Compounds

Compounds 2-10 were obtained from Pharminox Ltd, Biocity, Pennyfoot St, Nottingham NG 1 1GF, UK. Details of synthetic methods have been published (International Patent Application No. PCT/GB2011/051845 and PCT/GB2012/051467).

Biosensor-surface plasmon resonance (SPR) studies

Oligonucleotides 5′-biotin-d [AG₃ (T₂AG₃)₃] quadruplex and 5′-biotin-CGA₃T₃C(CT)₂GA₃T₃CG were purchased from Midland Certified Reagent Company (Midland, TX). Purification of DNA, preparation of solutions, collection of data, and analysis of results were conducted according to methods adopted in an earlier study [18].

Receptor inhibition

hEGR study and the M2 and the β2 receptor inhibition assay were performed as previously reported [17],[19].

Cells and culture conditions

Normal WI-38 diploid human lung fibroblasts and the human colorectal adenocarcinoma HT29 cells were obtained from American Type Culture Collection (Manassas, VA, USA). BJ fibroblasts expressing hTERT and SV40 early region (BJ-EHLT) were obtained as previously reported [8]. Cells were grown in Dulbecco Modified Eagle Medium (D-MEM, Invitrogen Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 2 mM L-glutamin and antibiotics.

Cell proliferation

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) assay was performed in treated and untreated cells for 96 hours. Cells were incubated with MTT solution (Sigma-Aldrich), and the purple formazan crystals were dissolved in isopropanol. Optical densities (OD) at 540 nm was determined on microplate reader.

Cytotoxic assay

The HT29 cells were seeded in 60 mM- Petri dishes at a density of 5x10⁴ cells/ plate in DMEM medium plus 10% serum FCS. After 24 hours cells were exposed to the following drugs: Ethyl-10-hydroxy-camptothecin (SN-38; 0.2, 0.4, 0.8 μM for 2 hrs), compound 8 (at 0.1, 0.2 and 0.4 μM for 96 hours) and compound 1 (0.5 and 1 μM for 96 hours). In the combination experiments the two different sequence of drug administration were evaluated: campthotecin followed by G-quadruplex ligands and the inverse sequence at fixed equipotent ratios. The medium containing the first drug was removed and replaced with fresh medium containing the second drug. Colony forming ability was evaluated as previously reported [20].

Immunofluorescence

Cells were fixed in 2% formaldehyde and permeabilized in 0.25% Triton X100 in PBS for 5 min at room temperature. For immunolabeling, cells were incubated with primary antibody, then washed in PBS and incubated with the secondary antibodies. The following primary antibodies were used: pAb and mAb anti-TRF1(Abcam Ltd.; Cambridge UK); mAb (Upstate, Lake Placid, NY) and pAb anti-γH2AX (Abcam); mAb anti-PCNA (Sigma Chemicals, Milano, Italy). The following secondary antibody were used: TRITC conjugated Goat anti Rabbit, FITC conjugated Goat anti Mouse (Jackson ImmunoResearch Europe Ltd., Suffolk, UK). Fluorescence signals were recorded by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera and elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany). This system permits to focus single planes inside the cell generating 3D highresolution images. For quantitative analysis of γH2AX positivity, 200 cells on triplicate slices were scored. For TIFs analysis, in each nucleus a single plane was analyzed and at least 50 nuclei per sample were scored.

Statistical analysis

Synergism, additivity, and antagonism were assessed by isobologram analysis as reported previously [21]. Combination index (CI) values <0.9, > 0.9 #x003C; 1.2, and >1.2 indicate synergism, additivity, and antagonism, respectively. The Student’s t-test (unpaired, two-tailed) was used for comparing statistical differences. Differences were considered statistically significant when P #x003C; 0.05.

On and off-target profiles of the new RHPS4-derivatives

The relative binding affinity of each compound showed in Figure 1 for quadruplex and duplex DNA structures were measured by the Surface Plasmon Resonance (SPR). This technique takes advantage of the refractive index change elicits by the binding of the drug with the h-Tel quadruplex DNA sequence 5′-d[AGGG(TTAGGG)₃]-3′ or an alternating hairpin duplex sequence immobilized on a sensor chip surface. As reported in Table 1, all the compounds analyzed, with the exception of compound 4, showed a comparable or an enhanced quadruplex on duplex ratio relative to that of compound 1, indicative of a global enhanced selectivity for G4 DNA structure.

Biological characterization of new RHPS4-derivatives

These encouraging results in terms of potentially improved toxicological profile and DNA binding selectivity, encouraged us to conduct a more comprehensive biological characterization of the new ligands. Firstly, we investigated if they were able to efficiently promote growth inhibition of tumor cells without affecting the survival of normal cells in vitro. In Table 1 we report for each compound the concentration causing 50% of growth inhibition (GI₅₀) calculated by performing a proliferation assay in the human colon cancer cell line HT-29 or in the human ‘normal’ lung WI-38 cells. The most efficacious agents in terms of normal/cancer cell selectivity were compounds 2, 6, 7 and 8, all exhibiting (analogously to compound 1) a low GI₅₀ in HT-29 and an high GI₅₀ in WI-38 treatment. On the basis of results described so far, we decided to exclude from the further analysis ligand 10 for its high value of hERG inhibition (95%) (Table 1).

