Targeting the cubilin receptor through the vitamin B(12) uptake pathway: cytotoxicity and mechanistic insight through fluorescent Re(I) delivery.

J.Med.Chem.2009,52,5253–5261 5253 DOI: 10.1021/jm900777v Targeting the Cubilin Receptor through the Vitamin B Uptake Pathway: Cytotoxicity and 12 Mechanistic Insight through Fluorescent Re(I) Delivery NerissaViola-Villegas,AmyE.Rabideau,MarkBartholoma,JonZubieta,andRobertP.Doyle* DepartmentofChemistry,SyracuseUniversity,Syracuse,NewYork13244-4100 ReceivedJune1,2009 Theintrinsicfactor(IF)vitaminB ileumanchoredreceptor,cubilin,mediatesendocytoticuptakeof 12 theIFcomplexofvitaminB tothebloodserum.Thisreceptorwastargetedfortheselectivedelivery 12 andaccumulationofanewbioprobe,aB conjugateofrhenium2,inthecubilinexpressingplacental 12 choriocarcinomaBeWocellline.Competitiveuptakeandcytotoxicityassaysof2wereinvestigatedand interactions with nuclear DNA explored. In addition, the mechanism of internalization of 2 was confirmed to proceed in an IF-cubilin mediated fashion via siRNA transfection experiments. These studiesshowthegreatpotentialofcubilinasanewtargetforthedeliveryofB basedconjugatesfor 12 cancerdiagnosticsand/ortreatment. Introduction malignantcellssuchashuman(K562andHL-60)11leukemic celllines,breast(MCF-7),12,13ovarian(NIH-OVCAR3),13and Deliveryofchemotherapeutics byselectivetropismoffers methionine-independent glioma (P60H)14 cells. The structure specificityandselectivitytowardmalignantcellsandfactors ofTCIIwasrecentlypublishedinaseminalpaperbyRandaccio thatdrivetheirtumorigenicity.1Releaseofadrugpayloadby etal.15SeveralreportshavetakenadvantageofTCII-Rover- such a selective biomarker directly in the tumor provides expression as a means of delivery of B conjugates.5,13,16,17 enhancedresponsetotreatmentwhileminimizingsideeffects 12 TCII-Rtargetshaveadisadvantage,however,asthisreceptoris normally experienced with chemotherapy (i.e., improved presentinhighconcentrationsinthekidneys,placenta,intes- therapeuticindex).2Becauseofthebenefitsoftargeteddeliv- tine,andliver.18TCIImediateduptakeofB involvesnotjust ery,pharmaceuticalparadigmshaveshiftedtowardcombin- 12 transport but also subsequent retention,19 so targeting this ingcytotoxicdrugswithmolecularvehicleswithsubsequent uptakepathwayhasadisadvantageespeciallyforradiophar- in-depthinvestigations ofthe concomitantpharmacokinetic maceuticaldrugs,whichrequirerapidclearancefromthebody. andpharmacodynamicpropertiesofsuchsystems.3Thisneed IFisa44kDaglycosylatedproteinproducedingastriccells to deliver treatments and/or diagnostics to a specific site of andhasthehighestspecificityforB (K ≈10-9)9,20ofthe interest has resulted in measures exploring such targeting 12 D threebindingproteins.21IFisresponsiblefortransportofB moieties as antibodies, peptides, hormones, and natural 12 acrosstheintestinalenterocyteviathereceptor,cubilin.8The cofactorssuchasvitamins.4 Site-specificdeliveryofpharmaceuticsviathevitaminB a structureofIFwassolvedrecentlybyAlpersetal.22Cubilinis 12 a ∼460 kDa protein composed of eight epidermal growth uptakepathwayhasgeneratedconsiderableinterestinrecent years.5,6B (seeFigure1)isahighlywater-soluble,nontoxic factordomainsand27CUBdomainproteins(seeFigure2). 12 CUBdomainsaretypically∼110-115aminoacidresiduesin vitamin that plays a key role in reactions ranging from the sizeandarederivedfromClr/Cls,Uegf,andbonemorpho- conversionofhomocysteinetomethionine,toDNAmethyla- tion.7Sinceitisanimportantrequirementforrapidlygrowing genicprotein-1complements.23BindingofIF-B 12 tocubilin occursprimarily at CUB domains 5-824andappears to be cancer cells, B makes for a plausible carrier for a drug 12 calcium dependent.25 This receptor is expressed in select payloaddeliverytothesetissues,assumingreceptorsinvolved tissuesincludingtheplacentalmembranes,26therenalprox- initsuptakecanbetargeted. imaltubularcells27andbrushborder,28gallbladder,29andthe B is absorbed during digestion and transported to the 12 gastrointestinal tract,30,31 making it an intriguing target for plasma via three binding proteins listed here: haptocorrin (HC,K ≈0.01pM),transcobalaminII(TCII,K ≈0.005pM), selectivedeliveryofcytotoxicorimagingagents. and int d rinsic factor (IF, K ≈ 1.0 pM).8,9 TCI d I and HC are TheuseofanIF-B 12 systemtospecificallytargetcubilinis, transportproteinsofB in d theplasma.10ThereceptorforTCII however, relatively unexplored. Some seminal studies, con- 12 ducted by C. B. Grissom and G. J. Russell-Jones, have (TCII-R) is reported to be overexpressed in a number of demonstrated the dissociation kinetics of IF and B 32 and 12 