Rapid Telomere Reduction in Cancer Cells Induced by G-Quadruplex

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Rapid Telomere Reduction in Cancer Cells Induced by G-Quadruplex-Targeting Copper Complexes Zhen Yu, Kevin Fenk, Derrick Huang, Sambuddha Sen, and J. A. Cowan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00215 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019

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Journal of Medicinal Chemistry

Rapid Telomere Reduction in Cancer Cells Induced by G-QuadruplexTargeting Copper Complexes

Zhen Yu, † Kevin D. Fenk, † Derrick Huang, ‡ Sambuddha Sen, † and J. A. Cowan*,†.

†

Department of Chemistry and Biochemistry, the Ohio State University, Columbus, OH 43210,

USA. ‡

Department of Biomedical Engineering, the Ohio State University, Columbus, OH 43210,

USA.

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ABSTRACT Telomere length determines the replicative capacity of mammalian cells. Successive telomere reduction to a critically short length can lead to cellular senescence that irreversibly prevents cells from further cell division. A series of Cu complexes has been designed as selective artificial nucleases that degrade G-quadruplex telomeric DNA and exhibit selective DNA binding affinity and cleavage reactivity towards G-quadruplex telomeric DNA over duplex DNA. In contrast to protein-based nucleases that usually lack membrane permeability, significant cellular uptake and nuclear localization of these Cu complexes was observed. Rapid telomere reduction of cancer cells was also observed after only 1-day incubation, while the absence of DNA fragmentation indicates a low level of non-selective DNA cleavage. Robust telomere reduction by the designed Cu complexes is an S-phase-specific event that is associated with increased formation of the Gquadruplex structure during DNA replication.


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INTRODUCTION Telomeric DNA located at the ends of chromosomes provides protection from deleterious DNA fusion and ensures the stability and integrity of the genome.1 Telomeres consist of telomere-binding proteins and a long tract of repeating guanine-rich sequence that has the potential to form the unique G-quadruplex secondary structure.2, 3 The length of telomeric DNA determines the cellular replicative capacity of mammalian cells.4 Cellular senescence, an irreversible arrest of cell growth, arises when telomere length is shortened to a critical point.5 Telomeres in various tumors have been reported to possess a shorter length than those in adjacent normal tissue from the same individual,6-8 therefore, cancer cells should be more vulnerable to telomere reduction, relative to normal cells.9,

10

Reduction of telomere length is

compensated by telomerase, a reverse transcriptase that is expressed in most cancer cells,11, 12 and the development of small-molecular ligands that inhibit telomerase has long been proposed as a means of interfering with telomere maintenance.13 However, inhibition of telomerase typically results in relatively slow reduction of telomeric DNA, therefore, prolonged incubation with these agents is required to induce a notable reduction of telomere length.14 For example, BIBR1532, an inhibitor against telomerase, reduces the telomere length of NCI-H460 cancer cells by ~ 60% only after 140 population doublings.15,

16

In fact, telomere length in normal

somatic cells, where telomerase activity is low or absent, can only be shortened by ~ 50-200 bp per cell division as a result of the inability of the DNA replication machinery to fully duplicate the lagging strand.17 Nucleases that degrade telomeric DNA can overcome the problem of slow telomere reduction induced by telomerase inhibitors and promote cellular senescence in cancer cells. Despite the current advanced application of nuclease technologies, a lack of membrane permeability still presents obstacles for application of engineered nucleases to biological systems.18 In fact, chimeric nucleases constructed from a telomere-binding protein and an endonuclease have been designed to induce telomeric DNA cleavage in cells.19 However, 3 ACS Paragon Plus Environment


permeabilization approaches or viral vectors are required to transport these constructs into cells, and raise the potential for cell damage and safety issues in clinical practice.20, 21 To address these problems, we have developed small-molecule metal complexes designed to mimic the function of nucleases that target telomeric DNA, because small molecules may readily cross the membrane by virtue of their low molecular weight and hydrophobicity. Moreover, to enhance their selectivity against cancer cells, we have designed selective artificial nucleases that target the G-quadruplex structure of telomeric DNA rather than more typical duplex structures. Previous studies have suggested a higher level of G-quadruplex formation during the S phase of the cell cycle, where DNA replication occurs, relative to the G1/G0 phase.22 Considering the frequent division that cancer cells undergo, nucleases that target G-quadruplex telomeric DNA should promote more selective telomere reduction in cancer cells relative to nucleases that degrade duplex telomeric DNA. Herein, we report the design of Cu complexes as artificial nucleases that induce prompt reduction of telomere length through targeted cleavage of G-quadruplex telomeric DNA.23, 24 To design such constructs we have used naphthalene diimide derivatives as a G-quadruplex binding moiety, and incorporated these into a DNA-cleaving moiety derived from the amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif.25-27 The latter can perform DNA cleavage under physiological conditions by formation of reactive oxygen species or Cu-bound oxygen species.2830

Due to the short lifetime of these oxygen species,31 only DNA residues in close proximity to

the Cu centers are affected.32 To study the localization of oxygen species generated by Cu complexes, three different sequences of ATCUN motif were coupled to the naphthalene diimide core, through either the C-terminal carboxylate, or an aspartate side chain, which positions the Cu centers in distinct orientations (Scheme 1). In addition, substitution isomers of a xylyl linker (m-xylyl and p-xylyl) were also designed, and the influence of Cu orientation on DNA cleavage reactivity and anticancer activity was assessed.


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Scheme 1. Synthesis of naphthalene derivatives. i. xylylenediamine. ii, Boc-peptide, DIC. iii, CuCl2 in tris-HCl (pH 7.4). Following HPLC purification, all ligand products were obtained with > 95% purity (Figure S1). NH2 O

O

Br N

O

O

N

N

O

O Br

i

O

HN N

N

O

O

N

N

O

O

2: p3: m-

O O

O O

N

N

N H

CuGGH

N H2

NH2

O O

Cu

N

4-9

O O

Cu N H2

N N H

CuGDH

O

N

N

O

O

N

N

O

O

O

Cu N H2

iii

N

N N H

NH2

R

HN N

H 2N

O

N

N

ii

HN

O

1

N H

O HN R

H N

N O

4: p-, R = CuGGH 5: p-, R = CuGDH 6: p-, R =CuDGH 7: m-, R = CuGGH 8: m-, R = CuGDH 9: m-, R = CuDGH

