On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts
- Sara Iachettini1,
- Malcolm FG Stevens2,
- Mark Frigerio3,
- Marc G Hummersone3,
- Ian Hutchinson3,
- Thomas P Garner4,
- Mark S Searle4,
- David W Wilson5,
- Manoj Munde5,
- Rupesh Nanjunda5,
- Carmen D’Angelo1,
- Pasquale Zizza1,
- Angela Rizzo1,
- Chiara Cingolani1,
- Federica De Cicco1,
- Manuela Porru1,
- Maurizio D’Incalci6,
- Carlo Leonetti1,
- Annamaria Biroccio1Email author and
- Erica Salvati1Email author
© Iachettini et al.; licensee BioMed Central Ltd. 2013
Received: 26 July 2013
Accepted: 29 August 2013
Published: 19 September 2013
Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment.
The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant β2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay.
Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with β2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities.
Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.
KeywordsTelomere targeting agents G-quadruplex Anti-cancer therapy
Telomeric DNA is protected and maintained at the ends of chromosomes by the action of the enzyme telomerase. Whilst the shortening of DNA telomeres during repeated cell division is a natural part of the cellular ageing mechanism, one of the hallmarks of cancer is the expression of telomerase by cancer cells which allows them to maintain telomeric length and adopt immortal characteristics[1, 2]. Telomerase requires a single-stranded DNA primer as substrate for the addition of telomeric repeats (TTAGGG), his terminal telomere G-rich single stranded tract, also called G-overhang, can fold into four-stranded G-quadruplex (G4) structures consisting of G-tetrads coordinated around a monovalent cation[4, 5]. G4 stabilization, deny access of telomerase to its substrate, representing a valid tool for telomerase targeted approach in cancer therapy. Nevertheless, for direct telomerase inhibition, a time-dependent response is observed, related to the basal length of the telomeres, due to the slow attrition of telomeres experienced after each cell division, thus limiting the efficacy of agents designed to inhibit telomerase alone[7–9]. The extremely rapid and potent cytotoxic effect triggered by G4 ligands interacting with telomeric DNA sequences (‘Telomere Targeting Agents’: TTAs) is explained by a dual mechanism of action. On one hand the inhibition of telomerase, and, on the other hand, disruption of the shelterin complex, a nulcleo-protein complex which stabilises and protects the ends of chromosomes from being recognized as double-strand breaks[7, 8]. The presence of G4 structures has been recently showed in non telomeric regions, as already hypothesized on the base of predictive studies. In particular, G4 forming regions were already found in the promoter of several cancer related genes (c-myc, bcl2, hif1, hTERT), and for some of those genes, a transcriptional inhibitory function was attributed to these structures. Consequently, G4 targeting molecules could have additional extra-telomeric features, which could improve their potential as anti-cancer agents.
3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium metho-sulfate 1 was prepared from 6-fluoro-1,2-dimethylquinolinium methosulfate 7 as described. 2-Acetylamino- (2) and 3-acetylamino-8,13-dimethyl-8H-quino[4,3,2-kl]-acridinium iodide (3) were prepared according to published methods.
13-Ethyl-3,11-difluoro-6,8-dimethyl-8H-quino[4,3,2-kl]acridinium trifluoromethosulfate (8)
Ethyl trifloromethosulfate (1 mL) was added to a solution of 3,11-difluoro-6,8-dimethyl-8H-quino[4,3,2-kl]acridine (6; 0.05 g, 0.15 mmol) in CHCl3 (2 mL) under nitrogen. The mixture was heated at 140°C in a sealed tube for 3 days, cooled and solvent evaporated. The residue was purified by column chromatography on silica gel (5% MeOH/DCM) to leave the salt (8) as a bright red solid (20%), mp >250°C (decomp.); IR (νmax) 1620, 1583, 1533, 1475, 1429, 1255, 1028 cm-1; 1H NMR (DMSO-d6) δ 8.58 (1H, dd, J = 10.0, 2.9 Hz), 8.43 (1H, s), 8.26 (2H, m), 8.21 (1H, dd, J = 9.4, 4.9), 8.04 (1H, m), 8.01 (1H, s), 7.78 (1H, m), 5.12 (2H, q, J = 6.8 Hz, N-CH2), 3.17 (3H, d, J = 5.1 Hz), 2.78 (3H, s, N-CH3), 1.15 (3H, t, J = 6.8 Hz, N-CH2CH 3 ); m/z 361.1 (M+).
