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

Internationale Ausgabe:DOI:10.1002/anie.201607965 G-Quadruplexes Deutsche Ausgabe:DOI:10.1002/ange.201607965 RuthenationofNon-stacked Guanines in DNAG-Quadruplex Structures:Enhancement of c-MYC Expression J8ssica Rodr&guez, JesfflsMosquera, Jos8R. Couceiro,M.Eugenio V#zquez,* and Jos8L. MascareÇas* Abstract: Guanine quadruplexes(GQs) are compact fourstranded DNAstructures that playakey role in the control of avariety of biological processes,including gene transcription. Bulkyruthenium complexes featuring abipyridine,aterpyridine,and one exchangeable ligand ([Ru(terpy)(bpy)X]n+)are able to metalate exposed guanines present in the GQ of the cMYC promoter region that are not involved in quadruplex base pairing.qRT-PCR and western-blot experiments indicated that the complexes promote aremarkable increase in the expression of this oncogene.Wealso show that exchangeable thioether ligands (X=RSR’,Met) allowregulation of the metalating activity of the complex with visible light. There is great interest in the development of metal-based DNAbinders that show improved selectivity and reduced toxicity relative to cis-platinum.[1] Ruthenium complexes are promising alternatives owing to their kinetic stability and rich photochemistry and redox properties.[2] In addition to noncovalent recognition of double-stranded DNA(dsDNA), several ruthenium complexes that form covalent DNA adducts,especially through reaction with the N7of guanines, have also been described.[3] While most of these complexes bind dsDNA, the functional relevance of G-quadruplexes (GQs)[4,5] calls for the development of probes capable of targeting these structures.[6] To our knowledge,there is only one precedent for the covalent metalation of aGQstructure with aruthenium complex, and the reaction presents low selectivity.[7] Herein, we demonstrate that coordination complexes of the type [Ru(terpy)(bpy)X]n+(X=Cl, RSR’,Met) can selectively metalate unpaired guanines present in parallel GQs,areaction that is enhanced upon irradiation. Importantly,wehave found that this selective metalation increases the expression of the oncogene c-MYC,apparently by disrupting the parallel GQ structure present in its promoter region. Our work was conceived after learning that while most DNA-metalating ruthenium agents are cytotoxic, the complex [Ru(terpy)(bpy)Cl]+(1)exhibits very low toxicity.[8] We reasoned that the bulky and relatively hydrophobic nature of this sort of complexes could offer excellent opportunities for the selective modification of accessible guanines,thereby promoting specific biological responses with reduced toxicity. We first studied the ability of chloro complex 1to metalate guanosine monophosphate (GMP). Mixing complex 1with 3equiv of GMP in phosphate buffer (pH 7.5), led to the partial formation of the aquo complex [Ru(terpy)- (bpy)H2O]+2(2;over 50%after 30 min at RT), while the guanosine remained essentially unreacted (Figure 1B, trace b). Further incubation for 2hafforded the metalated product 3and the aquo derivative 2(Figure 1A), with total consumption of the starting chloride (Figure 1B,trace d). Figure 1. A) GMP metalation reaction with [Ru(terpy)(bpy)Cl]PF6([Ru]- Cl, 1)and formation of the aquo derivative 2.B)HPLC of the reaction of 1(250 mm)and GMP (750 mm)in10mmphosphate buffer pH 7.5, 100 mmNaCl:inthe dark at t=0(trace a);after 30 min in the dark (trace b);initial mixture after 30 min of irradiation at 455 nm (trace c); and initial mixture after 2hin the dark (trace d). The injection peak is labeled with an asterisk. C) Signal in the MS spectrum corresponding to the monoadduct 3. [*] J. Rodr&guez,Dr. J. Mosquera,Dr. J. R. Couceiro, Dr.M.E.V#zquez, Prof. J. L. MascareÇas Centro Singular de InvestigaciknenQu&mica Biolkxica eMateriais Moleculares (CIQUS), and Departamento de Qu&mica Org#nica Universidade de Santiago de Compostela 15782 Santiago de Compostela (Spain) E-mail:j[email protected] [email protected] Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.doi.org/10. 1002/anie.201607965. T2016 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permitsuse and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. A ngewandte Chemie Zuschriften 15844 T2016 Die Autoren. Verçffentlicht vonWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem. 2016,128,15844 –15847 Interestingly,irradiation of the initial mixture for 30 min (l= 455 nm) led exclusively to the formation of the monoadduct 3 (approximate conversion of 80%based on the disappearance of GMP;Figure 1B,trace cand Figure S5 in the Supporting Information). Importantly,the reaction is fully orthogonal, so competitive control experiments in the presence of excess adenosine monophosphate (AMP), cytidine monophosphate (CMP), and thymidine monophosphate (TMP;Figure S6), as well as lysine or even cysteine (Figure S7), led exclusively to the formation of the GMP derivative.Moreover,experiments with double-stranded oligonucleotides (dsDNAs) presenting different arrangements of guanines revealed modest reactivity,but only with those featuring terminal guanines (Table S1, and Figures S10–S13 in the Supporting Information). This selectivity most likely stems from the bulkiness of complex 1, which cannot reach the sterically hindered nucleophilic N7 site in paired, internal guanines.