CuDGH

RESULTS and DISCUSSION To evaluate the G-quadruplex DNA binding affinity of all Cu complexes, and analogues lacking the Cu moiety, a fluorescence binding assay was designed where an oligonucleotide derived from telomeric DNA (22G4: 5-fluorescein-d(AGGG(TTA-GGG)3)) was utilized as a Gquadruplex model. As revealed in previous structural studies (PDB: 3UYH, 3SC8, 3T5E, 4DA3, 4DAQ),33, 34 the naphthalene diimide derivatives typically stack on the G-tetrad formed by G4, G10, G16, G22 residues from a G-quadruplex model. The 5-fluorescein is linked to Gquadruplex DNA through a flexible A1 residue that should allow fluorescein to detect ligand 5 ACS Paragon Plus Environment


stacking on the G-tetrad. Following interaction between DNA and the DNA-binding ligand, a quenching of fluorescein emission was observed at 520 nm (Figure S2), most likely reflecting a FRET response from fluorescein to the naphthalene diimide derivatives due to the overlap between the fluorescein emission spectrum and naphthalene diimide excitation spectrum (Figure S3). All Cu complexes 4-9 demonstrate significant binding affinity to the G-quadruplex DNA, with a KD ~ 120-160 nM (Table S1). The G-quadruplex binding affinities of our Cu complexes are consistent with other naphthalene diimide derivatives described in previous studies,35 implying that all Cu complexes should stack on the G-tetrad as analogues of naphthalene diimide derivatives. The compounds lacking the Cu-ATCUN center, 2 and 3, display similar binding affinity to G-quadruplex DNA (Table S1), implying that the interaction between G-quadruplex DNA and the ligand primarily arises from π-π stacking between the G-tetrad and naphthalene diimide core, while the interaction between the Cu-ATCUN moiety and DNA is negligible. In addition, the binding affinity for all Cu complexes 4-9 to calf-thymus DNA (CT-DNA) was also examined by use of a competition assay (Figure S4). All Cu complexes were found to exhibit ~ 350-510 fold selectivity for DNA binding towards G-quadruplex, relative to CT-DNA, while selectivity factors determined for the analogues lacking the Cu-ATCUN motif (2 and 3) were ~ 20 and ~ 24, respectively (Table S1). The introduction of bulky Cu-ATCUN motifs to the naphthalene diimide core apparently raises considerable steric strain that undermines nonselective interaction with dsDNA. In addition, the protonated NH2 group of 2 and 3 may readily interact with the negatively charged phosphate backbone of DNA. Cleavage of G-quadruplex DNA was evaluated for all Cu complexes. Biologicallyrelevant coreagents, ascorbate and peroxide were added to stimulate Cu redox chemistry and promote formation of Cu-bound reactive oxygen species,29 where the latter attack the deoxyribose ring and facilitate cleavage of the G-quadruplex DNA.24 In fact, following cleavage of 22G4 by Cu complexes in the presence of ascorbate and peroxide, a recovery of fluorescence intensity at 520 nm was observed. This increase of fluorescence intensity should be ascribed to 6 ACS Paragon Plus Environment

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either the dissociation of Cu complexes from the cleavage products, and/or a change of microenvironment. All Cu complexes demonstrate similar KM’s that range from ~ 0.7 to 2.2 µM (Table 1 and Figure S5), denoting significant binding affinity to substrate. The Cu complexes with m-xylyl linkers exhibit a larger kcat relative to their analogues with the p-xylyl linker, implying that the m-xylyl linker may direct the Cu centers spatially closer to substrate than the pxylyl linker. Moreover, among the m-xylyl series, the Cu complex with the DGH peptide motif, 9, displays the highest kcat relative to other analogues, whereas compound 4 shows the highest kcat among the p-xylyl series. These results demonstrate distinct orientations of the Cu centers resulting from the discrete ATCUN sequence and xylyl linkers, and their important role in DNA cleavage, since the orientation of the Cu center should essentially determine the relative position of Cu-bound oxygen species to substrate. In other words, tuning the ATCUN sequence and linker can allow appropriate positioning of these Cu-bound oxygen species towards substrate and improve the efficiency of DNA attack.

Table 1. Michaelis-Menten parameters for G-quadruplex telomeric DNA cleavage Compounds 4 5 6 7 8 9

kcat (min-1) 0.014 ± 0.001 0.012 ± 0.001 0.011 ± 0.001 0.020 ± 0.002 0.017 ± 0.002 0.022 ± 0.002

KM (µM) 0.69 ± 0.16 0.96 ± 0.28 0.86 ± 0.16 2.0 ± 0.5 1.4 ± 0.5 2.2 ± 0.3

kcat/KM (min-1µM-1) 0.020 ± 0.002 0.012 ± 0.002 0.013 ± 0.002 0.010 ± 0.002 0.013 ± 0.002 0.010 ± 0.003

To further investigate how cleavage reactivity is influenced by the relative orientation of the Cu center, cleavage products were separated and analyzed by denaturing PAGE. The cleavage rates for all nucleotides were determined. While the naphthalene diimide core stacks 7 ACS Paragon Plus Environment


selectively on the G-tetrad, Cu-ATCUN centers, with neutral charge, may not interact with the G-quadruplex significantly, and a broad spectrum of residues can thereby be cleaved by these catalysts. With the aid of a previously reported structure of the naphthalene core bound to Gquadruplex DNA,34 major cleavage sites were identified that most likely reflect the positions of the two Cu centers. One Cu center affects a broad spectrum of residues that include A1-A3, T6A7, and G20-G22, while the second Cu site is localized around G14, and G15 (Figure 1a and 1b). In fact, the cleavage rates at these cleavage sites are significantly influenced by the sequence of the Cu-bound ATCUN motif and by substitution of the xylyl linker. For example, one Cu center of compound 5 primarily impacts G4, while its analogues 4 and 6 show a greater cleavage rate at G2 and A7, respectively. The m-xylyl linker isomer also influences the cleavage rates, since the m-linker appears to place the Cu center in a different spatial position relative to the DNA. For example, in comparison to 5, compound 8 exhibits faster cleavage rates at G2 and G22, relative to position G4. All these results confirm distinct preferential cleavage sites for each Cu complex, and also reflect the different orientations of Cu-bound oxygen species formed by each complex. In addition, the cleavage reactivity towards an analogous oligonucleotide that represents duplex telomeric DNA ((ds12Telo: 5’-fluorescein-d(TTAGGG)-(CH2CH2O)6d(CCCTAA)) was also examined. All Cu complexes exhibit significantly more robust cleavage reactivity towards G-quadruplex telomeric DNA over duplex telomeric DNA (Figure 1c and 1d). This selectivity for DNA cleavage can be ascribed to the preferred binding of Cu complexes to G-quadruplex DNA over the duplex DNA.


