A potential rhodium cancer therapy: studies of a cytotoxic organorhodium(I) complex that binds DNA.

Bioorganic&MedicinalChemistryLetters23(2013)2527–2531 ContentslistsavailableatSciVerseScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl A potential rhodium cancer therapy: Studies of a cytotoxic organorhodium(I) complex that binds DNA Jeanette R. McConnell, Dimple P Rananaware, Deborah M. Ramsey, Kai N. Buys, Marcus L. Cole, ⇑ Shelli R. McAlpine DepartmentofChemistry,UniversityofNewSouthWales,Kensington,NSW2052,Australia a r t i c l e i n f o a b s t r a c t Articlehistory: Describedisanovelorganorhodium(I)complexthatiscytotoxictothecoloncancercelllineHCT116and Received10January2013 alters cell migration, DNA replication, and DNA condensation. Most importantly, the mechanism Revised23February2013 observed is not seen for the parent organorhodium dimer complex [{RhCl(COD)} ], RhCl , or the free 2 3 Accepted4March2013 ligand/proligands(CODand1-nbutyl-3-methylimidazoliumchloride).Thus,theactivityofthisorgano- Availableonline14March2013 rhodiumcomplexisattributabletoitsuniquestructure. (cid:2)2013ElsevierLtd.Allrightsreserved. Keywords: DNAbinding Cisplatin Metastasisinhibition Anticancer Rhodium TheFDAapprovedmetallodrugcisplatin1(Fig.1)isusedtotreat nanoparticles.22,23 There is little recent data on the biological morethan50%ofcancerpatients,makingitthemostwidelyused activityofRhodium(I)complexes.However,previousreportsshow oncologicaltherapy.2–6Theside-effectprofileandresistancedevel- thatRh(I)complexeshaveanticanceractivityagainstEhrlichasci- opmenttocisplatinhavedriventhedevelopmentofnewmetallo- tes, leukemia L1210, sarcoma 180, and metastatic lung drug therapies. However, the development of other clinical cancer.22,24,25 metallo-drugshasbeenslowandonlyoneiscurrentlyinclinical Hereinwereportthemechanisticcharacterizationofapotential trials,therutheniumbasedKP1019.7–12Othermetalsareindevel- lead structure, the organorhodium(I) complex; [RhCl(IBuMe)(- opment as anticancer agents including promising reports of rho- COD)] (1) (IBuMe=1-nbutyl-3methylimidazol-2-ylideneand diumspecies.13–15 COD=1,8-cyclooctadiene) (Fig. 1).26,27 Preliminary data indicate Rhodium(III)complexesarethemosthighlyresearchedorgano- thatcompound1(1)iscytotoxictoHCT-116coloncancercellline, rhodium.TheBartongrouphasledthewaywithRh(III)metalloin- whichledtoprofilingitsmechanismofaction.Mechanisticstudies sertors(Fig.1)thatbindDNAmismatches,disruptDNAsynthesis, involvedDNAbinding,DNAsynthesis,woundhealing,andcellcy- andhaveIC valuesinthelowmicro-molarrange.Theserhodium cle assays. Compound 1 displayed a mechanism similar to other 50 complexes are more potent in cells unable to repair DNA mis- organometallicdrugs,whereby1targetsDNA,inhibitsmetastasis, matches,whichmakesthemgoodcandidatesforcancer.16–18Other preventscelldivision,andreducesDNAcondensation. investigationsinvolvingRh(III)complexesincludecompoundsthat Cisplatin, 1 and several controls including the parent non- intercalate DNA via polypyridyl co-ligands. These Rh complexes IBuMe coordinated dimer [{RhCl(COD)} ], and RhCl were run in 2 3 have anti-topoisomerase II activity, and act as photo-inducible cytotoxicity assays against HCT116 cells. The controls were used agents.14,19–21,15 toevaluateifspecificligandswerenecessaryforcytotoxicity.The