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Cite this: DOI: 10.1039/c4dt02760d Received 10th September 2014, Accepted 22nd October 2014 DOI: 10.1039/c4dt02760d

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Platinum(II) clovers targeting G-quadruplexes and their anticancer activities† Xiao-Hui Zheng,‡a Qian Cao,‡a Yi-Liang Ding,a Yi-Fang Zhong,a Ge Mu,a Peter Z. Qin,b Liang-Nian Jia and Zong-Wan Mao*a

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Two porphyrin-bridged tetranuclear platinum(II) complexes are found to effectively stabilize various kinds of G-quadruplexes. Their clover-like shape endows them with the capability of targeting G-quadruplexes rather than the double-stranded structure. Their excellent anticancer activity is the result of a dual effect, inhibition of the telomerase activity and repression of oncogene expression.

G-quadruplexes (G4) are formed by G-rich DNA (or RNA) sequences through π–π stacking of G-tetrads, which are composed of four guanines through hydrogen bonds between the Watson–Crick edge of each base and the Hoogsteen edge from its neighbor.1 Such a structure is distinct from traditional double-stranded (ds) DNA and exists ubiquitously in functionally important regions of the eukaryotic genome, such as telomeres,2 gene promoters and the 5′-UTR end of mRNA,3 playing significant roles in regulation of cell aging, gene transcription and translation. Therefore small molecules targeting G4 have attracted widespread attention in the last decade.1,4 Numerous molecules have been found to possess anti-cancer activities based on stabilization of certain G4 structures, such as human telomere G4-DNA (leading to inhibition of the telomerase activity)5 and promoter G4-DNA (leading to oncogene down-regulation),6 finally resulting in inhibition of tumor cell proliferation.7 Among them cationic porphyrins are a kind of G4 stabilizer with high affinity.8 However, they have poor selectivity between G4 and dsDNA, such as 5,10,15,20-tetra(4-pyridyl) porphyrin (TMPyP4).9 Since the meso position of porphyrin can be flexibly substituted, porphyrin itself has the potential acting as a bridging ligand that coordinates with four metal ions and

a MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China. E-mail: [email protected]; Fax: (+86)2084112245 b Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA † Electronic supplementary information (ESI) available: Experimental details, supplementary tables and figures. See DOI: 10.1039/c4dt02760d ‡ Both authors contributed equally to this work.

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forms tetranuclear metal complexes.10 Platinum(II) complexes have long been considered as successful inorganic anticancer drugs due to their interaction with nucleic acids. Recently, we have reported a series of mononuclear, polypyridyl-bridged dinuclear, trinuclear and tetranuclear Pt(II) complexes acting as selective G4 binders.11 By altering bridging ligands, various multinuclear metal complexes with different geometry can be constructed, allowing their improved geometrical matching and interaction with G4 structures. Based on this previous work and in combination with the great structural difference between G4-DNA and dsDNA, porphyrin-containing platinum(II) complexes are considered to be potential G4-stabilizers with enhanced specificity and anti-cancer activity. In the present work, we reported two porphyrin-bridged tetranuclear platinum complexes (Fig. 1, Schemes S1† and S2†). A series of in vitro experiments demonstrates that both 1 and 2 selectively and effectively bind to G4-DNA. The fluorescence resonance energy transfer (FRET) thermal melting assay shows that both 1 and 2 display an excellent capability in raising the melting temperature of G4-DNA (ΔTm values >22 °C), particularly for the human telomere (HTG2112) and c-myc (Pu2713) (ΔTm values >35 °C), but they only have little effect on dsDNA (ΔTm values 11 nm) and a significant hypochromic effect (>40%) upon the titration of HTG21 and Pu27, respectively (Table S5†, Fig. S5†), indicating that a strong interaction, especially π–π stacking, occurs.15 In the fluorescence experiment, the emission of 1 and 2 displays moderate enhancement upon the titration of HTG21 or Pu27 (Fig. S6†), proving that the fluorescence from porphyrin is not quenched. Based on the previous reports that binding with a large amount of guanine will result in the quenching emission of porphyrin,16 the possibility of intercalation mode was ruled out in the present work. Both UV-vis and fluorescence experiments suggest that the major binding mode between Pt(II)

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Fig. 5 (a) Influence of 1 (8 μM) and 2 (40 μM) on the HeLa cell cycle evaluated by flow cytometry after 24 h of incubation; (b) apoptosis and cell death of HeLa cells treated with 1 (8 μM) and 2 (40 μM) for 48 h detected by flow cytometry after stained with Annexin V-FITC and PI. Cells that stained negative for both Annexin V-FITC and PI staining were classified as alive, while cells that stained positive for V-FITC and negative for PI were classified as apoptotic. Cells that stained positive for PI were classified as necrotic.

Fig. 4 (a) TRAP assay of 1 and 2, showing ladders generated by the action of telomerase on a TS primer (PCR amplified). The lower band is an internal control primer (IC); (b) Western blot on c-myc in HeLa cells treated with 1 and 2 for 24 h, β-actin used as an internal control (all the concentrations used in this test are less than their IC50 values obtained from the MTT assay).

clovers and G4 is end-stacking. Furthermore, Job-plot analysis was performed to get the maximum binding ratio of 4 : 1 ([complex]–[G4]) (Fig. S7†), indicating that other binding modes such as groove-stacking interactions could not simply be excluded. Finally, the ability of 1 and 2 to inhibit the human telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP) assay (Fig. 4a). The results illustrate that 1 and 2 are both effective telomerase inhibitors with IC50 values of 1.46 μM and 0.25 μM, respectively, which are lower than TMPyP4 (6.5 μM).17 On the other hand, the ability of 1 and 2 to repress oncogene expression was evaluated by the Western blot assay. The results illustrate that both 1 and 2 could downregulate c-myc oncogenes in HeLa cells (Fig. 4b). Furthermore, the MTT assays show that 1 and 2 display lower IC50 values than TMPyP4 in most cancer cells (Table S6†). Such high cytotoxicity is the result of a dual effect: inhibition of the telomerase activity and repression of oncogene expression. The flow cytometry study demonstrates that both 1 and 2 kill cancer cells by inducing apoptosis (Fig. 5a) and causing G2/M phase arrest (Fig. 5b). In conclusion, we synthesized two clover-like shaped, porphyrin-bridged tetranuclear platinum(II) complexes. Both of them show high affinity and excellent selectivity to the G4 structure rather than dsDNA. They also display high potential for selectively binding and stabilizing parallel G4 by end-stack-

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ing binding mode. The in vitro study indicates that their good anticancer activities result from a dual effect, inhibition of the telomerase activity and repression of oncogene expression. Combined with our previous work,11 the present work further suggests that adjusting the shape and configuration of multinuclear metal complexes by altering various bridging ligands is a strategy for constructing geometry-oriented G4 stabilizers and anticancer drugs. This study was supported by the National Natural Science Foundation of China (Nos. 21172274, 21231007 and 21328101), 973 Program (Nos. 2014CB845604 and 2015CB856301), 863 Program (No. 2012AA020305), the Ministry of Education of China (Nos. IRT1298 and 313058) and Science and Technology Department of Guangdong Province.

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Platinum(II) clovers targeting G-quadruplexes and their anticancer activities.

Two porphyrin-bridged tetranuclear platinum(II) complexes are found to effectively stabilize various kinds of G-quadruplexes. Their clover-like shape ...
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