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Figure 1. DNA cleavage sites for G-quadruplex telomeric DNA by Cu complexes showing cleavage selectivity. (a) and (b) The relative cleavage rates at different nucleotides of 22G4 by the stated Cu complexes were measured using denaturing-PAGE. The indicated Cu complexes (5 9 ACS Paragon Plus Environment


µM) were added to a solution containing 5 µM 22G4, 1 mM ascorbate, 1 mM H2O2, 100 mM KCl and 10 mM tris-HCl (pH 7.4). (c) and (d) The time-dependent DNA cleavage of Gquadruplex telomeric DNA 22G4 and duplex telomeric DNA ds12telo is shown.

A lack of membrane permeability remains a considerable challenge in current nuclease technology. To evaluate the internalization of these Cu complexes, the amount of Cu complex taken up by cells was quantified by LC-MS/MS, while confocal microscopy was used to study the location of Cu complexes in cells. Rapid internalization of all Cu complexes was observed after 12-h incubation, yielding ~ 0.3-1.2 mmol/mg complex in cell lysates (Figure 2a). The low molecular weight and hydrophobicity most likely promotes rapid cellular uptake. Interestingly, the change of ATCUN sequence also affects cellular uptake. Cu complexes bound by a GDH sequence show higher concentration in cells relative to their GGH and DGH analogues. The change of Cu orientation by the GDH peptide may better balance the polarity of the molecule and enhance cellular uptake. To further study the localization of Cu complexes in the cellular context, we took advantage of the intrinsic fluorescence of the naphthalene diimide core that can be excited by a 633 nm laser and used confocal microscope to visualize the Cu complexes in cells. The cellular nucleus was stained by DAPI dye following incubation with 5 or 8. Overlap between the cellular nucleus and naphthalene diimide fluorescence indicates the accumulation of these Cu complexes in the cellular nucleus (Figure 2b), and also implies that the biomolecules in cells targeted by these Cu complexes are in all likelihood genomic DNA instead of other nucleic acids in the cytosol or mitochondria. In fact, accumulation of compounds 5 and 8 in the cellular nucleus are similar to their analogues lacking DNA-cleaving motifs, 2 and 3. The localization of these naphthalene diimide derivatives into the cellular nucleus may result from DNA-targeting by the DNA-binding motif, while the DNA-cleaving motif has little contribution to localization. 10 ACS Paragon Plus Environment

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Figure 2. Demonstration of cellular uptake and colocalization of the naphthalene diimide derivatives and the nucleus. (a) MCF7 cells were incubated with 10 µM of the relevant Cu complex for 12 h. Concentrations of Cu complexes in cell lysates were measured by LC-MS/MS. (b) MCF7 cells were incubated with 0.25 µM indicated compounds for 12 h. Fluorescent imaging of the cellular nucleus (DAPI) and the intrinsic emission from the naphthalene core of the Cu complexes was performed by confocal microscopy.

Intracellular nuclease activity for these Cu complexes was also confirmed. Because the cleavage of telomeric DNA should result in the reduction of telomere length, a quantitative-PCR assay36 was employed to study the change of telomere length following incubation with the Cu complexes. DNA fragmentation is a distinct feature of cell death, since the upregulation of endogenous nuclease activity during apoptosis can also induce DNA cleavage of chromosomal DNA.37 To unambiguously evaluate the change of telomere length, cells were incubated with Cu complexes at nontoxic concentrations (0.5 µM) where apoptosis does not arise (data not shown). In addition, an alkaline comet assay to reveal overall DNA damage was also employed to ensure low levels of DNA fragmentation under these conditions (Figure 3a and S8). In fact, all Cu complexes at 0.5 µM were found to induce rapid telomere reduction by 20-50% after only a 3-



day incubation period, while 2 and 3, the analogues lacking the DNA cleavage motif, showed no change in telomere length (Figure 3b and 3c).

Figure 3. Telomere reduction and overall DNA fragmentation. (a) Overall DNA fragmentation level of MCF7 cells were measured after incubation with 0.5 µM naphthalene diimide for 3 days or 50 µM H2O2 for 30 min. (b) Time-dependent telomere reduction of MCF7 by 0.5 µM of the indicated compounds. (c) Telomere reduction of MCF7 by 0.5 µM of the indicated compounds for 3 days. (d) Time-dependent telomere reduction of HDFa by 0.5 µM of the indicated compounds. (e) Telomere reduction of synchronized S-phase and G1/G0-phase MCF7 cells following a 10-hr incubation with 0.5 µM Cu complexes. 12 ACS Paragon Plus Environment

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Similarly, a well-known G-quadruplex ligand BRACO19 lacking a DNA cleavage motif barely induces telomere reduction, even though BRACO19 has been reported to inhibit telomerase activity.38 In fact, a previous study has reported that BRACO19 can reduce the telomere length of MCF7 cells by only 17% after 39 days of incubation.39 Given that telomere attrition through telomerase inhibition required a prolonged period of time, the observed rapid telomere reduction by Cu complexes should arise from direct DNA cleavage rather than telomerase inhibition. The transient formation of G-quadruplex telomeric DNA can be recognized by G-quadruplex targeting nucleases, and thereby DNA cleavage promoted by these Cu complexes should lead to successive telomere reduction. A combination of Comet and telomere reduction assays confirmed the in vivo cleavage selectivity of theses Cu complexes. Namely, these G-quadruplex-targeting Cu complexes primarily promote cleavage of Gquadruplex telomeric DNA over the predominant duplex DNA. Compound 5, with a CuGDH DNA-cleaving motif exhibits more robust telomere reduction activity, relative to its analogues 4 and 6 with different DNA-cleaving motifs, as a result of higher cellular uptake of compound 5 over 4 and 6. Compound 8, with the CuGDH DNA-cleaving motif, induces similar telomere reduction to its analogues 7 and 9, although compound 8 has higher cellular uptake, which most likely reflects the fact that compound 8 demonstrates a lower kcat than 7 and 9. Besides Gquadruplex telomeric DNA, the possibility of cleavage of other G-quadruplex DNA or RNA by these Cu complexes should be excluded, since the low resolution of the Comet assay cannot reveal the cleavage of these G-quadruplex. In our work, we only focus on the telomere reduction activity of these Cu complexes, however, these Cu complexes may potentially target other Gquadruplex DNA/RNA and exhibit other modes of action in vivo. To examine cellular fate following telomere reduction, an assay evaluating upregulation of SA-β-galactosidase, a biomarker of cellular senescence, was employed, where senescent cells were colored blue and visualized by phase contrast microscopy (Figure S9).40 Cellular senescence of MCF7 cells increased significantly after incubation with 0.5 to 2.0 µM of 5 and 8. 13 ACS Paragon Plus Environment