uptake,amongotherstudies,ofnanoparticlesinCaco-2cells, *Towhomcorrespondenceshouldbeaddressed.Phone:(315)443 3584.Fax:(315)4434070.E-mail:rpdoyle@syr.edu. respectively.33 aAbbreviations:IF,intrinsicfactor;TCII,transcobalaminII;siRNA, Inspiredbythelimitedtissueexpressionofcubilinandits small interfering RNA; BeWO, placental choriocarcinoma line; CHO, putative importance for cancer cell growth, we decided to Chinesehamsterovarycancerline;BQBA,tert-butyl4-(bis((quinolin-2- yl)methyl)amino)butylcarbamate;CDT,1,10-carbonyl-di-(1,2,4-triazole); exploreIFasameanstomediatecubilintargetinganddelivery B 12 ,vitaminB 12 . ofB 12 .Wefocusedonthistransportmechanismasameansof r2009AmericanChemicalSociety PublishedonWeb07/22/2009 pubs.acs.org/jmc 5254 JournalofMedicinalChemistry,2009,Vol.52,No.16 Viola-Villegasetal. Figure2. The cubilin receptor has 27 CUB domains (red) and 8 epidermal growth domains (yellow). CUB domains 5-8 are involved primarily in IF-B binding: blue=megalin; RAP= 12 receptorassociatedprotein. Figure1. Thestructureofcyano-B includesaCo(III)boundtoa coordination. This has been reported for several rhenium 12 cyanogroupandtoN-donorsofatetradentatecorrinringand5,6- tricarbonyl complexes.35 The downfield shift is most dis- dimethylbenzimidazolewiththelattercontainingapendentphos- tinctive for the methylene protons in R-position to the phoriboseunit. tertiarynitrogenatomofthechelatemoietythatareshifted from3.92ppmto5.18and5.00ppm,respectively.Moreover, facilitating cellular entry of a rhenium fluorescent probe the proton NMR data show additional evidence of site tetheredtoB .Wepresenthereinthesynthesisandcharac- 12 specific binding of the rhenium tricarbonyl core to the terization of a rhenium(I) tricarbonyl core complexed to ligand.Asexpected,themethyleneprotonsintheR-position N,N0-bis(6,7-dihydroquinolin-2-ylmethyl)butane-1,4-diamine,1, to the tertiary nitrogen atom of the N,N-bis(quinolinoyl) anditsB conjugate,2.Invitroantiproliferativeassaysand 12 residue of the uncoordinated ligand show a singlet. After uptake studies via fluorescent confocal microscopy were coordination of the rhenium tricarbonyl precursor, these conductedontwodifferentcelllines:humanplacentalchor- resonances split into two sets of doublets with coupling iocarcinoma (BeWo) expressing cubilin and (cubilin (-)) constants consistent for geminal coupling (J=17.74 and Chinesehamsterovary(CHO)cells.siRNAmediatedinhibi- 17.81Hz),reflectinganABspinpattern.Theremovalofthe tionofcubilinwasconductedtodemonstratecubilinmediated Bocprotectinggroupwasaccomplishedinthefinalsynthetic uptakepathwayof2.FollowingIFmediatedB bioconjugate 12 step by reacting the intermediate in 3 M hydrochloric acid deliverythroughcubilinhasalsonotbeenextensivelyinves- overnight (Scheme 1). The infrared (IR) spectrum of 1 tigated.So,usingtherheniumconjugate,wealsoventuredto exhibits a sharp band at 2017 cm -1 and a broad, intense followtheprocessindepth. band around 1880 cm -1, which can be assigned to the fac- Re(CO) moiety.Whilethestretchingfrequencyat2017cm-1 ResultsandDiscussion 3 remains unchanged compared to the IR spectrum of the Synthesis and Characterization of 1. It was previously [Re(CO) (H O) ]Br precursor (2017 and 1936 cm-1), an 3 2 3 shown that the rhenium(I) tricarbonyl core coordinated to extensiveredshiftwasobservedforthe stretchingvibration a N,N-bis(quinolinoyl) moiety exhibits fluorescent proper- at1880cm-1.Inthemassspectra,thehighestm/zratioswere ties.34 The introduction of the rhenium fluorophore to B consistentwithcalculatedmassesofthecorrespondingcom- 12 was accomplished by the preparation of a bifunctional plex (Mþ) and the isotope patterns were in excellent agree- chelate having a N,N-bis(quinolinoyl) (BQBA) moiety for mentwiththecalculateddistributions. thecoordinationofthemetalcoreandanaminoterminusfor SynthesisandCharacterizationof2.Couplingof1toB 12 the coupling to B . The tridentate chelate functionality to focused on the ribose 50-hydroxy group because different 12 N-Boc-1,4-diaminobutane was introduced by a standard derivatizations on the corrin functional groups have been reductive amination technique. The protected bifunctional reportedtoprovideadecreaseinaffinityforbothTCIIand chelate was reacted with the rhenium metal precursor IF.36 Conjugation through the 50-hydroxyl group of the [Re(H O) (CO) ]Brat60(cid:1)Cinmethanolfor3h.Inthe1H ribose moiety of B , however, was reported to least affect 2 3 3 12 nuclear magneticresonance(NMR)spectrum, adownfield affinity. B wasconjugatedto1via atwo-stepprocedure. 