A Caged Ligand for a Telomeric G‐Quadruplex (2012)

Photoactivated telomerase inhibition: The biological activities of caged compounds are masked by photolabile protecting groups, and can be unmasked with spatiotemporal specificity by light. We describe the design and synthesis of a caged G-quadruplex (G4) ligand. On in situ activation by UV light irradiation the compound showed significant G4-stabilizing activity and telomerase-inhibitory activity. Human telomeric DNA, located at the ends of chromosomes, contains repeating single-strand (TTAGGG)n sequences.1 These G-rich strands form a characteristic stacked three-dimensional structure, called the G-quadruplex (G4).2 Stabilization of telomeric G4 has been shown to inhibit the activity of telomerase,3 an enzyme that is selectively expressed in most tumor cells and plays key roles in telomere maintenance and cellular immortalization.4 Furthermore, telomeric G4 stabilization also stimulates dissociation of POT1 and TRF2 (telomere-related proteins), which protect chromosome ends from degradation and inappropriate DNA repair and induce apoptosis of tumor cells.5 Stabilization of the telomeric G4 structure is thus considered to be a promising anticancer strategy. Various natural and synthetic small molecules that stabilize telomeric G4 have been investigated.6 However, additional features—that is, selectivity between telomeric G4 and duplex DNA, as well as selectivity between normal cells and cancer cells—are required for practically useful molecules. Although there has been recent progress in selective structural recognition of G4 by small molecules, stabilization of the G4 structure in specific target cells—cancer cells, for example—is still difficult. We considered that a caging strategy might be useful for overcoming these specificity issues and to aid the development of candidate drugs. Caged compounds are light-sensitive ligands in which one or more significant functional group(s) required for biological activity is/are masked with one or more photolabile group(s). Caged compounds are therefore biologically inactive. When they are “uncaged” by irradiation, however, the biological activity is unmasked, and so the latent biological activities of caged compounds can in principle be activated with high spatial and temporal specificity, or at specific molecular targets, by means of light irradiation.7 Here we describe the design, synthesis, and characteristics of the first example of a caged compound targeting telomeric G4. Uncaging by irradiation was confirmed to result in inhibition of telomerase activity in vitro and in growth inhibition of several cancer cell lines. We have recently reported a series of macrocyclic polyoxazoles—6OTDs (Scheme 1),8a, 8b 6M(4)OTDs,8c and 7OTD8d—as G4 ligands inspired by the highly potent natural G4 ligand telomestatin (1), isolated from Streptomyces annulatus 3533-SV4.9 The 6OTDs and 6M(4)OTDs each have two side chains on a macrocyclic core structure, and their G4-stabilizing abilities can be tuned by varying the functional groups on the side chains.8a Installation of amino functional groups in the side chains increases the G4-stabilizing abilities of the ligands, presumably through interaction with phosphate groups in DNA. However, the G4-stabilizing activity can be masked simply by protection of the amine with an acetyl group.8a With these considerations in mind, we designed the caged G4 ligand Y2Nv2-6M(4)OTD (2 b), which has phenolic moieties on the side chains of 6M(4)OTD. Phenolic hydroxy groups are known to interact with DNA through hydrogen bonding,10 and can easily be masked with the nitroveratryl (Nv) group as a photolabile protecting (caging) group. Structures of telomestatin (1) and 6OTDs 2 a and 2 b. The synthesis of Y2H2-6M(4)OTD (2 a, Scheme 1) was carried out from L-tyrosine by the previously developed procedure (see the Supporting Information).8c The phenolic hydroxy groups of 2 a were then masked with Nv groups by treatment with Nv bromide in the presence