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Cellular senescence is an irreversible state that arrests cells carrying critically short telomere length from further division. In fact, it is consistent with the cell aging process of normal cells where cellular senescence arises after telomeric DNA is shortened to a critical length.41 Given that cancer cells are immortalized by telomerase that is expressed to elongate telomere length, telomere reduction promoted by Cu complexes should be the origin of the observed cellular senescence of MCF7 cells.

Table 2. Inhibition of cell proliferation defined by IC50 values (µM) following a 3-day incubation with naphthalene derivatives.

Compounds

Huh7

MCF7

BxPC3

LS174T

Caco2

AsPC1

HDFa

2

2.2 ± 0.5

5.5 ± 0.1 2.2 ± 0.2

0.9 ± 0.3

9.7 ± 1.0 2.5 ± 0.2

1.5 ± 0.1

3

3.3 ± 0.3

6.5 ± 0.2 2.9 ± 0.5

2.7 ± 0.5

9.3 ± 1.3 4.7 ± 0.2

0.9 ± 0.1

4

3.6 ± 0.3

2.2 ± 0.3 1.8 ± 0.1

10.4 ± 0.9

3.4 ± 1.2 2.0 ± 0.1

9.7 ± 0.5

5

1.0 ± 0.1

1.2 ± 0.1 0.7 ± 0.1

1.9 ± 0.5

1.6 ± 0.4 1.7 ± 0.1

4.9 ± 0.5

6

11.0 ± 1.9

4.7 ± 0.2 3.5 ± 0.3

47.7 ± 6.1

3.4 ± 1.1 3.9 ± 0.2

12.0 ± 0.8

7

6.1 ± 0.2

2.7 ± 0.1 1.1 ± 0.3

5.4 ± 0.7

4.1 ± 0.2 2.6 ± 0.2

22.6 ± 0.7

8

4.8 ± 0.5

1.4 ± 0.2 1.1 ± 0.2

5.1 ± 1.0

3.2 ± 0.6 2.8 ± 0.1

14.4 ± 0.2

9

5.7 ± 0.6

3.8 ± 0.1 2.6 ± 0.2

15.1 ± 2.4

2.7 ± 0.8 3.1 ± 0.3

16.0 ± 0.4

Inhibition of cancer cell proliferation was studied by use of an MTT assay. In addition, human adult dermal fibroblasts (HDFa) were also studied as a normal cell control. All Cu complexes, and analogues lacking the bound Cu, can significantly inhibit proliferation of all tested human cancer cell lines (Table 2). For MCF7 cells, Cu complex 5 exhibits the most significant inhibition effect among all of the Cu complexes examined, which can be ascribed to 14 ACS Paragon Plus Environment

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efficient telomere reduction by 5 and high cellular uptake. The efficient internalization of these Cu complexes into MCF7 cells enables both DNA cleavage of telomeric DNA, and successive telomere reduction to a critically short length that eventually induces cellular senescence and stops cancer cell growth. In addition, all Cu complexes exhibit a higher inhibitory effect on cancer cells relative to normal HDFa cells, while the analogue lacking the Cu center exhibited little discrimination between cancer lines and fibroblasts. To further study the cell selectivity of these Cu complexes, the telomere length of normal cells was also measured following incubation with the test articles. Both Cu complexes 5 and 8 were found to promote less telomere reduction in normal fibroblast cells (Figure 3d). Since telomere reduction is associated with G-quadruplex formation, less robust telomere reduction may indicate less formation of G-quadruplexes in fibroblasts. Therefore, the effect of telomere reduction was further studied with synchronized cancer cells. MCF7 cells were synchronized in the G1/G0 phase and the S phase of the cell cycle, respectively. In fact, both 5 and 8 were observed to shorten telomeric DNA in the G1/G0 phase by ~ 3% while a faster telomere reduction of S-phase cells by ~ 15% was observed (Figure 3e). Previous studies have disclosed an increase of G-quadruplex formation during S-phase.22 The unwinding of the duplex structure during DNA replication should promote G-quadruplex formation, since single stranded telomeric DNA more readily forms G-quadruplex than duplex telomeric DNA.42 Given that cancer cells undergo more frequent cell division and DNA replication, this S-phase-specific telomere reduction event, induced by the Cu complexes, is in agreement with robust selective telomere reduction in cancer cells, and can promote selective inhibition against cancer cell proliferation.

CONCLUSIONS Copper complexes were designed as artificial nucleases that target G-quadruplex telomeric DNA. Selective DNA binding and cleavage by these Cu complexes was found toward 15 ACS Paragon Plus Environment


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G-quadruplex telomeric DNA over duplex DNA. Both 5 and 8, two copper complexes with the GDH ATCUN motif, exhibit the highest level of cellular uptake, accumulate in the cellular nucleus, and promote rapid telomere reduction in cancer cells. These results solve the low membrane permeability problems of traditional nucleases and slow telomere reduction problems of telomerase inhibitors. In addition, the designed Cu complexes promote more efficient telomere reduction during the S-phase of the cell cycle, relative to the G1/G0 phase, consistent with the fact that more G-quadruplexes form during DNA replication. Cancer cell lines were more vulnerable to these Cu complexes, relative to normal cells, since telomere reduction by these Cu complexes is mostly restricted to the S phase.