Rhodium(II) complexes have demonstrated efficacy in Ehrlich IC values(Table1)for1andcisplatinafter72harebothinthe 50 ascitessarcomaand P 388leukemia.22,23 Very recentlyRh(II)cit- lowmicro-molarrange,whereas[{RhCl(COD)} ]andRhCl aretoo 2 3 ratewasreportedtoprolongsurvivalandreducethesizeofbreast high to be measured. These data indicate that the cytotoxicity is cancer carcinoma in mice when administered with maghemite uniquetothecomplexasawhole.TheIC ofcompound1ismuch 50 lower than previously reported values of >200lM for Rh(I) complexes.28 ⇑ Abbreviations:DMSO,dimethylsulfoxide;IC50,inhibitoryconcentration(50%). Given that all rhodium complexes previously reported effect Correspondingauthor.Tel.:+61416728896/293855505;fax:+6129385 cells by interacting with the DNA,13 we evaluated compound 1’s 6111. E-mailaddress:s.mcalpine@UNSW.edu.au(S.R.McAlpine). abilitytobindDNA.Cisplatinand1wereincubatedwithpBR322 0960-894X/$-seefrontmatter(cid:2)2013ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.bmcl.2013.03.016 2528 J.R.McConnelletal./Bioorg.Med.Chem.Lett.23(2013)2527–2531 Figure 1. Structures of cisplatin, [Rh(HDPA)2-chrysi]3+ a lead Rh(III) anticancer moleculeandcompound1. Table1 Cytotoxicityofcisplatin,1,RhCl3and[{RhCl(COD)}2]againstthecoloncancercellline HCT116 Cisplatin 1 RhCl3 [{RhCl(COD)}2] HCT116 1.5lM±0.5 3lM±1 >100lMa >100lMa Datarepresentresultsfromfourindependentexperiments,andtheaverageIC50 valuesareshown.ConcentrationcurvesareavailableintheSupplementarydata. a Highestconcentrationmeasured. DNA, and the DNA binding patterns of 1 and controls were assessed (Fig. 2A). The profile of compound 1’s interaction with pBR322DNAisdifferentfromthatofcisplatin,andthenon-toxic ligandRhCl .Treatmentwithincreasingconcentrationsofcisplatin 3 showsaslowdecreaseinsupercoiledDNA,aswellasaslightde- creaseintheseparationofthebands.Thesedataconfirmthatcis- platin induces conformational changes in the DNA. In contrast, treatment of DNA with increasing concentrations of 1 leads to a significantdisappearanceofsupercoiledDNAandtheappearance of nicked DNA. This result indicates that 1 uncoils and damages theDNA.Incontrast,DNAisonlyminimallydegradedwhentrea- ted with RhCl . These data demonstrate that like cisplatin, and 3 otherorganorhodiumstructures,compound1bindstoDNA. To fully assess compound 1’s interaction with DNA, a BamHI digestionwascompleted.BamHIisarestrictionendonucleasewith a recognition site (G0GATCC) that hydrolyzes the phosphodiester bond between adjacent guanine sites.29,30 The pBR322 DNA con- Figure2. (A)pBR322DNAtreatedwithcompoundsatfollowingconditions:no treatment(control),1.25,2.5,5,10,20,40,60,80and100lM.Intensityofthe tains only one BamHI site. BamHI digestion of untreated pBR322 bandswasmeasuredbyImageJsoftware.(B)pBR322treatedwithcompoundsand plasmid DNA converts the supercoiled and singly nicked circular digested with 10 units of BamHI at following conditions: untreated undigested DNAtoalinearform.pBR322DNAwastreatedasdescribedabove (control),untreatedBamHIdigested(control)followedby1.25,2.5,5,10,20,40,60, withcisplatin,1,andRhCl .