Cardiovascular effects of anaesthetised Guinea pig
After anaesthesia with approximately 40 to 60 mg/kg (i.p.) sodium pentobarbitone, a jugular vein was cannulated for administration of the vehicle or test substance. Arterial blood pressure (systolic, diastolic and mean) was measured via a catheter inserted into the carotid artery, heart rate was derived electronically from the pressure waveform and a sample of arterial blood determined blood gases (PO2 and PCO2), O2 saturation, standard bicarbonate (HCO3), pH and base excess before the start of the experiment. Electrocardiogram (ECG) limb electrodes recorded the standard lead II configuration and QTcB interval (calculated as QTcB = QT/(√RR)). The animal was allowed to stabilise after completion of the surgical preparation for a period of at least 15 min. Then, after a further 10 min period of continuous recording of ECG and haemodynamic variables, the test substance or vehicle was administered intravenously as 3 iv infusions with each administration separated by 60 min.
For hERG study, HEK293 cells were cultured (1–7 days) in DMEM/GlutaMax-1 + 10% FBS and were plated on collagen-coated dishes (about 2×104 cells/dish). The cell was held at -80 mV. A 50-millisecond pulse to -40 mV was delivered to measure the leaking currents, which were subtracted from the tail currents online. Then the cell was depolarized to +20 mV for 2 seconds, followed by a second pulse to -40 mV for 1 second to reveal the tail currents. This paradigm was delivered once every 5 seconds to monitor the current amplitude. After the current amplitude stabilized, the test compound was delivered to the extracellular medium by a rapid solution changer perfusion system. During perfusion, the cell was repetitively stimulated with the protocol described above, and the current amplitude was continuously monitored. Data were acquired and analyzed by using pClamp (Axon Instruments), and Excel (Microsoft), and are reported as mean and individual values. The degree of inhibition (%) was obtained by measuring the tail current amplitude before and after drug superfusion (the difference current was normalized to control and multiplied by 100 to obtain the percent of inhibition). Concentration (log) response curves were fitted to a logistic equation (three parameters assuming complete block of the current at very high test compound concentrations) to generate estimates of the 50% inhibitory concentration (IC50). The concentration-response relationship of each compound was constructed from the percentage reductions of current amplitude by sequential concentrations. β2-adrenergic receptor CHO expressing cells were used for the receptor inhibition assay as described. The results are expressed as a percent of inhibition of control specific binding (100 - (measured specific binding/control specific binding) × 100)) obtained in the presence of the test compounds. The specific ligand binding to the receptors is defined as the difference between the total binding and the nonspecific binding determined in the presence of an excess of unlabelled ligand. All the in-vivo experiments were carried out at the Regina Elena Cancer Institute. All procedures involving animals and care were performed in compliance with our institutional animal care guidelines and with international directives (directive 2010/63/EU of the European parliament and of the council; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 2011).
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.
CD spectra were recorded on an Applied Photophysics Pi-Star-180 spectrophotometer (Applied Photophysics Ltd, Surrey, UK). The optical system was configured with a 75 W Xe lamp, circular light polarizer and end-mounted photomultiplier. The instrument had previously been calibrated with (D)-camphorsulfonic acid. Temperature was regulated using a Neslab RTE-300 circulating programmable water bath (Neslab Inc). CD spectra were recorded at 298 K in a 10 mm path length cell over a wavelength range of 215–345 nm in steps of either 1 0r 2 nm, with 3 nm entrance/exit slit widths: the number of counts was set to 10,000 with adaptive sampling set to 500,000. The spectra were corrected by subtracting the spectrum of the same buffer solution of 100 mM potassium chloride and 10 mM potassium phosphate at pH 7.0. Annealing and melting profiles were recorded using a thermoelectric temperature controller (Melcor) on 4 μ M DNA samples with and without 3.5 mol.equiv. of ligands using 0.5 K temperature increments and a cooling or heating rate of 0.2 K/min over the temperature range 298-368 K.
Cells and culture conditions
BJ fibroblasts expressing hTERT (BJ-hTERT) or 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.
