Ruthenation of Non‐stacked Guanines in DNA G‐Quadruplex Structures: Enhancement of c‐MYC Expression

In e na ionale Ausgabe:DOI:10.1002/anie.201607965 G-Quad uplexes Deu sche Ausgabe:DOI:10.1002/ange.201607965 Ru hena iono Non-s acked Guanines in DNAG-Quad uplex S uc u es:Enhancemen o c-MYC Exp ession J8ssica Rod &guez, JesfflsMosque a, Jos8R. Coucei o,M.Eugenio V#zquez,* and Jos8L. Masca eÇas* Abs ac : Guanine quad uplexes(GQs) a e compac ou - s anded DNAs uc u es ha playakey ole in he con ol o a a ie y o biological p ocesses,including gene ansc ip ion. Bulky u henium complexes ea u ing abipy idine,a e py - idine,and one exchangeable ligand ([Ru( e py)(bpy)X]n+)a e able o me ala e exposed guanines p esen in he GQ o he c- MYC p omo e egion ha a e no in ol ed in quad uplex base pai ing.qRT-PCR and wes e n-blo expe imen s indica ed ha he complexes p omo e a ema kable inc ease in he exp ession o his oncogene.Wealso show ha exchangeable hioe he ligands (X=RSR’,Me ) allow egula ion o he me ala ing ac i i y o he complex wi h isible ligh . The e is g ea in e es in he de elopmen o me al-based DNAbinde s ha show imp o ed selec i i y and educed oxici y ela i e o cis-pla inum.[1] Ru henium complexes a e p omising al e na i es owing o hei kine ic s abili y and ich pho ochemis y and edox p ope ies.[2] In addi ion o non- co alen ecogni ion o double-s anded DNA(dsDNA), se e al u henium complexes ha o m co alen DNA adduc s,especially h ough eac ion wi h he N7o guanines, ha e also been desc ibed.[3] While mos o hese complexes bind dsDNA, he unc ional ele ance o G-quad uplexes (GQs)[4,5] calls o he de elopmen o p obes capable o a ge ing hese s uc u es.[6] To ou knowledge, he e is only one p eceden o he co alen me ala ion o aGQs uc u e wi h a u henium complex, and he eac ion p esen s low selec i i y.[7] He ein, we demons a e ha coo dina ion complexes o he ype [Ru( e py)(bpy)X]n+(X=Cl, RSR’,Me ) can selec i ely me ala e unpai ed guanines p esen in pa allel GQs,a eac ion ha is enhanced upon i adia ion. Impo - an ly,weha e ound ha his selec i e me ala ion inc eases he exp ession o he oncogene c-MYC,appa en ly by dis up ing he pa allel GQ s uc u e p esen in i s p omo e egion. Ou wo k was concei ed a e lea ning ha while mos DNA-me ala ing u henium agen s a e cy o oxic, he com- plex [Ru( e py)(bpy)Cl]+(1)exhibi s e y low oxici y.[8] We easoned ha he bulky and ela i ely hyd ophobic na u e o his so o complexes could o e excellen oppo uni ies o he selec i e modi ica ion o accessible guanines, he eby p omo ing speci ic biological esponses wi h educed oxici y. We i s s udied he abili y o chlo o complex 1 o me ala e guanosine monophospha e (GMP). Mixing complex 1wi h 3equi o GMP in phospha e bu e (pH 7.5), led o he pa ial o ma ion o he aquo complex [Ru( e py)- (bpy)H2O]+2(2;o e 50%a e 30 min a RT), while he guanosine emained essen ially un eac ed (Figu e 1B, ace b). Fu he incuba ion o 2ha o ded he me ala ed p oduc 3and he aquo de i a i e 2(Figu e 1A), wi h o al consump