EXPERIMENTAL SECTION Materials and Instruments. All reagents were commercially available and of analytical grade. 1H-NMR spectra were recorded at 298 K using a Bruker DPX-400 spectrometer and standard pulse sequences. Electrospray mass spectra (ESI-MS) were measured by Bruker MicroTOF, and the predicted isotope distribution patterns were calculated using the prediction program provided by the manufacturer. Custom peptides were purchased from Biomatik. Compound

1,

2,9‐dibromo‐6,13‐bis[3‐(morpholin‐4‐yl)propyl]‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexadeca‐ 1,3,8,10,15‐pentaene‐5,7,12,14‐tetrone, was synthesized by use of a reported method, and characterization verified following literature procedures and analytical data.34 Oligonucleotides 22G4 (5’-fluorescein-d(AGGG-(TTAGGG)3)) and ds12Telo (5’-fluorescein-d(TTAGGG)(CH2CH2O)6-d(CCCTAA)) were purchased from Integrated DNA Technologies. Purity of compounds was evaluated by HPLC using a SunFire C18 column (5 μm, 150 mm length, 4.6 mm I.D.) at a flow rate of 1 mL/min and all compounds were found to have a purity above 95%.


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Synthesis

of

4,9-bis(4-(aminomethyl)benzylamino)-2,7-bis(3-

morpholinopropyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (2). A 150 mg quantity of compound 1 was mixed with 12 g of p-xylylenediamine and heated to 70°C with stirring under an anaerobic atmosphere of argon for 24 h at 70°C. After reaction, 240 mL diethyl ether was added to the reaction mixture. The precipitate was collected and dissolved in 1 M HCl, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.), with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 82%. 1H NMR (400 MHz, D2O) δ 7.46 (m, 8H), 7.33 (d, J = 17.6 Hz, 2H), 4.25 (s, 4H), 4.05 (m, 12H), 3.71 (t, J = 12.0 Hz, 4H), 3.42 (d, J = 12.3 Hz, 4H), 3.18 (t, J = 7.5 Hz, 4H), 3.05 (t, J = 11.6 Hz, 4H), 2.00 (s, 4H). HRMS (ESI) calculated for (M+H)+: m/z 789.4083, found 789.4036.

Synthesis

of

4,9-bis(3-(aminomethyl)benzylamino)-2,7-bis(3-

morpholinopropyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (3). A 150 mg quantity of compound 1 was dissolved in 12 g of m-xylylenediamine and the resulting solution was stirred under an anaerobic atmosphere of argon for 24 h at room temperature. After reaction, 240 mL diethyl ether was added to the reaction mixture. The precipitate was collected and dissolved in 1 M HCl, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.) with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 78%. 1H NMR (400 MHz, D2O) δ 7.49 (m, 8H), 7.37 (t, J = 3.6 Hz, 2H), 4.45 (s, 4H), 4.09 (s, 4H), 4.04 (d, J = 10.8 Hz, 8H), 3.74 (t, J = 12.4 Hz, 4H), 3.45 (d, J = 12.5 Hz, 4H), 3.27 (t, J = 9.9 Hz, 4H), 3.10 (t, J = 9.9 Hz, 4H), 2.05 (t, J = 7.4 Hz, 4H). HRMS (ESI) calculated for (M+H)+: m/z 789.4083, found 789.4099. 17 ACS Paragon Plus Environment


Synthesis

Page 18 of 33

of

(2S)‐2‐[2‐(2‐aminoacetamido)acetamido]‐N‐[(4‐{[(9‐{[(4‐{[(2S)‐2‐[2‐(2‐aminoacetamido)ace tamido]‐3‐(1H‐imidazol‐4‐yl)propanamido]methyl}phenyl)methyl]amino}‐6,13‐bis[3‐(morp holin‐4‐yl)propyl]‐5,7,12,14‐tetraoxo‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexadeca‐1,3,8,10,1 5‐pentaen‐2-yl)amino]methyl}phenyl)methyl]‐3‐(1H‐imidazol‐4‐yl)propanamide as ligand for 4. An 18.7 mg quantity of Boc-protected GGH-OH peptide was dissolved in 1 mL DMSO and allowed to be activated by 7.0 mg diisopropylcarbodiimide for 10 min at room temperature. A 6.4 mg quantity of N-hydroxysuccinimide was then added to the activated peptide. After 1 h incubation, the GGH-NHS ester was added to 10 mg of compound 2 and the resulting solution stirred under argon for 24 h at room temperature. After reaction, 10 mL of diethyl ether was added to the reaction mixture, followed by centrifugation. A 1 mL volume of concentrated HCl was added to the precipitate to deprotect the peptide over a period of 1 h, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.) with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 15%. 1H NMR (400 MHz, D2O) δ 8.54 (s, 2H), 7.43 (m, 8H), 7.28 (d, J = 7.2 Hz, 2H), 7.18 (s, 2H), 4.28 (d, J = 13.9 Hz, 4H), 4.17 (d, J = 15.6 Hz, 2H), 4.06 (m, 12H), 3.84 (s, 4H), 3.75 (t, J = 12.3 Hz, 4H), 3.41 (d, J = 11.1 Hz, 4H), 3.11 (m, 8H), 3.04 (t, J = 10.0 Hz, 4H), 2.65 (s, 4H), 2.02 (d, J = 5.1 Hz, 4H). HRMS (ESI) calculated for (M+2H)2+: m/z 646.3096, found 646.3002.

Synthesis

of

(2S)‐2‐(2‐aminoacetamido)‐N'‐[(4‐{[(9‐{[(4‐{[(3S)‐3‐(2‐aminoacetamido)‐3‐{[(1S)‐1‐carbam oyl‐2‐(1H‐imidazol‐4‐yl)ethyl]carbamoyl}propanamido]methyl}phenyl)methyl]amino}‐6,13 ‐bis[3‐(morpholin‐4‐yl)propyl]‐5,7,12,14‐tetraoxo‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexa deca‐1,3,8,10,15‐pentaen‐2‐yl)amino]methyl}phenyl)methyl]‐N‐[(1S)‐1‐carbamoyl‐2‐(1H‐im 18 ACS Paragon Plus Environment

Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


idazol‐4‐yl)ethyl]butanediamide as ligand for 5. A 21.6 mg quantity of Boc-protected GDHNH2 peptide was dissolved in 1 mL DMSO, and allowed to be activated by 7.0 mg diisopropylcarbodiimide for 10 min at room temperature. 6.4 mg of N-hydroxysuccinimide was then added to the activated peptide. After 1-hour incubation, the GDH-NHS ester was added to 10 mg of compound 2. The resulted solution was stirred under for 24 h at room temperature. After reaction, 10 mL diethyl ether was added to reaction mixture, followed by centrifugation. A 1 mL concentrated HCl was added to the precipitate to deprotect peptide for 1 h, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.), with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 20%. 1H NMR (400 MHz, D2O) δ 8.54 (d, J = 16.1 Hz, 2H), 7.46 (m, 8H), 7.30 (m, 2H), 7.23 (d, J = 16.3 Hz,2H), 4.24 (d, J = 15.0 Hz, 4H), 4.04 (m, 14H), 3.83 (dd, 2H), 3.71 (m, 8H), 3.45 (d, J = 10.2 Hz, 4H), 3.21 (m, 6H), 3.09 (m, 6H), 2.95 (s, 2H), 2.79 (s, 2H), 2.03 (d, J = 9.8 Hz, 4H). HRMS (ESI) calculated for (M+2H)2+: m/z 703.3311, found 703.3327.