12 shiftfortheprotonsadjacenttothemetalcorecomparedto Briefly, B was activated with 1.2 equiv of 1,10-carbonyl- 12 the spectrum of the free ligand was observed upon metal di-(1,2,4-triazole) (CDT) in dimethyl sulfoxide (DMSO) at Article JournalofMedicinalChemistry,2009,Vol.52,No.16 5255 Scheme 1. SynthesisoftheBifunctionalChelate1a aReagentsandconditions:(i)2-quinolinecarboxaldehyde,NaBH(OAc),DCE,roomtemp,overnight;(ii)[Re(HO)(CO)]Br,MeOH,60(cid:1)C,3h; 3 2 3 3 (iii)3Mhydrochloricacid,roomtemp,overnight. Scheme 2. 1,10-Carbonyl-di-(1,2,4-triazole) Coupled Reaction liquidchromatography.2elutedatretentiontimet ≈13.7 R ofB to1 min. The percentage yield of 2 ranged from 12% to 17%. 12 Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (see Supporting Information), 1H NMR spectroscopy, and inductively coupledplasma(ICP)wereemployedtoestablishtheiden- tityof2. The MALDI-TOF mass spectrum of 2 (see Supporting Information) displayed three sets of peaks with the parent peakcenteredat∼2022.3m/z.Theparentpeakat∼2022.3 m/z is consistent with the calculated theoretical molecular mass value of [M þ] ≈ 2022.09 for C H CoN O PRe. 91 112 18 18 Thebasepeakcenteredat∼1994.9m/zwasdeterminedtobe the parent peak less a carbonyl moiety (MW ≈ 28 m/z) CO with a calculated [Mþ - CO] ≈ 1994 m/z. The third set of peakscenteringat∼1966.9m/zwasinagreementwith[Mþ- 2CO]withatheoreticalvalueof∼1966m/z.Obtainingthe massspectrumofthefac-tricarbonylrhenium(I)coreproved to be a challenge, as the carbonyl groups are readily dis- charged from the complex upon ionization in situ. Closer inspectionoftheisotopicpatternsof2showspeaksinclose agreement with the theoretical isotope patterns. 1H NMR data were consistent with 2, and ICP results indicated the presenceofrhenium.Thecarbamatelinkerformedisstable overatleast24hbetweenpH5.5andpH7.4asfollowedby HPLC. Intrinsic Factor Bindingto 2. Bindingof the protein to 2 was monitored by electron absorption spectroscopy (see 60 (cid:1)C over 30 min in an oil bath under inert N . To this Figure3).UponeachadditionofIF,anincreaseinabsorp- 2 solution,1previouslydissolvedinDMSOwasaddeddrop- tion due to binding was observed consistent with previous wise.Thismixturewaslefttostandwhilebeingstirredunder literature.37,38SaturationoftheIFbindingsitewasreached N for6h(seeScheme2).RemovalofDMSOwasachieved whentheabsorbanceintensityremainedconstant.Thetotal 2 by precipitating the crude product in a 1:15 acetone/ether overallvolumewasnoted,andthefinalconcentrationof2 solution.Acrimsonsolidwascollectedviacentrifugationat boundtoIF(2-IF)wascalculated. ∼4000rpmover10min.Ifthesupernatantremainedpink, UptakeExperimentsviaFluorescentConfocalMicroscopy additionof1-2mLofacetoneaidsintheprecipitation.The of 2. To the BeWo cell line, a total volume of ∼1.5 mL of redsolidwasthenredissolvedin1:4acetonitrile/waterand 10μM2-IFwasadded.Thecellswerethenincubatedat37(cid:1)C subsequently purified via C reverse phase high pressure overtwoseparateperiodsof∼45minor6htoallowdrug 18 5256 JournalofMedicinalChemistry,2009,Vol.52,No.16 Viola-Villegasetal. Figure3. Intrinsicfactorbindingof2isindicatedbyanincreasein absorptionintensityduetofurtherloweringofsymmetryofB . 12 influx. The solution of 2-IF was then removed via pipet. Figure4. Cellbindingandinternalizationof2-IFafter∼45minof Subsequent washing with RPMI 1640 and 1(cid:2) PBS buffer incubationtakenat63(cid:2)showingimagesofBeWo(a)collectedbya (pH ≈ 7.4) in triplicate was performed with and without monochromatictransmittedlightphotomultipliertube(TMPT-1) and(b)afterexcitationat488nmwithfluorescentgreenemissionat ethanol fixation. Intracellular fluorescence was observed ∼560nm,consistentwithrhenium(I).(c)Themergedimagesafter after 45 min of drug exposure as shown in Figure 4. This simultaneousscansshowedilluminationinthenucleusandinthe indicates rapid entry of the drug into the cells and is cytosol. consistentwithreceptormediatedendocytosis. To further prove that internalization indeed occurred, Finally,tofurthersupportthehypothesisthat2-IFgained optical slicing at ∼1 μm per slice was conducted as shown cellular entry via receptor mediated endocytosis, a pH- inFigure5asagalleryview(seeSupportingInformationfor sensitivecyaninedyeCypHer5EwasconjugatedtoIF.This movie).Fluorescenceinthecellularmilieuwasonlyobserved cyaninedyehasapK of∼7.3andisfluorescent(red)onlyin a inthemiddlesections,confirmingtheinternalizationof2-IF. acidicconditions.Internalizationofthisdyethroughrecep- CompetitivebindingwithB against2-IFwasinvestigated tormediatedendocytosisprovidesanacidicenvironmentof 12 byaddingexcessofvitamin.ExcessB atconcentrationsof ∼5.5intheendosome.ConjugationoftheN-hydroxysucci- 12 ∼100 μM and ∼10 mM were added first