of potassium carbonate in DMF to generate Y2Nv2-6M(4)OTD (2 b) as a caged G4 ligand (see the Supporting Information). Firstly, stabilization of telomeric G4 with 2 a and 2 b was evaluated by a fluorescence resonance energy transfer (FRET) melting assay11 with use of a fluorescence-labeled single-strand oligonucleotide of telo21 {5′-FAM-d(GGG[TTAGGG]3)-TAMRA-3′}. The ΔTm values for 2 a and 2 b were calculated at concentrations of 2.0 μM, which corresponds to ten equivalents of ligand versus DNA, in the presence of potassium chloride (60 mM). Under these conditions, the ΔTm values for 2 a and 2 b were 16.6 °C and 4.0 °C, respectively, indicating that 2 a effectively stabilizes telomeric G4,12 but that the masked form (2 b) does not.13 Next, the time course of activation (uncaging) of 2 b by irradiation was analyzed by HPLC. Compound 2 b was irradiated at 365 nm, and 2 b and 2 a were quantified by their HPLC peak areas. As shown in Figure 1, 2 b disappeared in a time-dependent manner and was completely consumed within 20 min, with corresponding appearance of the deprotected form 2 a. This uncaging process—that is, conversion of 2 b into 2 a by light—was analyzed by a FRET melting assay. In this assay, a mixture of telo21 and 2 b (10 equiv relative to telo21) was irradiated (365 nm, 3 W), and the time-dependent change in ΔTm of telo21 was plotted. The ΔTm value increased from 4.0 °C to 17.0 °C in proportion to irradiation time, indicating that Y2-Nv2-6M(4)OTD (2 b) behaves as a caged G4 ligand, and telomeric G4 is increasingly stabilized as the uncaging reaction proceeds, generating 2 a (Figure 1 A). Time course of uncaging of 2 b. A) HPLC and fluorescence resonance energy transfer (FRET) melting assay. The FRET melting assay was conducted with telo21 (0.2 μM) and 2 b (2.0 μM) in the presence of K+ (60 mM). B) Electrophoretic mobility shift assay (EMSA). Telo24 (10 μM) was incubated with 2 a or 2 b (100 μM) in Tris⋅HCl buffer in the presence of K+ (250 mM). After irradiation at 365 nm for an appropriate time, the samples were mixed with Ficol (final 20 %) and run on a nondenatured polyacrylamide gel (20 %) with 1×TBE buffer. The caged G4 ligand Y2Nv2-6M(4)OTD (2 b) was further evaluated by an electrophoretic mobility shift assay (EMSA).9b, 14 In this assay, the uncaging of 2 b with light (365 nm) was carried out in the presence of telo24 for various reaction times, and the reaction products were subjected to electrophoresis. Under the conditions used, a new band corresponding to the telomeric G4 structure stabilized by 2 a appeared in increasing amounts as the irradiation time was extended. The irradiation-time-dependent increase in this band (lanes 3–8) reached saturation at 20 min [Figure 1 B, lanes 8 and 10 (control)]. These results are in good agreement with the uncaging results obtained by the FRET melting assay (Figure 1 A). The effects of 2 a and 2 b on telomerase activity were next examined in a cell-free system by a telomerase repeat amplification protocol (TRAP) assay4a with use of PC3 cell lysate (Figure 2). In this experiment, 2 a showed significant inhibitory activity at a concentration of 2.0 μM, whereas the caged ligand 2 b did not show any inhibitory activity under the same conditions (Figure 2, lanes 2 and 3). Next, the inhibitory activity of 2 b under irradiation was examined, and it was found that 2 b effectively inhibited the telomerase activity upon irradiation (lane 4). Because irradiation itself did not affect telomerase activity (lane 5), the uncaging reaction of 2 b to form 2 a must be the basis of the observed biological activity.15 Telomerase repeat amplification protocol (TRAP) assay of 2 a and 2 b. Lane 1: negative control (no compound). Lane 2: 2 a (2.0 μM). Lane 3: 2 b (2.0 μM). Lane 4: 2 b (2.0 μM)+irradiation. Lane 5: negative control+irradiation. Lane 6: positive control (no telomerase). These G4 ligands were further evaluated in a cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with A549 (lung cancer), DU145 (prostate