Synthesis

of

(2S)‐2‐amino‐N'‐[(4‐{[(9‐{[(4‐{[(3S)‐3‐amino‐3‐[({[(1S)‐1‐carbamoyl‐2‐(1H‐imidazol‐4‐yl)et hyl]carbamoyl}methyl)carbamoyl]propanamido]methyl}phenyl)methyl]amino}‐6,13‐bis[3‐( morpholin‐4‐yl)propyl]‐5,7,12,14‐tetraoxo‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexadeca‐1, 3,8,10,15‐pentaen‐2‐yl)amino]methyl}phenyl)methyl]‐N‐({[(1S)‐1‐carbamoyl‐2‐(1H‐imidazo l‐4‐yl)ethyl]carbamoyl}methyl)butanediamide as ligand for 6. A 21.6 mg quantity of Bocprotected DGH-NH2 peptide was dissolved in 1 mL DMSO, and allowed to be activated by 7.0 mg diisopropylcarbodiimide for 10 min at room temperature. 6.4 mg of N-hydroxysuccinimide was then added to the activated peptide. After 1-hour incubation, the DGH-NHS ester was added to 10 mg of compound 2. The resulted solution was stirred under for 24 h at room temperature. 19 ACS Paragon Plus Environment


Page 20 of 33

After reaction, 10 mL diethyl ether was added to reaction mixture, followed by centrifugation. A 1 mL concentrated HCl was added to the precipitate to deprotect peptide for 1 h, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.), with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 17%. 1H NMR (400 MHz, D2O) δ 8.51 (d, J = 6.1 Hz, 2H), 7.40 (m, 8H), 7.30 (d, J = 7.9 Hz, 2H), 7.17 (d, J = 9.3 Hz, 2H), 4.34 (d, J = 6.6 Hz, 4H), 4.26 (s, 4H), 4.01 (m, 14 H), 3.89 (s, 4H), 3.71 (t, J = 11.9 Hz, 4H), 3.41 (d, J = 12.0 Hz, 4H), 3.18 (m, 8H), 3.05 (dd, J = 20.7, 8.7, 4H), 2.65 (s, 2H), 2.02 (s, 4H). HRMS (ESI) calculated for (M+2H)2+: m/z 703.3311, found 703.3358.

Synthesis

of

(2S)‐2‐[2‐(2‐aminoacetamido)acetamido]‐N‐[(3‐{[(9‐{[(3‐{[(2S)‐2‐[2‐(2‐aminoacetamido)ace tamido]‐3‐(1H‐imidazol‐4‐yl)propanamido]methyl}phenyl)methyl]amino}‐6,13‐bis[3‐(morp holin‐4‐yl)propyl]‐5,7,12,14‐tetraoxo‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexadeca‐1,3,8,10,1 5‐pentaen‐2‐yl)amino]methyl}phenyl)methyl]‐3‐(1H‐imidazol‐4‐yl)propanamide as ligand for 7. An 18.7 mg quantity of Boc-protected GGH-OH peptide was dissolved in 1 mL DMSO, and allowed to be activated by 7.0 mg diisopropylcarbodiimide for 10 min at room temperature. 6.4 mg of N-hydroxysuccinimide was then added to the activated peptide. After 1-hour incubation, the GGH-NHS ester was added to 10 mg of compound 3. The resulted solution was stirred under for 24 h at room temperature. After reaction, 10 mL diethyl ether was added to reaction mixture, followed by centrifugation. A 1 mL concentrated HCl was added to the precipitate to deprotect peptide for 1 h, followed by purification by HPLC (Figure S1). Semipreparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.), with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). 20 ACS Paragon Plus Environment

Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yield: 17%. 1H NMR (400 MHz, D2O) δ 8.45 (s, 2H), 7.41 (m, 8H), 7.30 (s, 2H), 7.11 (m, 2H), 4.34 (d, J = 15.4 Hz, 4H), 4.23 (d, J = 15.3 Hz, 2H), 4.02 (m, 8H), 3.91 (s, 4H), 3.83 (s, 4H), 3.75 (t, J = 12.3 Hz, 4H), 3.43 (d, J = 12.3 Hz, 4H), 3.23 (t, J = 7.8 Hz, 4H), 3.08 (m, 8H), 2.65 (s, 4H), 2.02 (s 4H). HRMS (ESI) calculated for (M+2H)2+: m/z 646.3096, found 646.3053.

Synthesis

of

(2S)‐2‐(2‐aminoacetamido)‐N'‐[(3‐{[(9‐{[(3‐{[(3S)‐3‐(2‐aminoacetamido)‐3‐{[(1S)‐1‐carbam oyl‐2‐(1H‐imidazol‐4‐yl)ethyl]carbamoyl}propanamido]methyl}phenyl)methyl]amino}‐6,13 ‐bis[3‐(morpholin‐4‐yl)propyl]‐5,7,12,14‐tetraoxo‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexa deca‐1,3,8,10,15‐pentaen‐2‐yl)amino]methyl}phenyl)methyl]‐N‐[(1S)‐1‐carbamoyl‐2‐(1H‐im idazol‐4‐yl)ethyl]butanediamide as ligand for 8. A 21.6 mg quantity of Boc-protected GDHNH2 peptide was dissolved in 1 mL DMSO, and allowed to be activated by 7.0 mg diisopropylcarbodiimide for 10 min at room temperature. 6.4 mg of N-hydroxysuccinimide was then added to the activated peptide. After 1-hour incubation, the GDH-NHS ester was added to 10 mg of compound 3. The resulted solution was stirred under for 24 h at room temperature. After reaction, 10 mL diethyl ether was added to reaction mixture, followed by centrifugation. A 1 mL concentrated HCl was added to the precipitate to deprotect peptide for 1 h, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.), with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 16%. 1H NMR (400 MHz, D2O) δ 8.51 (d, J = 16.7 Hz, 2H), 7.32 (m, 8H), 7.20 (m, 4H), 4.24 (m, 8H), 3.98 (d, J = 9.8 Hz, 8H), 3.90 (s, 2H), 3.78 (d, J = 10.9 Hz, 2H), 3.73 (d, J = 11.0 Hz, 2H), 3.69 (t, J = 7.2 Hz, 4H) 3.37 (d, J = 8.4 Hz, 4H), 3.16 (m, 6H), 3.05 (m, 6H), 2.91 (s, 1H), 2.75 (s, 1H), 2.62 (s, 4H), 1.98 (d, J = 8.9 Hz, 4H). HRMS (ESI) calculated for (M+2H)2+: m/z 703.3311, found 703.3322.