5 × 104 cells were seeded in 60-mm Petri plates (Nunc, MasciaBrunelli, Milano, Italy) and 24 h after plating, 0.5 μM of freshly dissolved compound was added to the culture medium. Cell counts (Coulter Counter, Kontron Instruments, Milano, Italy) and viability (trypan blue dye exclusion) were determined daily, from day 2 to day 8 of culture.
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). 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 high-resolution images. For quantitative analysis of γH2AX positivity, 200 cells on triplicate slices were scored. For TIF’s analysis, in each nucleus a single plane was analyzed and at least 50 nuclei per sample were scored.
Fluorescence in situ hybridization (FISH)
For metaphase chromosome preparation cells were treated with demecolcine (Sigma, Milan, Italy) 0.1 mg/ml for 4 h and then harvested and washed in 75 mM KCl for 5 min at 37°C. After centrifugation cells were fixed in MeOH/acetic acid 3:1 overnight and then spread on slides. Hybridization with rhodamine-coupled PNA was performed as described. For each sample 20 metaphases per slice on triplicate were scored. Images of the metaphases were captured with a 100 × objective.
Chromatin immunoprecipitaion assay (ChIP)
BJ-EHLT fibroblasts were treated for 24 hrs with 0.5 μM of the compound. ChIP assay was performed as previously described. The following antibodies were used: pAb anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, Ca); mAb anti-TRF2 (Imgenex, San Diego, CA); pAb anti-POT1 (Abcam). mAb anti-β-actin (Sigma) was used as negative control of the ChIP assay.
Results and discussion
Synthesis of quino [4,3,2-kl] acridinium salts
Efforts to prepare higher alkyl homologues of 1 were only partially successful presumably because access by larger alkylating moieties at N-13 of pentacycle 6 were impeded by hydrogen atoms at positions 1 and 12 (for numbering system see Figure 1): thus whereas the N-ethyl quaternary salt 8 (20%) could be prepared with difficulty by heating 6 and ethyl trifluoromethane sulfonate in chloroform under nitrogen at 140°C in a sealed tube for 3 days, it was not possible to prepare n-propyl or i-propyl homologues of 6 under a range of forcing conditions. The isomeric N-acetyl compounds 2 and 3 were prepared starting from the 2-aminoquinoacridine 9 or 3-chloroquinoacridine 10, respectively, in several steps according to our previously published work.
Toxicity of quinoacridinium salt 1
Initial in vivo evaluation of 1, in human tumor xenografted nude mice, did not indicate any toxicity at efficacious doses, as no toxic deaths or body weight loss was observed during or after treatment. Furthermore, histological analysis, done at the end of treatment with 1, revealed no evidence of lesions or morphological alterations in the organs and tissues examined. Nevertheless, just after 1 administration, a marked but reversible hypotension was observable, accompanied by a heart rate and cardiac output decrease in the treated compared to the control mice.
On and off target profile of pentacyclic acridinium salts 1, 2 and 3
Off-target effects: cardiac receptor inhibition
On-target effects: ligand-quadruplex interaction
hERG % inhib. (10 μM)
B2 adrenergic % inhib. (10 μM)
Surface plasmon resonancea(K x 107 M-1)
CD study thermal stabilityd
Quadruplex (Q) DNAb
Duplex (D) DNAc
86 ± 3
89 ± 3
85 ± 3
Ligand redesign to minimize off target effects
The potent hERG inhibition compromised the acceptability of 1 as a clinical candidate, despite this agent having many of the attributes of an ideal pharmaceutical. Two strategies have been adopted in an attempt to minimize the hERG interaction: (i) sterically masking the (delocalized) positive charge on the acridinium cation by increasing the size of the substituent at position 13 as in compound 8; and (ii) evaluating compounds 2 and 3 as prototypes of two series of isomeric pentacyclic acridinium salts of the same chemotype as 1.
hERG tail current inhibition was used as a marker of potential off-target liabilities. The prototypic agent 1 potently inhibited hERG by 100% at 10 μM (IC50 0.2 μM) (Table 1); inhibition of hERG was reduced to 43% at 10 μ M (IC50 3.7 μM) in the 2-acetylaminoquinoacridinium iodide 2 and to 18% by 13-ethyl homologue 8, while the least potent hERG inhibitor (IC50 18 μM) was the 3-acetylamino isomer 3, a 90-fold improvement over 1. The marked improvement of 8 over 1, was paralled by a >10-fold reduction in the on-target effect against the h-Tel DNA sequence as measured by surface plasmon resonance (see below) suggesting that increasing the size of the onium head was not a fruitful developmental approach, for these reason the compound 8 was excluded from further studies.