ion o he s a ing chlo ide (Figu e 1B, ace d). Figu e 1. A) GMP me ala ion eac ion wi h [Ru( e py)(bpy)Cl]PF6([Ru]- Cl, 1)and o ma ion o he aquo de i a i e 2.B)HPLC o he eac ion o 1(250 mm)and GMP (750 mm)in10mmphospha e bu e pH 7.5, 100 mmNaCl:in he da k a =0( ace a);a e 30 min in he da k ( ace b);ini ial mix u e a e 30 min o i adia ion a 455 nm ( ace c); and ini ial mix u e a e 2hin he da k ( ace d). The injec ion peak is labeled wi h an as e isk. C) Signal in he MS spec um co esponding o he monoadduc 3. [*] J. Rod &guez,D . J. Mosque a,D . J. R. Coucei o, D .M.E.V#zquez, P o . J. L. Masca eÇas Cen o Singula de In es igaciknenQu&mica Biolkxica eMa e iais Molecula es (CIQUS), and Depa amen o de Qu&mica O g#nica Uni e sidade de San iago de Compos ela 15782 San iago de Compos ela (Spain) E-mail:j[email p o ec ed] [email p o ec ed] Suppo ingin o ma ion and he ORCID iden i ica ion numbe (s) o he au ho (s) o his a icle can be ound unde h p://dx.doi.o g/10. 1002/anie.201607965. T2016 The Au ho s. Published by Wiley-VCH Ve lag GmbH &Co. KGaA. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi suse and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica- ions o adap a ions a e made. A ngewand e Chemie Zusch i en 15844 T2016 Die Au o en. Ve ç en lich onWiley-VCH Ve lag GmbH &Co. KGaA, Weinheim Angew.Chem. 2016,128,15844 –15847 In e es ingly,i adia ion o he ini ial mix u e o 30 min (l= 455 nm) led exclusi ely o he o ma ion o he monoadduc 3 (app oxima e con e sion o 80%based on he disappea ance o GMP;Figu e 1B, ace cand Figu e S5 in he Suppo ing In o ma ion). Impo an ly, he eac ion is ully o hogonal, so compe i i e con ol expe imen s in he p esence o excess adenosine monophospha e (AMP), cy idine monophospha e (CMP), and hymidine monophospha e (TMP;Figu e S6), as well as lysine o e en cys eine (Figu e S7), led exclusi ely o he o ma ion o he GMP de i a i e.Mo eo e ,expe imen s wi h double-s anded oligonucleo ides (dsDNAs) p esen ing di e en a angemen s o guanines e ealed modes eac i - i y,bu only wi h hose ea u ing e minal guanines (Table S1, and Figu es S10–S13 in he Suppo ing In o ma ion). This selec i i y mos likely s ems om he bulkiness o complex 1, which canno each he s e ically hinde ed nucleophilic N7 si e in pai ed, in e nal guanines.[9] Rema kably,i adia ion o amix u e o he pa allel c- MYC quad uplex d[TTGAG3TG3TAG3TG3TA3][10] (10 mm) wi h 5equi o 1in 10 mmphospha e bu e (pH 7.5) and 100 mmKCl led o he clean o ma ion o ap oduc wi h amass co esponding o amonoadduc de i a i e (Fig- u e 2A, ace C, peak a 22 min, MYC-[Ru],81% con e - sion). We also obse ed eac ion in he absence o ligh (app ox. 41%con e sion a e 30 min a RT,Figu e 2A, ace b). Impo an ly,MSanalysis o he bo ine spleen phosphodies e ase (BSP) diges ion o he u hena ed oligo- nucleo ides lead o iden i ica ion o he i s guanine o he sequence (5’-TTGA…, Figu e 2C)as he me ala ion si e (Figu e S24).