Synthesis

Page 22 of 33

of

(2S)‐2‐amino‐N'‐[(3‐{[(9‐{[(3‐{[(3S)‐3‐amino‐3‐[({[(1S)‐1‐carbamoyl‐2‐(1H‐imidazol‐4‐yl)et hyl]carbamoyl}methyl)carbamoyl]propanamido]methyl}phenyl)methyl]amino}‐6,13‐bis[3‐( morpholin‐4‐yl)propyl]‐5,7,12,14‐tetraoxo‐6,13‐diazatetracyclo[6.6.2.04,16.011,15]hexadeca‐1, 3,8,10,15‐pentaen‐2‐yl)amino]methyl}phenyl)methyl]‐N‐({[(1S)‐1‐carbamoyl‐2‐(1H‐imidazo l‐4‐yl)ethyl]carbamoyl}methyl)butanediamide as ligand for 9. A 21.6 mg quantity of Bocprotected DGH-NH2 peptide was dissolved in 1 mL DMSO, and allowed to be activated by 7.0 mg diisopropylcarbodiimide for 10 min at room temperature. 6.4 mg of N-hydroxysuccinimide was then added to the activated peptide. After 1-hour incubation, the DGH-NHS ester was added to 10 mg of compound 3. The resulted solution was stirred under for 24 h at room temperature. After reaction, 10 mL diethyl ether was added to reaction mixture, followed by centrifugation. A 1 mL concentrated HCl was added to the precipitate to deprotect peptide for 1 h, followed by purification by HPLC (Figure S1). Semi-preparative HPLC was carried out by use of an HP 1100 Series HPLC apparatus with detection at 600 nm, and separation achieved with a C18 Vydac column (5 µM, 250 mm length, 10 mm I.D.), with a mobile phase of acetonitrile/H2O with 0.1% TFA (linear gradient was applied). Yield: 19%. 1H NMR (400 MHz, D2O) δ 8.52 (s, 2H), 7.36 (m, 8H), 7.20 (d, J = 7.6 Hz, 4H), 7.18 (s, 2H), 4.55 (t, J = 5.4 Hz, 2H), 4.34 (t, J = 6.6 Hz, 2H), 4.31 (s, 4H), 4.01 (m, 8H), 3.88 (s, 4H), 3.71 (t, J = 12.3 Hz, 4H), 3.41 (d, J = 11.7 Hz,4H), 3.18 (m, 6H), 3.06 (m, 6H), 2.95 (dd, J = 16.1, 5.2 Hz, 4H), 2.64 (s, 4H), 2.01 (s, 4H). HRMS (ESI) calculated for (M+2H)2+: m/z 703.3311, found 703.3290.

General methods for the synthesis of Cu complexes. Before further use in the synthesis of their corresponding Cu complexes, the concentrations of metal-free ATCUN-naphthalene diimide derivatives were quantified either by titrations with Cu(II) and monitoring the absorbance at 240 nm, or by fluorescence titrations with Cu(II) at 655 nm (λex = 620 nm). A


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solution of Cu complex was prepared in-situ by adding 2 equivalents of CuCl2 into the ligands in 10 mM Tris-HCl (pH = 7.4). Fluorescent DNA binding and cleavage assay. To study DNA binding affinity, G4 ligands were titrated to a solution of 1 µM 22G4 in 10 mM tris-HCl and 100 mM KCl (pH = 7.4). The change of emission intensity at 520 nm (ex: 495 nm) was monitored and used to calculate the dissociation constant KD for 22G4. The KD for CT-DNA was measured by use of a competition assay whereby G-quadruplex ligands were titrated to a solution containing 1 µM 22G4 and 100 µM CT-DNA in 10 mM tris-HCl and 100 mM KCl (pH = 7.4). DNA cleavage was performed by adding 1 mM ascorbate, 1 mM H2O2, and 0.5 µM of the indicated copper complex to varying concentrations of 22G4 DNA in 10 mM tris-HCl and 100 mM KCl (pH = 7.4) at 37°C, and the change of emission intensity was monitored at 520 nm (ex: 495 nm).

Denaturing PAGE. To maintain the same concentration of total nucleotides, cleavage reactions were performed with either 5 µM 22G4 or 11 µM ds12Telo. Aliquots from DNA stocks were then added to a solution to obtain a final concentration of 5 µM Cu complexes, 1 mM ascorbate and 1 mM H2O2 in 10 mM tris-HCl, followed by incubation at 37°C, while 100 mM KCl and 100 mM LiCl provided the counter cation in reaction buffer to stabilize 22G4 and 12dsTelo, respectively. Following reaction, a 15% gel with 6 M urea and a 25% gel with 7 M urea and 15% formamide were used to separate cleavage products of 22G4 and ds12Telo, respectively. The resulting gel was imaged by use of a Typhoon Trio Imager (GE). Integration of bands was performed by use of the software provided by the manufacturer.

Cellular uptake studied by LC-MS/MS. Cells were incubated with 10 µM copper complexes for 12 h, and then rinsed 3 times with PBS and lysed with 1 x protease inhibitor cocktail and 1% Triton X-100. Cell lysates were denatured at 95°C for 5 min and total protein concentration was then measured by use of a Bradford assay. Concentrations of the tested 23 ACS Paragon Plus Environment


articles were quantitated by LC-MS/MS. Separation was achieved by use of a C-18 column, while the mobile phase was a mixture of acetonitrile/water with 0.1% formic acid and a linear gradient was applied. An Agilent 6460 Triple Quadrupole Mass spectrometer was operated under the multiple reactions monitoring (MRM) mode with a collision energy 20-30 eV depending on the compound. The dwell time was 200 ms for each transition, and the following m/z transitions (precursor to product) were monitored: 323.6 to 110.1 for 4 and 7; 352.2 to 110.1 for 5, 6, 8, and 9.