[9] Remarkably,irradiation of amixture of the parallel cMYC quadruplex d[TTGAG3TG3TAG3TG3TA3][10] (10 mm) with 5equiv of 1in 10 mmphosphate buffer (pH 7.5) and 100 mmKCl led to the clean formation of aproduct with amass corresponding to amonoadduct derivative (Figure 2A,trace C, peak at 22 min, MYC-[Ru],81% conversion). We also observed reaction in the absence of light (approx. 41%conversion after 30 min at RT,Figure 2A, trace b). Importantly,MSanalysis of the bovine spleen phosphodiesterase (BSP) digestion of the ruthenated oligonucleotides lead to identification of the first guanine of the sequence (5’-TTGA…, Figure 2C)asthe metalation site (Figure S24).[11] No reaction was observed under the same conditions with amutated c-MYC GQ containing aCinstead of aGin position 3(MYCm,d[TTCAG3TG3TAG3TG3TA3]; Figure S18). Thechemoselectivity of the reaction can be rationalized by considering the secondary structure of the c-MYC GQ (Figure 3), which shows that G3 is exposed to the solvent and does not participate in the formation of the G-quartets.As aconsequence,ithas ahighly accessible N7nucleophile that can react with the bulky ruthenium complex. Importantly, circular dichroism experiments revealed that the GQ secondary structure is disrupted in the ruthenium adduct MYC- [Ru] (Figure S3, right). Control experiments with other related parallel quadruplex such as c-KIT1,which also presents non-stacked guanines,revealed asimilar reactivity pattern. Mass spectrometry shows that the promoter is modified by the ruthenium complex at the expected positions (Figures S21 and S22), although curiously,inthis case,CD analysis revealed that the quadruplex remained mostly intact (Figure S4).[12] With the above molecular information, we explored whether Ru complex 1could affect the expression of the oncogene c-MYC,since this oncogene is involved in many important cellular processes.[13] This was analyzed by realtime quantitative reverse transcription PCR (qRT-PCR) in HeLa and Vero cells (Figure 4and Figure S26, respectively), using porphyrin TMPyP4, aGQbinder that is known to repress the expression of c-MYC,[14] as acontrol. As shown in Figure 4, HeLa cells treated with 1(100 mm)inDulbeccoQs modified EagleQsmedium (DMEM) presented amodest but significant increase in the transcription of c-MYC compared to untreated cells (80%at16h and 200%at48h).[15] As expected, treatment with TMPyP4 led to a60% decrease in the cellular levels of c-MYC mRNAafter 48 h. We also analyzed the expression of c-MYC protein by western blot. In agreement with the qRT-PCR results,treatment of cells with 100 mmof 1,led to anoticeable increase in the levels of c-MYC protein (Figure 4, averaged 40% Figure 3. Structure of the GQ of c-MYC (PDB ID:1XAV), highlighting the exposed G3and the metalation selectivity.The large spheres in the quadruplex structure represent potassium ions. Figure 2. A) HPLC traces of amixture of the GQ of c-MYC (MYC, 10 mm)and 1(5 equiv) in 10 mmphosphate buffer pH 7.5, 100 mm KCl, at RT:inthe dark at t=0(trace a);after 30 min in the dark (trace b), initial mixture after irradiation for 30 min at 455 nm (trace c). B) MS of the metalated product (MYC-[Ru]) showing the peaks corresponding to the complex (m/z=8089) and the demetalated fragment(m/z=7600). C) MS of the products after BSP digestion of MYC-[Ru] showing the peaks corresponding to the digested complex (m/z=7489) and non-metalated digested product (m/z=7000) resulting from the cleavage at G3as shown by the dashed line in the structure. A ngewandte Chemie Zuschriften 15845Angew.Chem. 2016,128,15844 –15847 T2016Die Autoren. Verçffentlicht von Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim www.angewandte.de increase). These data confirm that, in contrast to most quadruplex targeting agents,complex 1promotes an increase instead of adecrease in the level of gene expression, thereby acting as atranscriptional activator.[16] ICP-MS measurements of isolated nuclei and chromatin obtained after treatment with complex 1confirmed the presence of relatively significant amounts of ruthenium, which is consistent with efficient cellular uptake and nuclear delivery of the complex (Tables S2 and S3). In agreement with the early studies,[8] cell viability assays confirmed that 1is essentially noncytotoxic (Figure S27). While the above data indicate that 1is capable of altering the expression of c-MYC,the development of derivatives that could be activated by using external irradiation would be highly attractive.