In order to investigate the ability of the new G4-stabilizing agents to cause telomere uncapping, a two-step analysis was performed to establish if the compounds were able to induce DNA damage and, more significantly, if the DNA damage was localized to the telomeres. By performing immunofluorescence analysis on human transformed BJ fibroblasts (BJ-EHLT) to evaluate the phosphorylation of H2AX, a hallmark of DNA double strand break, we observed that all the compounds induced DNA damage at both 0,1 and 0,5 μM doses with the exception of compounds 7 and 9 (Figure 2A). Unfortunately, we were not able to test compounds 4, 5 and the 0,5 μM dose of 3 as they were fluorescent in both red and green channels. The quantitative analysis revealed that the new ligands were more efficient in damaging DNA than compound 1, since they elicited a significant percentage of cells positive for γ-H2AX at a dose of 0,1 μM (Figure 2A). To ascertain whether γ-H2AX was phosphorylated in response to dysfunctional telomeres, double immunofluorescence experiments were processed by confocal microscopy. We observed that some of the γ-H2AX foci induced by G4-ligands colocalized with TRF1, a good marker for interphase telomeres [18], forming the so-called telomere dysfunction-induced foci (TIFs) [19] (Figure 2B). Of note, quantitative analysis identified compounds 3, 6 and 8 as the most potent in ability to specifically uncap telomeres: the percentage of cells with more than four γ-H2AX/TRF1 colocalizations reached, for compound 3, about 18% in 0,1 μM treated cells and 40% in 0,5 μM (compounds 6 and 8) (Figure 2B), with a mean of about seven TIFs per nucleus (Figure 2C). Intriguingly, even though the compounds 2, 7 and 9 seemed to be promising in terms of off-target profile, G4-affinity and cancer cell selectivity, they did not cause significant telomere dysfunction (Figure 2B).

With the intent of further confirming if the new compounds resembled the same molecular mode of action of the lead chemotype 1[9], we compared their capability to trigger a replication-dependent DNA damage - in particular, to determine which fraction of the cells formed γ-H2AX foci. We performed co-immunostaining to γ-H2AX and the proliferating cell nuclear antigen PCNA, which accumulates in the nucleus during S phase of cell cycle. In the case of compounds 1 and 8, γ-H2AX foci formation was almost exclusively restricted to PCNA-positive, and so replicating, cells: in the case of the other drugs γ-H2AX foci formed both in PCNA-positive and -negative cells (Figure 2D), indicating that the compounds induced a cell cycle-independent DNA damage. At the end of our screening, we can conclude that most successful novel molecules in terms of telomere targeting as well as of improved toxicological profile compared to the original compound 1 were the ligands 6 and 8. Of the new compounds, agent 8 showed a replication-dependent mode of action similar to compound 1.

Synergistc effect of compound 8 with a topoisomerase Inhibitor

We have previously reported that compound 1 in combination therapy with topoisomerase (TOPO) I inhibitors (Camptothecins) produced a synergistic antitumoral activitiy in in vitro and in vivo models [12],[13]. Our published results support the hypothesis that this synergism relies on the role of TOPO I in relaxing the topological stress normally occurring during the progression of the replication fork and drastically increased at telomeres by the presence of G4 stabilizing agents. Tumor cells exposed to a TOPO I inhibitor prior to the administration of a G4 ligand were prevented or impaired in repairing dysfunctional telomeres, becoming more susceptible to cell death than if they received the single treatments, or the opposite sequence, of drug exposure. The study of in vitro interaction between agent 8 and ethyl-10-hydroxy-camptothecin (SN-38), the active metabolite of camptothecin Irinotecan, was preceded by experiments in which the cell colony-forming ability of the human colorectal adenocarcinoma HT29 cells was evaluated at different doses of compound 1 or 8. Results reported in Figure 3A indicate that the new G4-ligand, at equal time of drug exposure (96 hours), inhibited cell survival in a dose-dependent manner like compound 1 but more efficiently so. Moreover, when HT29 cells were treated with different concentrations of SN-38 and compound 8, a strong synergistic effect, with a Combination Index (CI) #x003C; 0.5, was observed when the first agent was followed by the G4-ligand, both at already the lowest dosages tested (Figure 3B-C and data not shown). As expected from the previously reported combination between compound 1 and SN-38, the inverse sequence of drug administration was less effective in reducing the tumor cell survival, eliciting only an additive or slight synergistic interaction (Figure 3C), thus further confirming the high mechanicistic analogy between compounds 1 and 8 when applied in combination therapy with a TOPO I inhibitor.