Colocalization studied by confocal microscope. Cells were grown on a glass slide and incubated with the indicated compounds for 12 h, and then rinsed 3 times with PBS followed by fixation with 3% formaldehyde. A solution of 100 ng/mL DAPI was used to stain the cellular nucleus. Cells were then rinsed 3 times with PBS and visualized by use of Olympus Confocal Microscopy FV1000. The intrinsic emission of naphthalene diimide derivatives was excited by a 633 nm laser.

Alkaline comet assay. Cells were incubated with the indicated copper complexes for 3 days, while cells incubated with 50 µM H2O2 for 30 min were used as a positive control. Cells were harvested through trypsination, and then embedded into 0.75% low melting point agarose on Comet slides (Trevigen). Alkaline lysis buffer (pH > 13) was used to lyse cells in the dark for 15 h at 4°C. Gel electrophoresis was carried out for 10 min at 22 V in NaOH-EDTA buffer (pH > 12). After electrophoresis, the cells were rinsed with H2O and dried with ethanol, followed by staining with 10 µg/mL propidium iodide in PBS for 20 min. A Zeiss Axioskop microscope was used for fluorescence imaging. A total of 100 cells for each sample were scored by use of CASPlab software.


Page 24 of 33

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Telomere length measured by real time-PCR. A total of 1 × 105 cells were incubated with the test articles. After the indicated incubation time, the medium was withdrawn and the cells rinsed three times with PBS. Genomic DNA was extracted and the telomere length of each sample was subsequently measured by real time-PCR following a reported method36 by use of an Applied Biosystems 7900HT Fast Real-Time PCR System.

Cell synchronization. S-phase cells and G1/G0-phase cells were obtained by doublethymidine block and serum starvation, respectively. For S-phase cells, following retreatment with thymidine the cells were incubated with 0.5 µM compound and 100 ng/mL nocodazole for 10 h (control: 100 ng/mL nocodazole), since nocodazole can inhibit mitosis and prevent cells from advancing to the next round of the cell cycle. For G1/G0-phase cells, cells were incubated with 0.5 µM compound in serum-free medium. Cellular senescence. Cells were grown on a glass slide and incubated with the indicated compounds for 3 days. Cells were then rinsed with PBS, followed by fixation with 3% formaldehyde. After fixation, cells were rinsed with PBS and stained by use of an X-gal solution (1 mg/mL, pH 6) for 16 h at 37°C.43 A Zeiss Axioskop microscope was used to perform phase contrast imaging. Cytotoxicity. A total of 3,000 indicated cells were seeded into each well of a 96-well plate, and allowed to attach for 24 h. After 72-h incubation with the tested compounds, 80 μg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide was added to each well followed by 4-h incubation at 37°C. After the medium was carefully withdrawn, a 200 μL volume of DMSO was added to each well and the absorbance at 560 nm was measured by use of a SpectraMax M5 Multi-Mode Microplate Reader.



Page 26 of 33

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website: HPLC traces, DNA binding, Michaelis-Menten studies, denaturing PAGE, Comet assay, senescence assay, and Molecular Formula Strings.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Zhen Yu: 0000-0003-2092-9960 James A. Cowan: 0000-0002-4686-6825 Notes: The authors declare no competing financial interest. ABBREVIATIONS ATCUN, amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif; DIC, N,N′Diisopropylcarbodiimide; CT-DNA, calf-thymus DNA; DAPI, 4′,6-diamidino-2-phenylindole; HDFa,

human

adult

dermal

fibroblasts;

X-gal,

5-bromo-4-chloro-3-indolyl-β-D-

galactopyranoside; NHS, N-hydroxysuccinimide.

ACKNOWLEDGEMENTS This work was supported by grants from the National Institutes of Health [HL093446]. Z.Y. was supported by the Pelotonia Fellowship Program. 26 ACS Paragon Plus Environment

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Campisi, J.; di Fagagna, F. D. Cellular senescence: when bad things happen to good cells.

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10. Malumbres, M.; Barbacid, M. To cycle or not to cycle: a critical decision in cancer. Nat. Rev. Cancer 2001, 1, 222-231. 11. Shay, J. W.; Wright, W. E. Role of telomeres and telomerase in cancer. Semin. Cancer Biol. 2011, 21, 349-353. 12. Wyatt, H. D. M.; West, S. C.; Beattie, T. L. InTERTpreting telomerase structure and function. Nucleic Acids Res. 2010, 38, 5609-5622. 13. Neidle, S. Quadruplex nucleic acids as targets for anticancer therapeutics. Nat. Rev. Chem. 2017, 1. 14. Shay, J. W.; Wright, W. E. Telomerase therapeutics for cancer: challenges and new directions. Nat. Rev. Drug Discov. 2006, 5, 577-584. 15. Bryan, C.; Rice, C.; Hoffman, H.; Harkisheimer, M.; Sweeney, M.; Skordalakes, E. Structural basis of telomerase inhibition by the highly specific BIBR1532. Structure 2015, 23, 1934-1942. 16. Damm, K.; Hemmann, U.; Garin-Chesa, P.; Hauel, N.; Kauffmann, I.; Priepke, H.; Niestroj, C.; Daiber, C.; Enenkel, B.; Guilliard, B.; Lauritsch, I.; Muller, E.; Pascolo, E.; Sauter, G.; Pantic, M.; Martens, U. M.; Wenz, C.; Lingner, J.; Kraut, N.; Rettig, W. J.; Schnapp, A. A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J. 2001, 20, 6958-6968. 17. Harley, C. B.; Futcher, A. B.; Greider, C. W. Telomeres shorten during aging of human fibroblasts. Nature 1990, 345, 458-460. 28 ACS Paragon Plus Environment

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Table of Contents Graphic

Membrane-permeable small molecule copper complexes selectively target telomeric Gquadruplex DNA in cancer cells.


Rapid Telomere Reduction in Cancer Cells Induced by G-Quadruplex

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