to the cells and nimide ester of this dye to IF was conducted according to incubatedatroomtemperaturefor∼5min.Then,2-IFwas manufacturer’s (GE Sciences) instructions. A volume of added to the cells. The final concentration of 2-IF was 1mLwasthenaddedtoaplatecontaining∼200000BeWo ∼10 μM. The cells were then incubated for ∼45 min, after cells and incubated for 45 min. As shown in Figure 6, whichthemediumcontaining2-IFandB wasdiscarded.The fluorescence was observed. The red flouresence of the dye 12 cellswerethenwashedwithmedium1(cid:2) PBS(pH≈7.4) in counterstained with the green of 2 appears mostly yellow, triplicate to remove residual 2-IF. On the basis of observa- although some red can still be seen. This provides further tions,additionofa100-foldexcessof100μMB didlittleto evidencethatuptakeof2occursbyanIFreceptor(cubilin) 12 block binding of 2-IF complex to the cubilin receptor. At mediatedprocess. higherconcentrationsofB (10mM)however,cellularaccess In Vitro Antiproliferative Cell Assay. To determine 12 andaccumulationof2-IFwerecompletelyinhibited.Inaddi- whether cytotoxicity was significant for the transported tion, there was no observed fluorescence even after 6 h of conjugates, BQBA, 1, 2, and 2 bound to IF preincubation incubationundertheseconditions,indicatingthattheexcess (2-IF) were screened at a range of concentrations against B wasclearlyinhibitinguptakeof2-IF.Tofurthersupport BeWo and CHO cells. The cells were cultured to ∼90% 12 evidencethatIFiscriticalfortheB -conjugatetransportand confluence.Approximately,8000cells/wellwereinoculated 12 that entry is gainedthroughthe cubilinreceptor, the BeWo intoa96-wellplate.Theplatewasthenincubatedfor24hto cellswereexposedto2unboundtoIF(i.e.,2only)andwith assist in adherence. Stock solutions of the drugs were pre- prioradditionof10mMexcessB todetermineuptake.No paredwithRPMI1640media.Arangeofconcentrationsfor 12 fluorescencewasobservedat45minandevenafter6hforcells eachdrugwaspreparedwithavolumeof∼100μLofeach withpriorexposuretoexcessB .Bysaturatingthecellswitha addedtothecells.Thecellswerethenexposedtothedrugs 12 1000-foldexcessofB however,wenotedthattransportof2 overa6htimeperiod.Thedrugsolutionswerethenremoved 12 wasshutdown,presumablysincenownofreeIFisavailable from the plates, and the cells were washed with media to and the fact that the B -transport proteins have a greater removeanydrugresidues.A10%solutionofCCK-8dyein 12 affinity for the unmodified B .32 This lower affinity of RPMI 1640 supplemented with fetal bovine serum and 12 derivatized B was previously established in the binding penicillinstreptomycinsolutionwasprepared.Avolumeof 12 kinetics studies of Fedosov et al., wherein a B analogue 100μLofthisdyewasthenaddedtotheplatesandincubated 12 modifiedwithrhodamineatthe50-OHribosylgroupofB for24h.ThecalculatedIC valuesareshowninTable1. 12 50 bindstoIFataloweraffinityby2ordersofmagnitude(K ≈ NoIC valueswereobtainedforBQBAupto5mM.The D 50 10-13)thancyano-B (K ≈10-15).32 rheniumcomplex1,however,displayedanIC of376μM, 12 D 50 Chinese hamster ovary cells were investigated for any 10-foldmoretoxicthantheB conjugate2.1isrationalized 12 uptakeof2-IFasacontrol.Therewasnoobservedfluores- tohavegreater, morerapidaccesstotheintracellularpool cenceinthiscelllinefor2-IF,indicatingalackofuptakeof due to passive diffusion.40 Passive diffusion of nutrients in thedrugviatheIFreceptor.Hence,thispointstoanabsence this cell line was reported to be indirectly proportional to ofthecubilinreceptor,assupportedbytheliterature.39 size,withthepermeabilitycoefficient,P ,displayingalinear e Article JournalofMedicinalChemistry,2009,Vol.52,No.16 5257 Figure5. Randomdepthlaseropticalslicesat∼1μmpersliceofBeWocellsshowfluorescenceinthemiddleslicesofthecell,confirming internalizationof2-IFafter45minofincubationat37(cid:1)C. Table1. IC Values(mM)ofBeWoandCHOCellsafter6hofDrug 50 Exposure IC (mM) 50 drug BeWo CHO BQBA a 2.979(0.210 1 0.376(0.028 2.627(0.280 2 3.180(0.258 a 2-IF 1.844(0.478 4.860(0.283 aNotcytotoxicatconcentrationsofupto5mM. cubilinreceptor.siRNAmediatedinhibitionofcubilinwith corresponding loss of uptake of 2-IF would provide con- clusive evidence that internalization of 2-IF in BeWo cells Figure6. RedfluorescencewasdisplayeduponexposureofanIF- proceedsviathecubilinreceptoruptakepathwayandestab- CypHer5EconjugatetoendosomalpHof∼5.5.2wasboundtothis lish2asaspecificbioprobeforthisreceptor.Afluoroscein conjugate via IF (confirmed by electronic absorption assay as in conjugateofrandomsequencesiRNA,knownnottoinhibit Figure3(datanotshown)). anymRNA,wasemployedasatransfectionuptakemarker. decrease with