cancer), HeLa (carcinoma), and MCF7 (breast cancer) cells. Y2H2-6M(4)OTD (2 a) showed significant antiproliferative activity within 2 days (Table 1). In contrast, 2 b did not show any antiproliferative activity (>30 μM), but activity emerged after irradiation (Table 1). This caged system thus appears to be effective in cancer cells in vitro.16 IC50 [μM] Cell line 2 a 2 b 2 b+light A549 0.82±0.07 >30 0.95±0.08 DU145 0.38±0.23 >30 0.69±0.29 HeLa 0.068±0.01 >30 0.12±0.05 MCF7 0.21±0.13 >30 0.25±0.06 In summary, we have designed, synthesized, and characterized the activity of the first example of a caged telomeric G4 ligand: Y2Nv2-6M(4)OTD (2 b). This caged ligand 2 b was activated by irradiation (365 nm) to afford the uncaged compound 2 a, which showed significant telomeric G4-stabilizing activity in EMSA and in FRET melting assay. On exposure to light irradiation, compound 2 b showed telomerase-inhibitory activity in a cell-free TRAP assay and exhibited high antiproliferative activity against cancer cell lines in a cell-based MTT assay. Further efforts to apply this concept in vivo are in progress. FRET melting assay: The FRET melting assay was carried out according to reported methods.11 It was performed with an excitation wavelength of 470–505 nm and a detection wavelength of 523–543 nm and use of the DNA Engine Opticon 2 Real-Time Cycler PCR detection system (BioRad). The dual fluorescently labeled oligonucleotides Flu-ss-telo21 {5′-FAM-d[GGG(TTAGGG)3]-TAMRA-3′} and Flu-ss-ds-26-mer {5′-FAM-d[(TA)2GC(TA)2T6(TA)2GC(TA)2]-TAMRA-3′} were used in this protocol. The donor fluorophore was 6-carboxyfluorescein (FAM) and the acceptor fluorophore was 6-carboxytetramethylrhodamine (TAMRA). All purified nucleotides (Sigma Genosys) were dissolved as stock solutions (100 μM) in MilliQ water to be used without further purification. Further dilutions of the oligonucleotides were performed with potassium cacodylate buffer (pH 7.4, 60 mM), and FRET experiments were carried out with oligonucleotide solutions (400 nM). Dual-labeled DNA was annealed by heating at 96 °C for 2 min and was allowed to cool to room temperature. Original compounds were prepared as DMSO stock solutions (10 mM) and diluted to 200 μM with DMSO. The rest of the dilutions were performed with FRET (cacodylate) buffer. FRET experiments were carried out with a 4.0 μM starting solution of the compound. Next, the annealed DNA (20 μL) and the compound solution (20 μL) were distributed across 96-well plates (Bio-Rad), with a total reaction volume of 40 μL, with labeled oligonucleotide (200 nM) and the compound (2.0 μM). The plates were incubated at 25 °C. Subsequent experiments used the following temperature procedure in RT-PCR, finishing as follows: 25 °C for 30 min, then a stepwise increase of 1 °C every minute from 25 °C until 99 °C. During the procedures, we measured the FAM fluorescence after each step. The change in melting temperature at 2.0 μM compound concentration—ΔTm (2.0 μM)—was calculated from at least three experiments by subtraction of the blank from the averaged melting temperature of each compound (2.0 μM). CD experiments: CD spectra were recorded with a JASCO-810 spectropolarimeter (Jasco, Easton, MD) in a quartz cell of 1 mm optical path length and with an instrument scanning speed of 100 nm min−1 and a response time of 1 s, over the wavelength range of 230–320 nm. CD spectra are representative of ten averaged scans taken at 25 °C. The nucleotide, telo24 (5′-d[TTAGGG]4-3′, purified, FASMAC Co., Ltd. Kanagawa, Japan), was dissolved as a stock solution (1 mM) in MilliQ water to be used without further purification. The nucleotide was further diluted with KCl (100 mM) and Tris<M.