedicinalChemistry,2008,Vol.51,No.12 KostrhunoVaetal. measurements that the modified oligonucleotides contained one mL for DNA and 0.04 mg/mL for EtBr, which corresponded to osmiumatompermole.ItwasalsoverifiedusingDMSfootprint- thesaturationofallintercalationsitesofEtBrinDNA.41 ing13 that one molecule of osmium complex was coordinated to Other Physical Methods. Absorption spectra were measured theN7atomofthesingleGinthetopstrandofeachduplex. withaVarianCary4000UV-visspectrophotometerequippedwith DNATranscriptionbyRNAPolymeraseinVitro.Transcrip- athermoelectricallycontrolledcellholderandquartzcellswitha tionofthe(NdeI/HpaI)restrictionfragmentofpSP73KBDNAwith pathlengthof1cm.Purificationofoligonucleotideswiththeaid T7RNApolymeraseandelectrophoreticanalysisofthetranscripts ofHPLCwascarriedoutonaWatersHPLCsystemconsistingof was performed according to the protocols recommended by Waters 262 pump, Waters 2487 UV detector, and Waters 600S Promega (Promega Protocols and Applications, 43-46 (1989/90)) controllerwithMonoQHR5/5column.Theanalysiswiththeaid andpreviouslydescribedindetail.13,14TheDNAconcentrationused of ICP OES was perfomed using Jobin Yvon, Ultrace 170 in this assay was 3.9 × 10-5 M (related to the monomeric equipment.TheFAASmeasurementswerecarriedoutonaVarian nucleotidecontent). AA240ZZeemanatomicabsorptionspectrometerequippedwitha Chemical Modifications. The modification of the metalated GTA120graphitetubeatomizer.ForICPOESandFAASanalyses, oligonucleotideduplexesbyKMnO 4 ,DEPC,andKBr/KHSO 5 was DNA was precipitated with ethanol and dissolved in 0.1 M HCl. performedasdescribedpreviously.18,35–37Thetoporbottomstrands The gels were visualized by using a BAS 2500 FUJIFILM of the oligonucleotide duplexes were 5′-end labeled with bioimaging analyzer, and the radioactivity associated with bands [γ-32P]ATPandT4polynucleotidekinase. wasquantitatedwiththeAIDAimageanalyzersoftware(Raytest, LigationandElectrophoresisofOligonucleotides.Unmodified Germany). 21-mersinglestrand(bottomstrandoftheduplexdescribedinthe Cytotoxicity.ThehumanovariantumorcelllinesA2780(parent, Results section, DNA Unwinding and Bending paragraph) were cisplatin-sensitive) and A2780cisR (with acquired cisplatin resis- 5′-end-labeledwith[γ-32P]ATPbyusingT4polynucleotidekinase. tance)wereculturedinRPMI1640medium(Gibco),supplemented Thentheywereannealedwiththeirphosphorylatedcomplementary with10%FBS,2mMglutamine,50µg/mLgentamycinat37°C strands (unmodified or containing the monofunctional osmium inanatmosphereof95%airand5%CO.Celldeathwasevaluated adduct).TheduplexeswereallowedtoreactwithT4DNAligase. 2 byusingasystembasedonthetetrazoliumcompoundMTT[3-(4,5- Theresultingsamplesalongwithligatedunmetalatedduplexeswere dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide]which subsequentlyexaminedon8%nativePAA[mono:bis(acrylamide) isreducedbylivingcellstoyieldasolubleformazanproductthat ratio)29:1]electrophoresisgels.Otherdetailsoftheseexperiments can be detected colorimetrically.43 Cells were seeded in 96-well wereasdescribedinpreviouspapers.19,34,38 sterile plates at a density of 104 cells/well in 100 µL of medium Unwinding of Negatively Supercoiled DNA. Unwinding of and were incubated for 16 h. Osmium complexes were dissolved closedcircularsupercoiledpUC19plasmidDNAwasassayedby in DMSO; the stock solutions were freshly prepared before use. an agarose gel mobility shift assay.22 The unwinding angle Φ, The final concentration of DMSO in cell culture medium did not induced per osmium-DNA adduct, was calculated upon the exceed0.25%.Thecompoundswereaddedtofinalconcentrations determinationofther valueatwhichthecompletetransformation b from0to128µMinavolumeof100µL/well.Seventy-twohours of the supercoiled to relaxed form of the plasmid was attained. Samples of plasmid DNA at the concentration of 1.6 × 10-4 M later 10 µL of a freshly diluted MTT solution (2.5 mg/mL) was pipettedintoeachwell,andtheplatewasincubatedat37°Cina (relatedtothemonomericnucleotidecontent)wereincubatedwith complexes 1-4 at 37 °C in the dark for 24 h. All samples were humidified5%CO 2 atmosphere.After5hthemediumwasremoved andtheformazanproductwasdissolvedin100µLofDMSO.The precipitated by ethanol and redissolved in the TAE (Tris-aceate/ cellviabilitywasevaluatedbymeasurementoftheabsorbanceat EDTA)buffer.Onealiquotoftheprecipitatedsamplewassubjected toelectrophoresison1%agarosegelsrunningat25°Cinthedark 570nm,usinganAbsorbanceReaderSUNRICETECANSCHOEL- LER. All experiments were made in triplicate. IC values withTAEbuffer,andthevoltagewassetat25V.Thegelswere 50 (compound concentration that produces 50% of cell growth thenstainedwithEtBr,followedbyphotographywithtransillumi- inhibition)werecalculatedfromcurvesconstructedbyplottingcell nator.Theotheraliquotwasusedforthedeterminationofr values b byICPOES. survival(%)versusdrugconcentration(µM).Allexperimentswere DNA Melting. The melting curves of CT DNAs at the madeintriplicate. concentration of 32 µg/mL were recorded by measuring the CellularOsIIAreneComplexUptake.CellularuptakeofOsII absorbance at 260 nm. The melting curves of unmodified or arenecompoundsandcisplatinwasmeasuredinA2780cells.The metalated DNA were recorded in the medium containing 0.01 M cells were seeded in 60 mm tissue culture dishes (30000/cm2). NaClO with 1 mM Tris-HCl/0.1 mM EDTA, pH 7.4. The value Afterovernightincubation,thecellsweretreatedwiththeosmium 4 of t was determined as the temperature corresponding to a compoundorcisplatinfor6hatequimolarconcentration(10µM); m maximumonthefirst-derivativeprofileofthemeltingcurves.The thisconcentrationwasverifiedbythemeasurementofosmiumor t valuescouldbethusdeterminedwithanaccuracyof(0.3°C. platinuminthegrowingmediumbyICPOES.Theattachedcells m FlowLinearDichroism(LD).FlowLDspectrawerecollected werewashedtwicewithPBS(4°C)andcentrifugedat2500rpm, by using a flow Couette cell in a Jasco J-720 spectropolarimeter and the pellet was stored at -80 °C. Afterward, the pellets were adapted for LD measurements. Long molecules, such as DNA digestedwith12MHNO 3 ,30%H 2 O 2 ,and12.1MHCl.Osmium (minimumlengthof∼250bp),canbeorientatedinaflowCouette andplatinumcontentwasdeterminedbyICPOES. cell.Theflowcellconsistsofafixedoutercylinderandarotating Acknowledgment. This research was supported by the solidquartzinnercylinder,separatedbyagapof0.5mm,giving atotalpathlengthof1mm.LDspectraofDNAattheconcentration MinistryofEducationoftheCR(MSMTLC06030,ME08017, of 0.1 µg/mL modified by the osmium complexes were recorded OC08003), the Academy of Sciences of the Czech Republic at25°Cin10mMNaClO plus20mMNaCland10mMsodium (Grants 1QS500040581, KAN200200651, IAA400040803, 4 cacodylate,pH7.0.39,40 AV0Z50040507, and AV0Z50040702), the Grant Agency of Fluorescence Measurements. These measurements were per- theCR(203/06/1239)andtheGrantAgencyoftheMinistryof formed on a Shimadzu RF 40 spectrofluorophotometer using a 1 Health of the CR (NR8562-4/2005), EPSRC (studentship for cm quartz cell. Fluorescence measurements of DNA modified by A.F.A.P.),andtheWellcomeTrust(InternationalCollaboration osmiumattheconcentrationof32µg/mLinthepresenceofEtBr AwardforP.J.S.andV.B.).Theauthorsalsoacknowledgethat were performed at an excitation wavelength of 546 nm, and the emitted fluorescence was analyzed at 590 nm. The fluorescence their participation in the EU COST Action D39 enabled them intensitywasmeasuredat25°Cin0.4MNaCltoavoidsecondary to exchange regularly the most recent ideas in the field of binding of EtBr to DNA.41,42 The concentrations were 0.01 mg/ anticancer metallodrugs with several European colleagues. 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