[11] No eac ion was obse ed unde he same condi ions wi h amu a ed c-MYC GQ con aining aCins ead o aGin posi ion 3(MYCm,d[TTCAG3TG3TAG3TG3TA3]; Figu e S18). Thechemoselec i i y o he eac ion can be a ionalized by conside ing he seconda y s uc u e o he c-MYC GQ (Figu e 3), which shows ha G3 is exposed o he sol en and does no pa icipa e in he o ma ion o he G-qua e s.As aconsequence,i has ahighly accessible N7nucleophile ha can eac wi h he bulky u henium complex. Impo an ly, ci cula dich oism expe imen s e ealed ha he GQ secon- da y s uc u e is dis up ed in he u henium adduc MYC- [Ru] (Figu e S3, igh ). Con ol expe imen s wi h o he ela ed pa allel quad uplex such as c-KIT1,which also p esen s non-s acked guanines, e ealed asimila eac i i y pa e n. Mass spec ome y shows ha he p omo e is modi ied by he u henium complex a he expec ed posi ions (Figu es S21 and S22), al hough cu iously,in his case,CD analysis e ealed ha he quad uplex emained mos ly in ac (Figu e S4).[12] Wi h he abo e molecula in o ma ion, we explo ed whe he Ru complex 1could a ec he exp ession o he oncogene c-MYC,since his oncogene is in ol ed in many impo an cellula p ocesses.[13] This was analyzed by eal- ime quan i a i e e e se ansc ip ion PCR (qRT-PCR) in HeLa and Ve o cells (Figu e 4and Figu e S26, espec i ely), using po phy in TMPyP4, aGQbinde ha is known o ep ess he exp ession o c-MYC,[14] as acon ol. As shown in Figu e 4, HeLa cells ea ed wi h 1(100 mm)inDulbeccoQs modi ied EagleQsmedium (DMEM) p esen ed amodes bu signi ican inc ease in he ansc ip ion o c-MYC compa ed o un ea ed cells (80%a 16h and 200%a 48h).[15] As expec ed, ea men wi h TMPyP4 led o a60% dec ease in he cellula le els o c-MYC mRNAa e 48 h. We also analyzed he exp ession o c-MYC p o ein by wes e n blo . In ag eemen wi h he qRT-PCR esul s, ea - men o cells wi h 100 mmo 1,led o ano iceable inc ease in he le els o c-MYC p o ein (Figu e 4, a e aged 40% Figu e 3. S uc u e o he GQ o c-MYC (PDB ID:1XAV), highligh ing he exposed G3and he me ala ion selec i i y.The la ge sphe es in he quad uplex s uc u e ep esen po assium ions. Figu e 2. A) HPLC aces o amix u e o he GQ o c-MYC (MYC, 10 mm)and 1(5 equi ) in 10 mmphospha e bu e pH 7.5, 100 mm KCl, a RT:in he da k a =0( ace a);a e 30 min in he da k ( ace b), ini ial mix u e a e i adia ion o 30 min a 455 nm ( ace c). B) MS o he me ala ed p oduc (MYC-[Ru]) showing he peaks co esponding o he complex (m/z=8089) and he deme ala ed agmen (m/z=7600). C) MS o he p oduc s a e BSP diges ion o MYC-[Ru] showing he peaks co esponding o he diges ed complex (m/z=7489) and non-me ala ed diges ed p oduc (m/z=7000) esul - ing om he clea age a G3as shown by he dashed line in he s uc u e. A ngewand e Chemie Zusch i en 15845Angew.Chem. 2016,128,15844 –15847 T2016Die Au o en. Ve ç en lich on Wiley-VCH Ve lag GmbH &Co. KGaA, Weinheim www.angewand e.de inc ease). These da a con i m ha , in con as o mos quad uplex a ge ing agen s,complex 1p omo es an inc ease ins ead o adec ease in he le el o gene exp ession, he eby ac ing as a ansc ip ional ac i a o .