(Fig.2B).TheBamHIrestrictiondiges- 80and100lM. 3 tionassays(Fig.2B)showthatbothcisplatinand1inhibitdiges- tionby BamHI. These results indicate that cisplatin and 1 induce a DNA conformational change, inhibit the binding of BamHI to Rhodium(I)complexeshavealsodemonstratedactivityagainst the DNA, and partially prevent DNA digestion. Cisplatin shows metastaticcancers.Thus,weevaluatedtheabilityof1andcisplatin maximuminhibitionat10lMcomparedto40lMfor1. toinhibitcelldivisionandmigrationusingwound-healingassays ThroughthisinteractionwithDNA,organorhodiumcomplexes with HCT116 cells, a metastatic cancer cell line.13,34 Cells were havebeenshowntodisruptDNAsynthesis,andtherebyhaltcell seededintowoundcreatingculture-inserts,treatedwith1orcis- division, acting as cytostatic drugs before causing cell death.17 platin, and imaged every 30min over 24h. Analysis of these WecompletedaBrdUincorporationassayinHCT116cellstodeter- time-lapse images showed that 1 inhibits wound closure (Fig. 4, mine if 1 disrupts DNA synthesis. BrdU is a thymidine analogue andSupplementarydataS1).Thewoundstreatedwith1andcis- that is incorporated into DNA of replicating cells.31 The relative platin remained 59% and 70% open respectively, indicating that amountsofBrdUuptakewerethenvisualizedbyprobingforBrdU thecellsweremigratinganddividingatasignificantlyslowerrate with a Phycoerythrin (PE) conjugated antibody and the nucleus thanthatofthevehiclecontrol(DMSO)after24h.Nuclearstaining wascounter-stainedwithHoescht33342.32,33ExtensiveBrdUup- of 1-treated cells at this time point indicate that the reduced take indicates cells are activelysynthesizingDNA,whereas mini- migration rate is not due to cell death. Along with the halt in mal staining of BrdU shows that cells are not replicating their DNA synthesis these data demonstrate that compound 1 is anti- DNA. The decrease in BrdU fluorescence after treatment with 1 proliferativeandinhibitscelldivisionandmigrationafter24h. and cisplatin shows that both compounds halt DNA synthesis Since1haltsDNAsynthesisandcausesadecreaseincellmobil- and have anti-proliferative effects on HCT116 cells (Fig. 3A and ity,weusedcellcyclephaseanalysisofsynchronizedcellstoquan- B)17 These data are consistent with evidence reported by others titate the number of cells in cell cycle phases. Employing usingRh(I)analogs.17 propidiumiodide(PI)asastaintoquantitatethenumberofcells J.R.McConnelletal./Bioorg.Med.Chem.Lett.23(2013)2527–2531 2529 Figure3. Compound1inhibitsDNAreplication.(A)FixedandpermeabilizedHCT116cellswereprobedforBrdUwithaPEconjugatedantibody.Thedramaticdecreasein fluorescencedemonstratesthat1andcisplatinhaltDNAreplication.Hoescht33342actstoconfirmthenuclearlocalizationofBrdU.(B)Cellsweretreatedwith2lMcisplatin and5lM1.TheamountofBrdUincorporationwasdeterminedfrom10fieldsofview,countingallcells,andrepeatedinduplicate.ValuesarereportedasDMSOrepresenting 100%BrdUincorporation(⁄denotesp<0.0001whencomparedtoDMSOusingStudent’sttest) anincreaseinG -phase.16SynchronizedHCT116cellswereleftun- 1 treated,treatedwith1%DMSO,2lMof1,or7.5lMcisplatinfor 40h. Analysis using flow cytometry we observed treatment with either1orcisplatinresultsinadisproportionateshiftofthecells into S-phase relative to the DMSO control (Supplementary data Fig.S6). QuantificationofPIintensityindicatesthat1andcisplatinin- creasethenumberofPI-brightcellsby43%and78%respectively, compared to 23% increase in DMSO treated cells (Fig. 5A). These datasuggesttwopossibilities:(i)thatthecellsaredividing,which isinconsistentwiththewoundhealingandBrdUincorporationas- says (Figs. 2 and 