>HCl buffer (pH 7.2, 50 mM), and CD spectra were measured with a 10 μM oligonucleotide solution. The solution was annealed by heating at 96 °C for 2 min and then allowed to cool slowly to room temperature. The nucleotide solution (200 μL) and the compound solution (2.0 μL) were mixed and injected into a quartz cell. Electrophoresis mobility shift assay (EMSA): EMSA was performed as follows: telo24 oligonucleotide (50 μM, 2.0 μL) in MilliQ water was added to Tris⋅HCl buffer (50 mM, pH 7.1, 5.0 μL) with KCl (250 mM). This solution was heated at 94 °C for 2 min and was then allowed to cool slowly to 25 °C. Compound 2 a or 2 b in DMSO (1 mM, 1.0 μL) was added to the DNA solution. After a 14 h incubation period, with irradiation of the solution mixture with λ=365 nm light for 0–30 min, the sample was mixed with Ficol 400 solution (100 mg mL−1, 2.0 μL). Each mixture (1.5 μL) was run on nondenatured polyacrylamide gel (20 %) in 1×TBE buffer (4 °C, 250 V for 55 min) and stained with Stains-all®. The gels were imaged with a phosphorimager (Typhoon 8600, Molecular Dynamics) using 580–640 band pass filters. The ligand–G4 complexes of lanes 2–9 in Figure 1 B were quantified by use of ImageQuant 5.2 software. Telomerase repeat amplification protocol (TRAP) assay: Telomerase repeat amplification protocol (TRAP) assays were conducted as follows: PC3 cells (1×105) were lysed with TRAP lysis buffer (200 μL),17 placed on ice for 30 min, and centrifuged for 20 min (15 000 rpm, 4 °C). This TRAP lysate (2.0 μL) was mixed with 10×T-PCR buffer (5.0 μL), dNTPs (2.5 mM , 1.0 μL), TS primer (5′-d[AATCC GTCGA GCAGA GTT]-3′, 50 ng μL−1, 2.0 μL), TSNT (5′-d[AATCC GTCGA GCGAG TTAAA AGGCC GAGAA GCGAT]-3′, 0.2 pM, 0.5 μL), DEPC (diethyl pyrocarbonate) solution in MiiliQ water (30 μL), and compounds (DMSO solution, 5.0 μL), and placed on ice for 30 min (UV light irradiation was conducted during this incubation period). After the telomerase reactions (30 min, 20 °C), the reacting solution was placed on ice, and NT primer (5′-d[ATCGC TTCTC GGCCT TTT]-3′, 10 μM, 2.0 μL), ACX primer {5′-d[GCGCGG (CTTACC)3 CTAACC]-3′, 10 μM, 2.0 μL}, and Gene Taq (Nippon Gene, 0.5 μL) were added. Three-step PCR was performed: 27 cycles of 94 °C for 25 s, 50 °C for 25 s, and 72 °C for 45 s. PC3 cell lysates were used as negative controls, with lysis buffer as a positive control. Each PCR reacting solution (approximately 5.0 μL) and 10×loading buffer (Takara Bio Inc., 1.0 μL) were loaded onto a nondenaturing polyacrylamide gel (10 %, Perfect NT Gel, D.R.C. Co., Ltd., Tokyo, Japan) and run for 10 min at 100 V and then for 55 min at 200 V. Gels were visualized with SYBR Green I (Takara Bio Inc. Shiga, Japan). Cell culture: A549, DU145, HeLa, and MCF7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, 1.0 g L−1 glucose, nacalai tesque) containing fetal bovine serum (10 %) and kanamycin (0.1 mg mL−1). In brief, A549, DU145, HeLa, and MCF7 cells were seeded (500 cells per well) into 96-well plates in DMEM (80 μL) and grown for 24 h. Subsequently ligand (2 a, 2 b, or 3) solutions (20 μL) of appropriate concentration were added [when caged compounds were added, light irradiation (20 min) was conducted just after the addition of the ligand] and incubation was carried out for a further 48 h. MTT (Sigma–Aldrich) solution (0.5 mg mL−1, 10 μL) was then added to each well and incubated for 4 h at 37 °C. After removal of the solvent, dimethyl sulfoxide (nacalai tesque, 100 μL) was added. Corolimetric reading at 570 nm and 630 nm (as a control) was performed with a microplate reader (xMark Microplate Reader, Bio-Rad). The results are expressed as the mean percentages of cellular growth inhibition from triplicate cultures. We thank Dr. Kyotaro Hirashima and Dr. Sachiko Okabe (JFCR) for advice on TRAP assay and cellular proliferation assay. This work was supported in part by the Novartis Foundation for the Promotion of Science (Japan), the Mochida Memorial Foundation ,and the Naito Foundation, as well as by Grants-in-Aid for Scientific Research (B) from JSPS (No. 23 310 158) and Exploratory Research (21 655 060) and a grant under the Industrial Technology Research Grant Program (01A04006b) from theNew Energy and Industrial Technology Development Organization of Japan (NEDO). Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.