[16] ICP-MS measu emen s o isola ed nuclei and ch oma in ob ained a e ea men wi h complex 1con i med he p esence o ela i ely signi i- can amoun s o u henium, which is consis en wi h e icien cellula up ake and nuclea deli e y o he complex (Tables S2 and S3). In ag eemen wi h he ea ly s udies,[8] cell iabili y assays con i med ha 1is essen ially non- cy o oxic (Figu e S27). While he abo e da a indica e ha 1is capable o al e ing he exp ession o c-MYC, he de elopmen o de i a i es ha could be ac i a ed by using ex e nal i adia ion would be highly a ac i e.[17] Towa ds his aim, we p epa ed complexes 4and 5,which ea u e a hioe he ligand (Figu e 5) and a e kine ically s able bu unde go apid ligand exchange upon i adia ion wi h isible ligh .[18] In con as o he chlo ide complex 1,in i o expe imen s wi h he hioe he de i a i e 4showed ha i does no eac wi h he c-MYC quad uplex in he da k a e 30 min, bu yields he desi ed GQ monoadduc upon i adia ion (71%con e sion, Figu e S17). Theace yl- me hionine de i a i e 5p esen s e en highe kine ic s abili y han complex 4,and no aces o he me ala ion adduc s,o e en o he aquo de i a i e 2,we e obse ed a e se e al hou s in he da k. Howe e ,i adia ion o 30 min igge s e icien co alen me ala ion o he GQ (Figu e S20). We also analyzed he e ec o complex 5on ansc ip ion o he c- MYC gene by qRT-PCR in Hela cells.Asshown in he Figu e 5(le ), c-MYC mRNAle els inc eased a e i adi- a ion, becoming simila o hose obse ed a e ea men wi h he aquo complex 2.Wes e n-blo analysis con i med ha enhancemen o he gene exp ession only ook place in he p esence o ligh , while he le els o he p o ein c-MYC did no change when he cells we e kep in he da k (Figu e 5 and Figu e S29). As expec ed, bo h qRT-PCR and wes e n- blo expe imen s showed ha he aquo compound 2is ac i e bo h in he da k and unde i adia ion. Mo eo e ,con ol expe imen s con i med ha he i adia ion does no ha e any measu able e ec on cell iabili y (Figu e S28), and ha he complex does no gene a e signi ican amoun s o single oxygen upon i adia ion (Figu e S25). In conclusion, we ha e demons a ed he selec i e modi- ica ion o accessible guanines lanking he GQ o c-MYC wi h designed bulky u henium complexes.Impo an ly, he bioo hogonal me ala ion enhances he exp ession o he oncogene c-MYC.Gi en ha an inc ease in c-MYC an- sc ip ion has been shown o be impo an in se e al cance s,in pa icula o he enewal abili y o cance s em cells,[19] ou disco e y migh lead o in e es ing biological applica ions. Acknowledgemen s We hank he suppo by he Spanish g an s SAF2013-41943- R, CTQ2015-70698-R, and CTQ2013-49317-EXP, he Xun a de Galicia GRC2013-041, he ERDF,and he Eu opean Resea ch Council (Ad anced G an No.340055). Suppo o COST Ac ion CM1105, COST CM1306 and he o eo-cinqa ne wo k a e also kindly acknowledged. J.R. hanks he Xun a de Galicia o aPh.D. ellowship.We hank Rebeca Menaya Figu e 4. A) qRT-PCR analysis o c-MYC (ligh ba s) and ALAS1 (black ba s) ansc ip ion. Hela cells we e incuba ed o 16 ho 48hwi h ei