increasing molecular weights (MW: = 400- After transfection of the cubilin siRNA, the cells were 70000).41Inaddition,thepositivechargeofrhenium(I)may incubated at 37 (cid:1)C. Uptake experiments were conducted beprovidingelectrostaticinteractionswithmembranepro- after allowing the cells to grow over 24 h. The growth teins,providinganenhanceduptakeof1insynergywiththe medium was aspirated and 10 μM 2-IF was added to the passive permeation of the drug. Cell membrane studies transfected cells and control cells (no siRNA of any type conducted by Patillo et al. showed the BeWo cell lines as added) and incubated over 45 min. After incubation, the possessing a negative transmembrane potential of about drug-containing medium was discarded and the cells were -35mV.42Thisfasterdiffusionof1inBeWocellsmayalso then washed with 1(cid:2) PBS in triplicate to remove residual explain the difference in IC values compared to CHO 2-IF.Atbothtimepoints,confocalmicroscopyexperiments 50 (2.627mM)forthesamecompound,althoughrapidefflux showedilluminationforbothcontrolandtransfectedcells. may also play a role. 2 displayed an IC of 3.180 mM in Fluorescence intensity values differed markedly however. 50 BeWocells,whilenotoxicitywasobservedfor2intheCHO The control plate displayed a mean intensity of ∼4.0, line.Thisissupportedbyconfocalmicroscopyexperiments whereas the transfected cells had an average recorded in- of2withpriorexposuretolowerB concentrationsinBeWo tensityvalueof∼0.4.Thissignificantdecrease(∼10-fold)in 12 cellsshowingminimalfluorescence,indicatingloweruptake fluorescenceintensityinthetransfectedcellsisindicativeofa of 2. In CHO cells, there was no observed fluorescence, significant reduction of drug uptake and correlates with consistent with 2 not permeating the plasma membrane. knockdownofthecubilingene.Wealsoconducteduptake TheIC valuesfor2-IFinBeWocellsarecalculatedtobe experimentsafter48hoftransfection.Opticalslices(∼1μm) 50 ∼1.844mMhowever,significantlylowerthanthatfor2not of the transfected BeWo cells revealed internalization with previously bound to IF. This increased toxicity of 2-IF is varyingfluorescenceintensitiesrangingfrom0.81toasmuch most likely due to the greater uptake through the cubilin as3.7,consistentwiththecubilinreceptorswitchingbackto receptor. ThehigherIC concentration displayed inCHO expression upon multiple passages (the BeWo line has a 50 cells is most likely a consequence of this cell’s inability to doublingtimeof∼20h). internalize2-IF,inagreementwiththelackoffluorescence Propidium Iodide Counterstaining. To look at a possible observedinCHOcellsuponexposureto2-IF. mechanism of toxicity, propidium iodide (PI) counterstain siRNA Gene Knockdown of Cubilin Receptor. To fully wasutilizedtodeterminewhethernuclearlocalizationof2, establishtherouteofuptakeascubilinbased,smallinterfer- once transported, occurred. PI is a known intercalator for ing RNA (siRNA) specific for cubilin mRNA was trans- DNA43 and RNA.44 In addition, this dye is an excellent fectedintotheBeWocellstoknockdownexpressionofthe nuclear stain with cells that have been previously stained 5258 JournalofMedicinalChemistry,2009,Vol.52,No.16 Viola-Villegasetal. Figure7. Counterstainingwithpropidiumiodidealongwith2-IFshowsnuclearandcytosolicaccumulationof2asshowninthemerged(left) andseparatefluorescentimagesofcellsmarkedwithpropidiumiodide(center)and2(right). with a fluorescent dye such as 2. RNase A digestion for Allinvitrocellexperimentswereperformedinanair-filtered ∼5 min at room temperature was conducted along with and UV-irradiated Labconco Purifier I laminar flow hood. staining with PI to prevent staining of RNA and the cyto- Chinesehamsterovariancells(CHO)cellswereobtainedfrom plasm.45 Since this fluorophore is not able to permeate the American Type Culture Collection (ATCC) culture code CHO-K1.TheA2780/ADcelllineusedfortestingwasprovided throughthecellmembrane,PIisdissolvedinasolubilizing bytheFoxChaseCancerCentre,Philadelphia,PA.TheBeWo detergentsuchasTritonX-100andaddedtothecellpool. choriocarcinomahumancellline(ATCCnumberCCL-98)was Confocalimages,obtainedsubsequently,oftheBeWocells purchasedfromATCC.Fetalbovineserum(FBS)andCellgro showaggregationof2inthecytosolbutalsointhenucleus Cellstripper were purchased from Mediatech from Manassas, (see Figure 7). It is probable that the positively charged VA.Penicillin-streptomycinsolutionwith10000unitspenicil- rheniumchelatecomponentcaninteractwiththenegatively lin and 10 mg/mL streptomycin in 0.9% NaCl was obtained charged DNA backbone, and this may play a role in the fromSigma.InvitrogensuppliedRPMI16401(cid:2)growthmedia observedtoxicity. containingL-glutamineandphenolredwithandwithoutfolic acid. 