[17] Towards this aim, we prepared complexes 4and 5,which feature athioether ligand (Figure 5) and are kinetically stable but undergo rapid ligand exchange upon irradiation with visible light.[18] In contrast to the chloride complex 1,invitro experiments with the thioether derivative 4showed that it does not react with the c-MYC quadruplex in the dark after 30 min, but yields the desired GQ monoadduct upon irradiation (71%conversion, Figure S17). Theacetylmethionine derivative 5presents even higher kinetic stability than complex 4,and no traces of the metalation adducts,or even of the aquo derivative 2,were observed after several hours in the dark. However,irradiation for 30 min triggers efficient covalent metalation of the GQ (Figure S20). We also analyzed the effect of complex 5on transcription of the cMYC gene by qRT-PCR in Hela cells.Asshown in the Figure 5(left), c-MYC mRNAlevels increased after irradiation, becoming similar to those observed after treatment with the aquo complex 2.Western-blot analysis confirmed that enhancement of the gene expression only took place in the presence of light, while the levels of the protein c-MYC did not change when the cells were kept in the dark (Figure 5 and Figure S29). As expected, both qRT-PCR and westernblot experiments showed that the aquo compound 2is active both in the dark and under irradiation. Moreover,control experiments confirmed that the irradiation does not have any measurable effect on cell viability (Figure S28), and that the complex does not generate significant amounts of singlet oxygen upon irradiation (Figure S25). In conclusion, we have demonstrated the selective modification of accessible guanines flanking the GQ of c-MYC with designed bulky ruthenium complexes.Importantly,the bioorthogonal metalation enhances the expression of the oncogene c-MYC.Given that an increase in c-MYC transcription has been shown to be important in several cancers,in particular for the renewal ability of cancer stem cells,[19] our discovery might lead to interesting biological applications. Acknowledgements We thank the support by the Spanish grants SAF2013-41943R, CTQ2015-70698-R, and CTQ2013-49317-EXP,the Xunta de Galicia GRC2013-041, the ERDF,and the European Research Council (Advanced Grant No.340055). Support of COST Action CM1105, COST CM1306 and the orfeo-cinqa network are also kindly acknowledged. J.R. thanks the Xunta de Galicia for aPh.D.fellowship.Wethank Rebeca Menaya Figure 4. A) qRT-PCR analysis of c-MYC (light bars) and ALAS1 (black bars) transcription. Hela cells were incubated for 16 hor48hwith either 100 mmRuCl, TMPyP4, or 50 mm5-fluorouracil(5-FU, adisruptor of RNA synthesis). Amounts are relative to the expression levels of the housekeeping gene GAPDH.Values are the average of three experiments, and error bars indicate the standard error.B)Analysis of the expression of c-MYC by western-blot. HeLa cells were incubatedfor 16 hwith 100 mmcompound 1and then lysed, and c-MYC was detected by SDS-PAGE,followed by western blot with an anti-MYC Antibody (upper panel). The relative amount of protein in two independentexperimentswas quantified by densitometry (lower panel). Data are represented as the fold change with respect to untreatedcontrols. Error bars indicate the standard deviationofthe fold change with respect to untreated controls. Figure 5. A) Thioether complexes 4and 5.B)c-MYC transcription as measured by qRT-PCR in the dark (black bars) or after irradiation (60 min, light bars). Hela cells were incubated for 16 hwith 100 mm RuMet (5)oraquo complex 2.RNA levels are given relative to the expression levels of the housekeeping gene GAPDH.C)Expression of c-MYC as measured by western blot in the presence or absence of compound 5.The relative amount of protein with respect to that of bactin was quantified by densitometry (lower panel). The experimental procedures are as describedinFigure 4. A ngewandte Chemie Zuschriften 15846 www.angewandte.de T2016Die Autoren. Verçffentlicht von Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Angew.Chem. 2016,128,15844 –15847 for her help with the biological experiments and Verlnica PiÇeiro of the Unidad de An#lisis Elemental (RIAIDT-USC) for the ICP analysis. Keywords: bioinorganic chemistry ·DNA damage ·metalation · photochemistry ·ruthenium Howtocite: Angew.Chem. Int. Ed. 2016,55,15615–15618 Angew.Chem. 2016,128,15844–15847 [1] a) C. X. Zhang,S.J.Lippard, Curr.Opin. Chem. Biol. 2003,7, 481;b)A.Levina, A. Mitra, P. A. 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Takahashi, S. Yamanaka, Cell 2006,126,663. Manuscript received:August 17, 2016 Revised: September 29, 2016 Final Article published: November 17, 2016 A ngewandte Chemie Zuschriften 15847Angew.Chem. 2016,128,15844 –15847 T2016Die Autoren. Verçffentlicht von Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim www.angewandte.de