The interaction with β2-adrenergic receptor was determined by a binding assay of 1, 2 and 3 to the transgenic β2-adrenegic receptor expressed on the surface of CHO cells. Inhibition of receptor was reported as inhibition of control specific binding (100 - (measured specific binding/control specific binding) × 100) obtained in the presence of the test compounds. A decay of 75% and 70% of receptor inhibition is observed comparing 1 to 2 and 3 compounds respectively (Table 1). These results indicate that potential toxicities in this chemotype, as predicted by hERG and β2-adrenergic receptor interactions, can be addressed by suitable molecular modification.
On target-effects: ligand-quadruplex interactions
The ability of the three ligands to induce structure in the single stranded h-Tel sequence in aqueous solution in the absence of significant concentrations of K+ ions was also investigated. The unfolded h-Tel sequence at 298 K gives a low intensity positive band in the CD spectrum at 265 nm (Figure 4b). However, in the presence of 3.5 molar equivalents of ligand, emergence of the characteristic band at 290 nm was observed, consistent with the ligand-induced formation of the anti-parallel structures evident in the K+ buffered solution. Thus, under both sets of conditions (with and without stabilising K+ ions), evidence is adduced for ligand selectivity for the anti-parallel quadruplex structure[12, 13].
This analysis was extended to examine the effects of ligand binding on thermal stability by measuring the unfolding curves at 290 nm of the complexes formed in K+ solution, corresponding to the CD spectra shown in Figure 4a. Monitoring the thermal unfolding transition for h-Tel produces a sigmoidal unfolding curve with a transition mid-point Tm value of 72 ± 3°C (Figure 4c). All three ligands show significant effects in enhancing the stability of the quadruplex by shifting the Tm values to higher temperatures (∆Tm ~ 15-19°C compared to h-Tel without bound ligands) (Table 1).
Biological effects of quinoacridinum salts
To directly evaluate telomere damage elicited by the different ligands, the telomere status of drug-treated BJ-EHLT was analysed by a fluorescence in situ hybridization on metaphase spreads with a telomere specific fluorescent probe. The cytogenetic analysis revealed that all the compounds induced a significant increase of frequency of telomere doublets (characterized by a double telomere signal at chromosome ends) and sister telomere fusions (in which two sister chromatids telomeric signals are fused into one single spot), while other telomere aberrations (telomere losses and/or deletions) were not found. However, again telomere aberrations induced by 2 are quantitatively similar to the lead compound, while a lower effect was observed upon treatment with 3. As a result of chromosome ends fusion consequent to telomere damage, chromatin bridges are occasionally observed between daughter cells after mitosis (also called anaphase bridges). In 1 treated BJ-EHLT, anaphase bridges frequency in a cycling population was ten-fold increased. With a minor extent 2 and 3 were both able to induce anaphase bridges when administered at the same dose, closely comparing the effects of the lead compound (Figure 7e).
This response is typical of the telomere deprotection occurring during cellular senescence or upon the loss of telomeric proteins[34–40]. The ability of G-quadruplex ligands to uncap telomeres and to possess anti-tumor activity has been already described for other agents,[41–45] reinforcing the notion that these agents can act as inhibitors of a telomere-related process and therefore the rationale for the development of this class of inhibitors as anti-tumor agents must be found elsewhere other than in higher telomerase expression in cancer cells.
Taken collectively our results clearly demonstrate that compounds 2 (but less efficiently 3) rapidly disrupt telomere architecture of cells, by delocalizing the telomeric protein POT1, resulting in a potent DNA damage response characterized by the formation of several telomeric foci.
Furthermore, it is apparent that the 2-substitued quinoacridinium salt 2 more closely mimics the overall pharmaceutical profile of the prototypic compound 1 than the regioisomer 3. Our recent synthetic work has therefore focused on the 2-substituted series and our efforts to maximize on-target and minimize off-target properties will be reported separately.
Molecular modification of quinoacridinum salts 1 have shown to reduce undesired cardiotoxic effects while maintaining the on-target features as telomere targeting agents. This findings provide a strong rational for development of this class of compounds as tools for a G-quadruplex targeted anti-cancer therapy.
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