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-di- Conclusion sulfophenyl)-2H-tetrazolium,monosodiumsaltsolution(WSK-8), waspurchasedfromDojindo. We have successfully demonstrated targeted delivery of a Purification using reverse phase high-pressure liquid chro- rheniumbasedbioprobetothecubilinreceptor.ThenewB 12 matography(RP-HPLC)wasconductedusinganAgilent1100 conjugateofrhenium,2,requiresintrinsicfactorandusesthe with manual injection and automated fraction collector. A IF-cubilin import mechanism of B . Confocal microscopy ZorbaxC analyticalcolumn(42mm(cid:2)10mm)wasutilized 12 18 results show internalization and accumulation of 2 bound to foranalyticalrunswithaflowrateof0.7mL/min.Purification IFinthenuclearandcytosolicregionoftheplacentalBeWocell of2wasmadewithaC 18 semipreparativecolumn(9.4mm(cid:2) 250 mm) at a flow rate of 2 mL/min. Detection was by line.SaturationofthisuptakemechanismwithexcessB shuts 12 ultravioletmonitoringat254or280nm.Elutionwasperformed downtherouteofentryof2.Thisisindicativeandconsistent using water (A) and MeCN (B) as solvents and with the withtheendocytoticmechanismofcubilin.Inaddition,siRNA followinggradient:(1)30%Band70%Ato40%Band60% inhibition of the cubilin gene significantly knocked down Aover8min,(2)40%Band60%Ato50%AandBover8 uptake of 2-IF. This conclusively points to the key role that min,4250%AandBto100%Bover5min(t =13.7min). R thecubilinreceptorisplayingintheinternalizationof2-IF.IF Protonnuclearmagneticresonance(1HNMR)wasperformed mediateddeliveryofB conjugatestocubilinoverexpressing usingBrukerAvanceDPX500MHzandBrukerAvanceDPX 12 malignanciesisachievableandshowsimmensepotential.This 300 machines. Electrospray ionization (ESI) and matrix as- receptor in particular makes for a suitable biomarker for sosted laser desorption ionization time-of-flight (MALDI- targeting cancers in the gastrointestinal26 and renal tissues,27 TOF) mass spectrometry analyses were carried out on a Shi- and the bioprobe itself can be readily switched to a 99mTc madzuLCMS-2010AmassspectrometerandBrukerAutoflex derivativeinspiredbytheB -99mTcconjugateofAlbertoetal.46 MALDI-TOF, respectively. A Perkin-Elmer ELAN 6100 was 12 usedtoconductinductivelycoupledplasmaanalysis(ICP).A Varian Cary 50 Bio UV-vis spectrophotometer recorded the ExperimentalSection electronic absorption spectra of the compounds and was also Materials.Allreactionswereperformedunderaninertatmo- used for intrinsic factor binding studies. Centrifugation was sphereofargonornitrogenusingstandardSchlenktechniques. performedfor10minat4000rpmat4(cid:1)CwithaSorvallLegend Dimethyl sulfoxide (DMSO, 99%, Sigma) was dried by char- RTcentrifuge.Circulardichroism(CD)experimentswerecon- gingthroughacolumnof4A˚ molecularsieves(Mallinckrodt) ducted with an Aviv model 202 spectrometer at 25 (cid:1)C in the previouslydriedovernightat120(cid:1)C.Reagentslistedbelowwere wavelength range of 200-350 nm. The CD experiments were purchasedandusedwithoutfurthermanipulations:vitaminB performedusingaquartzcuvettewithapathlengthof2mm. 12 (B , 99%, Sigma), rhenium(I) pentacarbonyl bromide (98%, ConfocalmicroscopyexperimentswereconductedwithaZeiss 12 Sigma) 1,10-carbonyl-di-(1,2,4-triazole) (g90%, CDT, Fluka), LSM 700 Pascal confocal microscope with Zen 2008 image quinoline-2-carboxaldehyde(98%,AlfaAesar),sodiumtriace- analysis software equipped with argon ion and HeNe lasers. toxyborohydride (95%, Sigma), 1,2-dichloroethane (DCE, IRspectrawereobtainedonaPerkin-Elmerseries1600FT-IR g99%,Sigma),1,2-dichloromethane(DCM,g99.5%,Sigma), spectrometerintheregion400-4000cm-1withpolystyreneasa methanol (MeOH, g99.8%, Sigma), acetonitrile (MeCN, reference. g99.8%,Sigma),sodiumsulfate(g99.9%,anhydrous,Sigma), Synthesis of [BQBA(Re(CO) ]þ (1). Amounts of 0.1968 g 3 intrinsicfactor(IF,MPBiomedicalsLLC).siRNAtransfection (1.045 mmol) of N-Boc-1,4-diaminobutane and 0.3450 g reagentswerepurchasedfromSantaCruzBiotechnology.Calf- (2.195 mmol) 2-quinolinecarboxaldehyde were dissolved in thymus(CT-DNA)andpropidumiodide(PI)werepurchased 30 mL of dichloroethane under argon and allowed to stir for from Calbiochem. Compounds were confirmed to be >95% 30min.Then,0.6646g(3.136mmol)ofsodiumtriacetoxyboro- purebyHPLC,1HNMR,and/orICP. hydridewasaddedtothepurplereactionmixture,andstirring Article JournalofMedicinalChemistry,2009,Vol.52,No.16 5259 wascontinuedovernight.Aftercompletionofthereaction,the solutioncontainingIF.Anincreaseinabsorbanceindicatesfor- solvent was removed followed by the addition of 30 mL of mationofabinaryB -IFcomplex.34,43Theresultingfinalsolu- 12 methanoltodestroyexcessreducingagent.Afterthegasevolu- tionwithIF-B binarycomplexformedwassubsequentlyused 12 tionceasedthesolventwasremovedoncemoreandthecrude forconfocalmicroscopystudiesandcytotoxicityassays. reaction mixture purified by basic alumina chromatography ConfocalMicroscopyUptakeExperiments.Thehumanchor- usingethylacetateaseluent.Thefirstfractionelutedcontained iocarcinoma placental cell line (BeWo) and Chinese hamster thepurpleproduct.1HNMR(300MHz,CDCl ):δ=8.04- ovarycells(CHO)weregrownandculturedasadherentmono- 3 7.96(m,4H),7.70-7.56(m,6H),7.40(m,2H),4.67(s,1H),3.92 layersinaventedflaskwitha175cm2culturearea,filtervented (s,4H),2.96(m,2H),2.54(t,J=7.07Hz,2H),1.53(m,2H), closecap,andangledneck.Thesecells weregrownto >85% 1.32(m,11H)ppm.Yield:74%(0.3640g). confluence. Amounts of 0.3640 g (0.774 mmol) of tert-butyl 4-(bis- BeWo placental cells and Chinese hamster ovary cells ((quinolin-2-yl)methyl)amino)butylcarbamate(BQBA)and (∼200000cellsperdish)wereplatedon35mm(cid:2)100mmglass 0.4042g(0.774mmol)of[Re(H O) (CO) ]Brwereheatedunder bottom vented dishes (Martek). The cultures were allowed to 2 3 3 argonat60(cid:1)Cin30mLofmethanolfor3h.Thesolventwas adhereontothedishsurfaceover24hinRPMI-1640mediaat removed and the crude mixture purified by basic alumina 37(cid:1)C.Tothesecells,2-IFwithafinalconcentrationof∼10μM chromatography with a gradient of ethyl acetate/methanol. with and without prior addition of excess B (100 μM and 12 Unreactedligandwaselutedwithethylacetatefollowedbythe 10mM)wasadded.Cellswerealsoincubatedwith10μM2(i.e., elutionofthemetalcomplexwithethylacetate/methanol,10:1. unboundtoIF)andexcessB (10mM).Thedisheswerethen 12 1HNMR(300MHz,MeOH-d ):δ=8.46(t,J=8.92Hz,4H), incubatedat37(cid:1)Cover45minand6h.Thesolutionswerethen 4 7.94 (dd, J = 8.09, 1.37 Hz, 2H), 7.80 (ddd, J = 8.70, 7.06, removed,andthecellswerewashedwith1mL1(cid:2)PBS(pH≈ 1.57Hz,2H),7.64(m,2H),5.18(d,J=17.74Hz,2H),5.00(d, 7.4)intriplicate.Fluorescencewasthenobservedwithafluor- J=17.81Hz,2H),3.09(t,J=6.72Hz,2H),1.95(m,2H),1.57 escent confocal microscope with intensities recorded at 63(cid:2) (m,2H),1.33(s,9H)ppm.ESIMS:740.65m/z[Mþ],calculated magnification. 741.21m/z.Yield:38%(0.241g). PropidiumIodideStaining.BeWocellswerepreviouslyplated Anamountof0.241g(0.294mmol)ofrheniumtricarbonyl (∼200000 cells per dish) in 35 mm (cid:2) 100 mm glass bottom tert-butyl 4-(bis((quinolin-2-yl)methyl)amino)butylcarbamate venteddishes(Martek)andincubatedovernight.Avolumeof bromide was dissolved in 50 mL of 3 M hydrochloric acid, 1mLof10μM2-IFwasaddedtothecellsandincubatedat37(cid:1)C andthemixturewasstirredatroomtemperatureovernight.The for45min.Themediumcontainingthedrugwasdiscardedand solvent was removed under reduced pressure to give 1 as a replacedwith∼1mLofethanol(chilledat∼20(cid:1)C).After3min, brownishpowder.1HNMR(300MHz,MeOH-d ):δ=8.45 theethanolwasremoved.Asolutioncontaining20μLof1mg/ (dd,J=13.56,8.62Hz,4H),7.95(d,J=7.34Hz,2 4 H),7.80(m, mLPI,200μLof10μg/mLRNaseA,5μLofTritonX-100,and 2H),7.66-7.59(m,4H),5.23(d,J=17.80Hz,2H),5.09(d,J= 4.78mLof1(cid:2)PBSbufferwasprepared.Avolumeof1mLof 17.96Hz,2H),3.88(m,2H),3.01(m,2H),2.07(m,2H),1.80(m, thissolutionwasaddedtotheBeWocells,andthemixturewas 2H)ppm.ESIMS:640.90m/z[Mþ],calculated641.16m/z.IR incubatedinthedarkfor5min.Imageswerecollectedat63(cid:2) (KBrpellet):ν=3447,2017,1880cm-1.Yieldwasquantitative. magnificationusingafluorescentconfocalmicroscope. Synthesis of B -[BQBA(Re(CO) ]þ (2). B (0.0250 g, DrugCytotoxicity.Theproliferationoftheexponentialphase 12 3 12 0.0369 mmol) was activated with 1.2 mol equiv of CDT cultures of BeWo and CHO cells was assessed by WSK-8 (0.0440 g, 0.0244 mmol) in 3 mlL of DMSO. The solution colorimetric assay. This assay follows ATP production and washeatedwhilebeingstirredat60(cid:1)CunderN for30min.7 indicatesmitochondrialhealth.WSK-8wasperformedaccord- 2(g) (0.0142g,0.0221mmol)in2mLofDMSOwasaddeddropwise ing to manufacturer’s instructions (Dojindo). Adherent cell tothissolution,andthemixturewasstirredatroomtempera- cultures were harvested by stripping of culture flasks using a turefor6 h.A crimson solidwas collectedafterthesolution nonenzymaticcellstripperandaftera30minincubationperiod. wasprecipitatedina1:15acetone/ethermix.Thepowderwas To eachwell, aliquotsof 100μLwereinoculated,resulting in thenpurifiedusingC RP-HPLCwitharetentiontimet = 8000cellsperwell.Theplateswerethenincubatedovera24h 18 R 13.7min.1HNMR(300MHz,DMSO-d ):δ=8.83(d,2H), periodtofacilitateadherence.Serialdilutionsofdrugs1,2,2-IF, 6 