he 100 mmRuCl, TMPyP4, o 50 mm5- luo ou acil(5-FU, adis up o o RNA syn hesis). Amoun s a e ela i e o he exp ession le els o he housekeeping gene GAPDH.Values a e he a e age o h ee expe i- men s, and e o ba s indica e he s anda d e o .B)Analysis o he exp ession o c-MYC by wes e n-blo . HeLa cells we e incuba ed o 16 hwi h 100 mmcompound 1and hen lysed, and c-MYC was de ec ed by SDS-PAGE, ollowed by wes e n blo wi h an an i-MYC An ibody (uppe panel). The ela i e amoun o p o ein in wo independen expe imen swas quan i ied by densi ome y (lowe panel). Da a a e ep esen ed as he old change wi h espec o un ea edcon ols. E o ba s indica e he s anda d de ia iono he old change wi h espec o un ea ed con ols. Figu e 5. A) Thioe he complexes 4and 5.B)c-MYC ansc ip ion as measu ed by qRT-PCR in he da k (black ba s) o a e i adia ion (60 min, ligh ba s). Hela cells we e incuba ed o 16 hwi h 100 mm RuMe (5)o aquo complex 2.RNA le els a e gi en ela i e o he exp ession le els o he housekeeping gene GAPDH.C)Exp ession o c-MYC as measu ed by wes e n blo in he p esence o absence o compound 5.The ela i e amoun o p o ein wi h espec o ha o b- ac in was quan i ied by densi ome y (lowe panel). The expe imen al p ocedu es a e as desc ibedinFigu e 4. A ngewand e Chemie Zusch i en 15846 www.angewand e.de T2016Die Au o en. Ve ç en lich on Wiley-VCH Ve lag GmbH &Co. KGaA, Weinheim Angew.Chem. 2016,128,15844 –15847 o he help wi h he biological expe imen s and Ve lnica PiÇei o o he Unidad de An#lisis Elemen al (RIAIDT-USC) o he ICP analysis. Keywo ds: bioino ganic chemis y ·DNA damage ·me ala ion · pho ochemis y · u henium How oci e: Angew.Chem. In . Ed. 2016,55,15615–15618 Angew.Chem. 2016,128,15844–15847 [1] a) C. X. Zhang,S.J.Lippa d, Cu .Opin. Chem. Biol. 2003,7, 481;b)A.Le ina, A. Mi a, P. A. Lay, Me allomics 2009,1,458; c) A. Casini, C. G. Ha inge , A. Naza o ,P.J.Dyson, Top. O ganome . Chem. 2010,32,57. Fo e iews on DNAbinding, see:d)M.E.V#zquez, A. M. CaamaÇo,J.L.Masca eÇas, Chem. Soc.Re . 2003,32,338;e)E.Pazos,J.Mosque a, M. E. V#zquez, J. L. Masca eÇas, ChemBioChem 2011,12,1958. [2] a) C. A. Pucke ,J.K.Ba on, J. Am. Chem. Soc. 2009,131,8738; b) S. B. F icke , Dal on T ans. 2007,4903;c)W.H.Ang,A. Casini, G. Sa a, P. J. J. Dyson, O ganome . Chem. 2011,696,989; d) J. G. Vos, J. M. Kelly, Dal on T ans. 2006,4869;e)A.Ju is,V. Balzani,F.Ba igelle i, S. Campagna,P.Belse ,A. on Zelewsky, Coo d. Chem. Re . 1988,84,85. [3] a) S. Be anzos-La a, L. Salassa, A. Hab ema iam, O. No ako a, A. M. Piza o,G.J.Cla kson, B. Lisko a,V.B abec,P.J.Sadle , O ganome allics 2012,31,3466;b)A.F.A.Peacock,P.J.Sadle , Chem. Asian J. 2008,3,1890. [4] a) E. Hende son,C.C.Ha din, S. K. Walk, I. Tinoco,J .,E.H. Blackbu n, Cell 1987,51,899;b)P.Mu a , S. Balasab umanian, Cu .Opin. Gene . De . 2014,25,22; c) M. Ta sounas,M. Tijs e man, J. Mol. Biol. 2013,425,4782. [5] L. H. Hu ley, Biochem. Soc.T ans. 2001,29,692. [6] H. Be and,S.Bomba d, D. Monchaud,M.