- Open Access
Identification of novel RHPS4-derivative ligands with improved toxicological profiles and telomere-targeting activities
© Rizzo et al.; licensee BioMed Central Ltd. 2014
- Received: 11 August 2014
- Accepted: 22 September 2014
- Published: 6 October 2014
The Erratum to this article has been published in Journal of Experimental & Clinical Cancer Research 2015 34:9
The pentacyclic acridinium salt RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate, compound 1) is one of the most interesting DNA G-quadruplex binding molecules due to its high efficacy in tumor cell growth inhibition both in in vitro models and in vivo against human tumor xenografts in combination with conventional chemotherapeutics. Despite compound 1 having desirable chemical and pharmaceutical properties, its potential as a therapeutic agent is compromised by off-target effects on cardiovascular physiology. In this paper we report a new series of structurally-related compounds which were developed in an attempt to minimize its off-target profile, but maintaining the same favorable chemical and pharmacological features of the lead compound. By performing a comparative analysis it was possible to identify which derivatives had the following properties: (i) to show a reduced capacity in respect to compound 1 to inhibit the hERG tail current tested in a patch clamp assay and/or to interact with the human recombinant β2 receptor; (ii) to maintain both a good G4-binding affinity and cancer cell selectivity; and (iii) to trigger DNA damage with specific telomere uncapping. These studies allowed us to identify a novel G4-stabilizing molecule, compound 8, being characterized by reduced off-target effects and potent telomere on-target properties compared to the prototypic compound 1. Moreover, compound 8 shares with compound 1 the same molecular mode of action and an anti-tumour activity specifically restricted to replicating cells, as evident with its particularly efficient activity in combination therapy with a topoisomerase I inhibitor. In conclusion, we have identified a new pentacyclic derivative 8 having suitable properties to be the focus of further investigations as a clinical candidate for cancer therapy.
- Telomere-targeting agents
DNA has played an historic role as a molecular target for the development of some effective chemotherapeutics producing a significant improvement in the survival of patients. However, unfortunately, adverse side effects have limited their clinical potential. Consequently, much effort has been invested into finding novel agents that are more selective for cancer-specific DNA targets. Secondary DNA structures, such as G-quadruplex (G4), higher-order four-stranded structures, which can form in guanine-rich nucleic acid sequences, have recently emerged as a new class of molecular targets for developing DNA-interactive compounds as therapeutics in oncology and in other diseases . Interest in the more general therapeutic significance of G4 has expanded during the past decade to include G4 structures not only at chromosome ends but also in the promoter sequences of a wide range of genes important in cell signalling, recognized as hallmarks of cancer. The broad concept of G4 DNA being therapeutically-susceptible hot-spots has recently been validated by their direct visualization in human cells  and by the finding that these structures can be stabilized in cells by small molecules ,.
As a result of research on telomeric G4 and the cellular consequence of targeting them with small molecules that stabilize these structures, their biological and therapeutic significance is well appreciated and continues to be an active field of drug discovery to identify appropriate modulators to be tested in patients. In this context, several chemotypes with different chemical structures have been developed showing good anti-tumor properties both in vitro and in xenografts ,. However, notwithstanding the promising results obtained in preclinical models, the synthetic compound quarfloxin, CX-3543, is the sole G4-binding small molecule that has progressed to date to phase II clinical trial  and very recently Tetragene (www.tetragene.com) has in-licensed it for further clinical development.
Our pioneering studies have clearly reported that G4-interacting agents are more than simple telomerase inhibitors and that their direct target is rather the telomere per se than telomerase ,. In particular, we have investigated thoroughly the antitumor properties and the molecular mechanism(s) of action of a G4 ligand, the pentacyclic acridine RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate, compound 1). We observed initially that, in addition to its telomerase-inhibitory properties, this drug exerts an anticancer effect by impairing telomere replication with consequent telomeric chromatin alteration leading to the activation of a strong DNA damage response at telomeres -. Compound 1 is also one of the most effective and selective G4 ligands, showing single agent antitumoral activity with a good toxicological profile in a variety of human tumor xenografts in mice, and able to potentiate the antitumoral efficacy of topoisomerase I inhibitors and, spectacularly so, in a triple combination with irinotecan and a PARP-1 inhibitor -.
Recently, we identified a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G4 structure that can be efficiently stabilized by RHPS4 with the consequence reduction of VEGFR-2 expression, thus resulting in the impairment of the angiogenic process . Notwithstanding the fact that compound 1 has been documented in preclinical studies as a promising G4 ligand having many of the attributes of an ideal pharmaceutical , this compound did not progress to clinical trials since our recent study demonstrated some undesirable off-target effects. In fact, experiments performed on guinea pig showed cardiotoxicity probably related to the interaction of compound 1 with the β2 adrenergic receptor and M1, M2 and M3 muscarinic receptors, together with a potent inhibition of the hERG (human Ether-a-go-go Related Gene) tail current . Through careful structural modifications, two second-generation molecules with significantly improved off-target profiles were identified  giving hope that it may be possible to develop a new agent from this pentacyclic class with minimal off-target liabilities. In this paper we report that a new series of compounds with antitumor properties comparable to compound 1, coupled with improved toxicological profiles, thus identifying new possible candidates for clinical application.
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 .
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 . 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.
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.
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 .
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.
Synergism, additivity, and antagonism were assessed by isobologram analysis as reported previously . 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.
In previous papers we have detailed the chemical and pharmacological properties of the telomere-targeted agent 3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulphate (RHPS4, compound 1). Unfortunately, its desirable potential as a therapeutic agent is compromised by cardiovascular effects that could be ameliorated to some extent in other related structures containing the pentacyclic acridinium pharmacophore .
On and off-target profiles of the new RHPS4-derivatives
On and off target profile of novel 1-derivative ligands
Off target receptor profile
DNA Affinity & Selectivity (Measured by SPR)
Cancer cell activity
”Normal” Cell activity
Muscarinic (M2) % inh @1μM
β2 adrenergic %inh @1 μM
hERG % inh @10 μM
Quadruplex DNA affinity Kx106M-1
Duplex DNA affinity Kx106M-1
Ratio PMX WI38 /HT29
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).
With the intent of further confirming if the new compounds resembled the same molecular mode of action of the lead chemotype 1, 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
In conclusion, the modifications of the prototype pentacyclic acridinium salt 1 allowed the synthesis and the selection of a novel promising G4-stabilizing telomere targeting agent (compound 8), being superior to compound 1 both in toxicological profile and on-target properties, which could be a suitable compound for progression into clinical trials.
Costs of experiments described within this manuscript were funded by Pharminox Ltd. The costs of the biological experiments were funded by Italian Association for Cancer Research (AIRC # 11567). Dr. A. Rizzo and E. Salvati are recipient of fellowships from the Veronesi Foundation.
We wish to thank Dr. I. Hutchinson, Dr. Marc Geoffrey Hummersone, Dr D. Cousin and Dr. M. Frigerio for the synthesis of the new compounds 2-10.
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