8.50(d,2H),8.32(d,2H),8.22(d,2H),8.08(m,3H),7.88(t, andBQBAwereprepared.Avolumeof100μLofthedifferent 2H),7.83(s,1H),6.93(s,1H),6.68(s,1H),6.60(s,1H),6.06(s, drugconcentrationswasthenaddedtothewellsandincubated 1H) ppm. MALDI-TOF MS: 2022.37 m/z [M þ], calculated for6h.Thedrugswereremovedfromthewellsandrinsedwith 2022.09m/z.Yield:15.3%(3.8mg)basedonB . growth medium. A solution containing 10% WSK-8 dye in 12 Cell Culture and Growth. Cells were grown in Millipore growthmediumwithavolumeof100μLwasthenaddedtoeach 250mLculturebottleswithventedlids.Chinesehamsterovary ofthemicroculturesandincubatedover24h.Opticaldensities (CHO,ATCCcodeCCL-61)celllineandhumanBeWochor- were measured using a plate reader (Thermo Multiskan EX iocarcinoma cell line (ATCC code CCL-98) were cultured as plate readerequippedwithAscent software,version2.6).The adherent monolayers in RPMI 1640 growth media (Gibco) percentageofcellviabilitywasdeterminedrelativetountreated supplemented with 10% fetal bovine serum (Hyclone) and controlmicrocultures.TheIC concentrationswerecalculated 50 10000unitsofpenicillinand10mg/mLstreptomycin(Sigma). onthebasisofanexponentialfitusingOriginLabs8software CellswereharvestedusingCellstripper(Mediatech,Manassas, withR2g0.80inallcases.Thisassaywasconductedintriplicate VA). The cells were then collected via centrifugation, and witheachpointintriplicateonseparate“batches”ofcompound the solution containing Cellstripper and media was disposed. oncells. Resuspension of the cells proceeded by addition of 5 mL of siRNA Gene “Knockdown” of the Cubilin Receptor. The completegrowthmediumandaspiratedgentlybypipetting.All siRNA transfection experiment was conducted according to cell cultures were grown and incubated in mammalian cell manufacturer’sinstructions.ThecubilinsiRNAwasdissolved incubatormaintainedat37(cid:1)Cwith5%CO and95%humidity. in 330 μL of RNase-free buffer. For each transfection, the 2 Allcell-basedassayswereperformedusingcellsinexponential following solutions were prepared. Solution A was prepared growth. by mixing 6 μL of the cubilin siRNA with 100 μL of siRNA IntrinsicFactorBindingto2.IFweighing10mgwasdissolved transfection medium. Solution B contained 6 μL of siRNA in2mLof1(cid:2)phosphatebufferedsaline(PBS)(pH≈7.4)and transfection reagent and 100 μL of transfection medium. The rotatedovernightin4(cid:1)C.Theundissolvedsolidswereremovedvia twosolutionsweremixedandincubatedatroomtemperature centrifugation.Theelectronicabsorptionspectraof2(20-50μM) for30min.Avolumeof2mLoftransfectionmediumwasused werethenmonitoreduponadditionof5-10μLincrementsofthe to wash a plate of cells (∼200000) previously prepared and 5260 JournalofMedicinalChemistry,2009,Vol.52,No.16 Viola-Villegasetal. allowedtogrowinpenicillin-streptomycinfreegrowthmedium 103(12),4386–4391.(b)Garau,G.;Fedosov,S.N.;Petersen,T.E.; overnight.TothemixturecontainingsolutionsAandB,0.8mL Geremia, S.; Randaccio, L. Crystallization and preliminary X-ray of transfection medium was added. This solution was then diffractionanalysisofhumantranscobalamin,avitaminB 12 -transport- addedtothecellsandincubatedfor5-7hat37(cid:1)C.Avolume ingprotein.ActaCrystallogr.,Sect.D:Biol.Crystallogr.2001,57 (Part12),1890–1892. of1mLofgrowthmediumcontaining2(cid:2)FBSandpenicillin- (16) Bauer, J. A. Synthesis, characterization and nitric oxide release streptomycin was added to the cells without removing the profileofnitrosylcobalamin:apotentialchemotherapeuticagent. transfection solution. The cells were then incubated for an Anti-CancerDrugs1998,9(3),239–244. addition 18 h, after which the medium was discarded and (17) Smeltzer,C.C.;Cannon,M.J.;Pinson,P.R.;Munger,J.D.,Jr.; replacedwithfresh1(cid:2)growthmedium.After24h,thetrans- West, F. G.; Grissom, C. B. Synthesis and characterization of fected cellswerethenexposedto10μM2-IFfor45min. The fluorescentcobalamin(CobalaFluor)derivativesforimaging.Org. Lett.2001,3(6),799–801. mediumcontaining2-IFwasremoved,andthecellswerewashed (18) Bose,S.;Seetharam,S.;Seetharam,B.Membraneexpressionand with1(cid:2)PBSintriplicate.Uptakeof2-IFoncubilinknocked interactionsofhumantranscobalaminIIreceptor.J.Biol.Chem. downversuscubilinexpressingBeWocells(i.e.,notransfection 1995,270(14),8152–8157. performed) was compared via fluorescence confocal micro- (19) Seetharam, B.; Bose, S.; Li, N. Cellular import of cobalamin scopy. (vitaminB-12).J.Nutr.1999,129(10),1761–1764. (20) Seetharam,B.;Levine,J.S.;Ramasamy,M.;Alpers,D.H.J.Biol. Chem.1988,263,4443–4449. Acknowledgment. The authors thank Colin Fuss (CESE, (21) Moestrup, S. K.; Kozyraki, R.; Kristiansen, M.; Kaysen, J. 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