4), or (ii) that cells treated with cisplatin or 1 incorporatemorePIrelativetotheDMSOcontrol.Sinceanincrease Figure4. Woundclosurecomparisonofnon-treated,cisplatinand1treatedHCT 116cells.Cellswereseededintowoundcreatingculture-inserts(Ibidi).After24h inPIincorporationcanserveasanestimateofthenumberofcells theinsertswereremoved.Thecellsweretreatedwiththerespectivecompounds inS-phaseorindicateachangeinDNAcondensation,36itislogical (cisplatin2lM,15lM)1hbeforeimaging,andthenimagedevery30minfor24h thattreatmentofDNAwithbindingagentssuchascisplatinand1, onaNikon(cid:3)BiostationIM.Compositeimagesforspecifictimepointswereusedto which partially opens DNA, will result in higher PI fluorescence. comparetheimpactofeachcompoundonwoundclosure.(SupplementarydataS3) ThewidthofthewoundwasmeasuredwithImageJsoftware.Thirtypointsalong ThisissupportedbytheobservedtotalPIfluorescenceforcisplatin the wound were used to analyse wound closure, values reported here are (ca.3(cid:2)DMSO)and1(ca.2(cid:2)DMSO)intreatedcells(Supplemen- mean±SEM.(⁄denotesp<0.0001whencomparedtothecorrespondingtimepoint tary data Fig. S6). Our data lead us to propose the uptake model forDMSO). illustratedinFigure5B. Takentogether,ourwoundhealingassays(Fig.4),DNAbinding assays(Fig.2A),andcelldivisionanalysis(Figure3)allsupportthe locatedintherestingphase(i.e.,G 0 /G 1 -phase)versusthoseprepar- hypothesisthattreatmentofHCT116cellswith1haltDNAreplica- ingtodivide(i.e.,S-phase)wegenerateddataonhowcompound1 tionbybindingDNAandopeningthedoublehelix.Inconclusion, affects the cell cycle.35 Organorhodium complexes have been re- wehaveidentifiedanovelorganorhodium(I)complexthat:iscyto- portedtocauseacellcycleshiftwithareductioninS-phaseand toxic to HCT116, has an IC value much lower than other Rh(I) 50 2530 J.R.McConnelletal./Bioorg.Med.Chem.Lett.23(2013)2527–2531 Figure5. (A)SynchronizedHCT-116cellsweretreatedfor40hunderthefollowingconditions:notreatment(control),1%DMSO,2lM1,or7.5lMcisplatin.Cellswere harvestedandanalyzedbyflowcytometry,asdescribedinSupplementarydata.TheDNAcontent(PIfluorescence;y-axis)versustreatment(x-axis)areplotted.Thenumber ofPIbrightcellsinSphasearecomparedpertreatmentgroup.ResultsareshownforsamplesundergoingDMSO-treatment(blackbars),cisplatintreatment(greybars),and 1—treatment(whitebars).Errorbarsindicatedstandarddeviation,andresultsarerepresentativeoffour(n=4)independentexperiments.Statisticalanalysiswasperformed usingtheStudent’sttest.The‘⁄⁄⁄’indicatesp<0.0001,‘⁄⁄’indicatesp<0.001,and‘⁄’indicatesp<0.04.(B)ModelsystemillustratingthatthedrugfacilitatesPIincorporation. complexes,haltsDNAreplication,alterscellmigration,andinduces Supplementarydata DNAcondensation.Mostimportantly,themechanismobservedis uniqueto1andisnotseeninstudieswiththeparentorganorho- Supplementarydata(experimentaldetailsforbiologicalassays dium dimer complex [{RhCl(COD)} 2 ], RhCl 3 , or the free ligand/ and imaging experiments) associated with this article can proligands (COD and 1-nbutyl-3-methylimidazolium chloride). be found, in the online version, at http://dx.doi.org/10.1016/ TherecentsuccessesofRh(II)and(III)organorhodiumsasantican- j.bmcl.2013.03.016. ceragentssupportexplorationofRhcomplexs.Thedatareported here are expected to provoke new interest in Rh(I) complexes as Referencesandnotes anpotentialoncogenicagents. 