-P.Teulade-Fichou, J. Biol. Ino g.Chem. 2007,12,1003. [7] K. Wu,S.Liu, Q. Luo,W.Hu, X. Li, F. Wang,R.Zheng,J.Cui, P. J. Sadle , J. Xiang,Q.Shi, S. Xiong, Ino g.Chem. 2013,52, 11332. [8] O. No ako a, J. Kaspa ko a, O. V ana, P. M. an Vlie , J. Reedijk, V. B abec, Biochemis y 1995,34,12369. [9] Ru henium isbipy idyland ela ed complexes ha do no ea u e la ge a oma ic ligands,such as dppz, ypically do no display signi ican DNA-binding p ope ies:C.V.Kuma ,J.K. Ba on, N. J. Tu o, J. Am. Chem. Soc. 1985,107,5518–5523. [10] A. Amb us,D.Chen, R. A. Jones,D.Yang, Biochemis y 2005, 44,2048 (NMR s uc u e o his c-MYC GQ,PDB ID:1XAV). [11] BSP 5’-exonucleaseshows a es ed hyd olysis a o be o e u hena ed si es:K.Wu, W. B. Hu, Q. Luo,X.C.Li, S. X. Xiong, P. J. Sadle ,F.Y.Wang, J. Am. Soc.Mass Spec om. 2013,24,410. [12] c-KIT1 pa allel GQ:(d[TAG3AG3CGCTG3AGGAG3TT], 71%con e sion). The eac ion wi h c-KIT1 led o he o ma ion o wo new peaks,iden i iedbyMALDIasmono- and bis-adduc (see Figu es S4, S21, S22, and S23). No eac ion was obse ed when using amu a ed c-KIT quad uplex con aining Cins ead o Ginposi ions 17 and 20 (KITm,d[TAG3AG3CGCTG3AC- GACGGTT],Figu e S23), hus con i ming ha he me ala ion akes place in hese non-s ackedguanines. A e e ence o c- KIT1:M.C.Hein ich, C. L. Co less,G.D.Deme i, C. D. Blanke,M. on Meh en,H.Joensuu, L. S. McG ee ey,C.J. Chen, A. D. Vanden Abbeele,B.J.D uke ,B.Kiese,B.Eisen- be g,P.J.Robe s,S.Singe ,C.D.Fle che ,S.Silbe man,S. Dimi ije ic, J. A. Fle che , J. Clin. Oncol. 2003,21,4342. [13] a) C. Y. Lin, J. Lo en, P. B. Rahl, R. M. Pa anal, C. B. Bu ge, J. E. B adne ,T.I.Lee,R.A.Young, Cell 2012,151,56; b) D. Dominguez-Sola, C. Y. Ying,C.G ando i,L.Ruggie o,B.Chen, M. Li, D. A. Galloway,W.Gu, J. Gau ie ,R.Dalla-Fa e a, Na u e 2007,448,445;c)M.A.G ego y,S.R.Hann, Mol. Cell. Biol. 2000,20,2423. [14] C. L. G and, H. Han, R. M. MuÇoz, S. Wei man, D. D. VonHo , L. H. Hu ley,D.J.Bea ss, Mol. Cance The . 2002,1,565. [15] Simila esul s we e ob ained wi h he Ve o mammalian cell line (see Figu e S26). [16] Z. A. Walle ,S.A.Sewi z, S. T. Hsu, S. Balasub amanian, J. Am. Chem. Soc. 2009,131,12628. [17] a) A. M. CaamaÇo,M.E.V#zquez, J. Ma &nez-Cos as,L. Cas edo,J.L.Masca eÇas, Angew.Chem. In . Ed. 2000,39, 3104; Angew.Chem. 2000,112,3234;b)A.Jim8nez-Balsa, E. Pazos,B.Ma &nez-Alba donedo, J. L. Masca eÇas,M.E. V#zquez, Angew.Chem. In . Ed. 2012,51,8825; Angew.Chem. 2012,124,8955;c)J.Mosque a,M.I.S#nchez, J. L. Masca eÇas, M. E. V#zquez, Chem. Commun. 2015,51,5501. [18] a) S. Bonne , B. Limbu g,J.D.Meeldijk, R. J. M. K. Gebbink, J. A. Killian, J. Am. Chem. Soc. 2011,133,252;b)R.E. Goldbach, I. Rod iguez-Ga cia,J.H. an Len hem, M. A. Sie- gle ,S.Bonne , Chem. Eu .J.2011,17,9924. [19] K. Takahashi, S. Yamanaka, Cell 2006,126,663. Manusc ip ecei ed:Augus 17, 2016 Re ised: Sep embe 29, 2016 Final A icle published: No embe 17, 2016 A ngewand e Chemie Zusch i en 15847Angew.Chem. 2016,128,15844 –15847 T2016Die Au o en. Ve ç en lich on Wiley-VCH Ve lag GmbH &Co. KGaA, Weinheim www.angewand e.de