1. Rosenberg,B.;Vancamp,L.;Trosko,J.E.;Mansour,V.H.Nature1969,5191,385. 2. Jakupec,M.A.;Glanaski,M.;Arion,V.B.;Hartinger,C.G.;Keppler,B.K.Dalton Acknowledgments Trans.2008,183. 3. Strodal,B.;Davey,M.Life2007,11,696. TheauthorswishtothankDr.FazlulHuqfromtheUniversityof 4. Siegel,R.;Naishadham,D.;Jemal,A.CancerJ.Clin.2012,62,10. Sydney for protocols and helpful comments. We also thank Mat- 5. Allardyce,C.S.;Dyson,P.J.PlatinumMet.Rev.2001,45,62. 6. Ang,W.H.;Daldini,E.;Scolaro,C.;Scopelliti,R.;Juillerat-Jeannerat,L.;Dyson,P. thewR.Gytonforsynthesizingcompound1(1).WethanktheUni- J.Inorg.Chem.2006,45,9006. versityofNewSouthWalesforfinancialsupportofDMRandSRM, 7. Dale,L.D.;Tochter,J.H.;Dyson,T.M.;Edwards,D.I.T.D.A.;AntiCancerDrug and the Australian Research Council (DP110104759) for financial Des.1992,7,3. 8. Yan,Y.K.;Melchart,M.;Habtemariam,A.;Sadler,P.J.Chem.Commun.2005,38, support(MLC). 4764. J.R.McConnelletal./Bioorg.Med.Chem.Lett.23(2013)2527–2531 2531 9. Hartinger,C.G.D.P.JChem.Soc.Rev.2009,38,391. 23. Craciunescu,D.;Molina,C.;Parrondo-Iglesias,E.J.Braz.Chem.Soc.1994,5,23. 10. Hartinger,C. G.; Jakupec, M. A.;Zorbas-Seigfried, S.; Groessl, M.; Egger, A.; 24. Giraldi,T.;Zassinovich,G.;Mestroni,G.Chem.Biol.Interact.1974,9,389. Berger,W.;Zorbas,H.;Dyson,P.J.;Keppler,B.K.Chem.Biodivers.2008,5,21. 25. Craciunescu,D.;Scarcia,V.;Furlani,A.;Iglesias,E.;Ghirvu,C.;Papaioannou,A. 11. Gasser,G.;Ott,I.;Metzler-Nolte,N.J.Med.Chem.2011,54,3. AnticancerRes.1989,3,781. 12. Pelletier,F.;Comte,V.;Massard,A.;Wenzel,M.;Toulot,S.;Richard,P.;Picquet, 26. Park,K.H.;Kim,S.Y.;Son,S.U.;Chung,Y.K.Eur.J.Org.Chem.2003,22,4341. M.;LeGendre,P.;Zava,O.;Edafe,F.;Casini,A.;Dyson,P.J.J.Med.Chem.2010, 27. Cole,M.L.;Gyton,M.R.;Harper,J.B.Aus.J.Chem.2011,64,1133. 53,6923. 28. Dorcier,A.;Ang,W.;Bolano,S.;Gonsalvi,L.;Juillerat-Jeannerat,L.;Laurenczy, 13. Katsaros,N.A.Crit.Rev.Oncol.Hematol.2002,42,297. G.;Peruzzini,M.;Philips,A.;Zanobini,F.;Dyson,P.Organometallics2006,25, 14. Schaefer,S.;Sheldrick,W.S.J.Organomet.Chem.2007,692,1300. 4090. 15. Scharwitz,M.A.;Ott,I.;Geldmacher,Y.;Gust,R.;Sheldrick,W.S.J.Organomet. 29. Roberts,R.J.;Wilson,G.A.;Young,F.E.Nature1977,265,82. Chem.2008,693,2299. 30. Chowdhury,M.A.;Abdullah,A.;Beale,P.;Fisher,K.J.Inorg.Biochem.2005,99, 16. Ernst,R.K.A.;Barton,J.Biochemistry2011,50. 1098. 17. Ernst,R.J.;Song,H.;Barton,J.K.J.Am.Chem.Soc.2009,121,2359. 31. Defazio,A.;Leary,J.A.;Hedley,D.W.;Tattersall,M.H.J.Histochem.Cytochem. 18. Komor,A.C.;Schneider,C.J.;Weidmann,A.G.;Barton,J.K.J.Am.Chem.Soc. 1987,35,571. 2012,134,19223. 32. Rossi, D. J.; Londesborough, A.; Korsisaari, N.; Pihlak, A.; Lehtonen, E.; 19. Barry,C.T.E.;Day,C.;Saluta,G.;Kucera,G.;Bierbach,U.Inorg.Chem.2002,41, Henkemeyer,M.;Makela,T.P.EMBO2001,20,2844. 7159. 33. Chechlacz,M.;Vemuri,M.C.;Naegele,J.R.J.Neurochem.2001,78,141. 20. Singh,S.;Joshi,S.;Singh,A.;Saxena,J.;Dandey,D.Inorg.Chem.2007,46,10869. 34. Park,S.-A.;Na,H.-K.;Kim,E.-H.;Cha,Y.-N.;Surth,Y.J.CancerRes.2009,69, 21. Harlos,M.;Ott,I.;Gust,R.;Alborzinia,H.;Wolfi,S.;Kromm,A.;Sheldrick,W.J. 2416. Med.Chem.2008,51,3924. 35. Crissman,H.A.;Steinkamp,J.A.J.CellBiol.1973,59,76. 22. Carneiro,M.;Piexoto,R.;Joanitti,G.;Oliveira,R.;Telles,L.;Miranda-Vilela,A.; 36. Demarcq,C.;Bunch,T.R.;Creswell,D.;Eastman,A.CellGrowthDiffer.1994,5, Bocca,A.;Vianna,L.;Silva,I.;Souza,A.;Lacava,Z.;Bao,S.J.Nanobiotechnol. 983. 2013,11,4.