A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic canc

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We report here that a tetra-substituted naphthalene-diimide derivative (MM41) has significant in vivo anti-tumour activity against the MIA PaCa-2 pancreatic cancer xenograft model. IV administration with a twice-weekly 15 mg/kg dose produces ca 80% tumour growth decrease in a group of tumour-bearing animals. Two animals survived tumour-free after 279 days. High levels of MM41 are rapidly transported into cell nuclei and were found to accumulate in the tumour. MM41 is a quadruplex-interactive compound which binds strongly to the quadruplexes encoded in the promoter sequences of the BCL-2 and k-RAS genes, both of which are dis-regulated in many human pancreatic cancers. Levels of BCL-2 were reduced by ca 40% in tumours from MM41-treated animals relative to controls, consistent with BCL-2 being a target for MM41. Molecular modelling suggests that MM41 binds to a BCL-2 quadruplex in a manner resembling that previously observed in co-crystal structures with human telomeric quadruplexes. This supports the concept that MM41 (and by implication other quadruplex-targeting small molecules) can bind to quadruplex-forming promoter regions in a number of genes and down-regulate their transcription. We suggest that quadruplexes within those master genes that are up-regulated drivers for particular cancers, may be selective targets for compounds such as MM41.

MM41 shows anticancer activity in vivo

The maximum tolerated dosage (MTD) for iv administration of MM41 was found to be ca 30mg/kg. A preliminary pharmacokinetic study at 20mg/kg has determined the in vivo half-life to be ca 4hrs. Since the targets (individual genomic DNA quadruplexes) are present in low copy numbers per cell, this half-life value suggests that even with twice-weekly dosing of MM41, there would be sufficient bioavailability to produce transcriptional inhibition of particular genes by means of quadruplex stabilisation. A therapeutic schedule of two doses were explored with the MIA PaCa-2 pancreatic tumour xenograft model, conducted at 10 and 15 mg/kg, each twice weekly, over a period of 5½ weeks (40 days: 12 doses). At this point the mice were sacrificed, apart from two from the 15 mg/kg group, which were maintained for a further 239 days without any further MM41 dosing. These two mice were chosen since their xenografts had completely regressed during the dosage period.

The synthesis and characterisation of MM41 (4,9-bis((3-(4-methylpiperazin-1-yl)propyl)amino)-2,7-bis(3-morpholinopropyl) benzo[lmn][3,8] phenanthroline-1,3,6,8(2H,7H)-tetraone: molecular weight 831.08) has been previously reported23,28. MM41 was analytically pure as shown by lc-ms and NMR methods and was formulated for biological studies as the freely water-soluble formate salt.

FRET studies

FRET DNA melting assays on MM41 were performed using a fluorescence resonance energy transfer (FRET) assay modified as a high-throughput screen in a 96-well format.49 The labelled oligonucleotides had attached the donor fluorophore FAM: 6-carboxyfluorescein and the acceptor fluorophore TAMRA: 6-carboxytetramethyl-rhodamine. The FRET probe sequences were diluted from stock to the correct concentration (400nM) in a 60mM potassium cacodylate buffer (pH 7.4) and then annealed by heating to 95 °C for 10 min, followed by cooling to RT in the heating block (3–3.5hrs). Solutions were prepared using 60 mM potassium cacodylate buffer (pH 7.4). 96-well plates (MJ Research, Waltham, MA) were prepared by aliquoting 50 μl of the annealed DNA into each well, followed by 50 μl of the compound solutions. Measurements were made on a DNA Engine Opticon (MJ Research) with excitation at 450–495nm and detection at 515–545 nm. Fluorescence readings were taken at intervals of 0.5 °C in the range 30–100 °C, with a constant temperature being maintained for 30sec prior to each reading to ensure a stable value. Final analysis of the data was carried out using a script written in the program Origin 7.0 . The advanced curve-fitting function in Origin 7.0 was used for calculation of ΔTm values.

Molecular dynamics simulations

The starting point for the modelling study was the NMR structure of the BCL-2 promoter quadruplex, with mixed parallel/antiparallel G-strands forming the core of the quadruplex29, and a docked MM41 molecule (details of the docking procedure are given in the Supplementary Information). The G-stem bases exhibit both syn and anti-orientations. Previous studies have shown that parmXOL4 (XOL4) modification of the Cornell et al. force field refines the syn-anti balance as it facilitates syn-anti transitions through the 120° X region by decreasing the energy barrier for this transition, increases it through the 350° X region and refines the shape and depth of the syn minimum36. Previous simulations of G-quadruplexes in XOL4 have also shown an improvement in structures with respect to the simulations with earlier force field versions36. The XOL4 refinement has recently been included as DNA default force field in the AMBER code. Therefore, we carried out the present simulations using the parmbsc0 version of the Cornell et al. force-field with the modifications35,36,50,51 Solvation and addition of more ions to the quadruplex were performed with the help of the xleap module of the AMBER12 program. The quadruplex was neutralized using K+ ions and TIP3P water molecules were used for solvation. The system was placed in a periodic box whose boundaries extended at least 10 Å from any solute atom. The parameters for K+ ions were adapted for AMBER from a previous study (radius 1.593Å and well depth 0.4297054kcal mol1)52. The parameters for the MM41 ligand were generated via the Antechamber module of the AMBER software using the GAFF force field.

Standard equilibration protocols were used for initial minimization of the structure. The first round of equilibration with explicit solvent and ions involved 1000 steps of steepest descent, followed by 1000 steps of conjugate gradient energy minimization. A 300ps MD equilibration was performed in which the quadruplex was constrained, whereas the solvent and ions were allowed to equilibrate. The system was gently heated from 0K to 300K with a time step of 0.5 ps. This was followed by subsequent rounds of MD simulation, at constant pressure and 300K for 1ns. The constraints were gradually relaxed, until no constraints were applied to the system. The final MD simulations were carried out for 350ns using ACEMD53. The periodic boundary conditions were defined by the PME algorithm and non-bonded the cut-off was set to 10Å54. Covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm with a tolerance of 0.0001Å, which allowed the use of an integration time step of 2fs55. All the simulations were carried out at constant pressure of 1atm and constant temperature of 300K. The temperature and pressure was maintained using a Berendsen weak coupling thermostat56. The final production run without restraints was carried out for a continuous 350 ns and the frames were collected every 20ps. Analyses of trajectory were performed using the ptraj module of AMBER57 and the VMD58 and PYMOL programs were used for visualization.

Confocal studies

The human cancer cell line, MIA PaCa-2, was purchased from ATCC. Cells were maintained in DMEM culture medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1:100 dilution of penicillin-streptomycin solution Hybri-MaxTM. The cell line was maintained at 37°C, 5% CO2 and routinely passaged. Cells were seeded on poly-D-lysine coated coverslips in 12-well plates, 24 hours prior to addition of anticancer compound. To prepare the coverslips they were sterilised with 100% ethanol and rinsed with 1X PBS. Coverslips were then coated with 25 μg/ml poly-D-lysine for 1 hr at room temperature, rinsed in 1X PBS, and left to fully dry in the Laminar Flow Cabinet overnight before use. MM41 was added to the cells at a concentration of 333.2nM (equivalent to 20 X IC50) in culture medium. Following a 30 min incubation period with MM41 at 37°C, cells were rinsed with 1 x PBS and then fixed with ice-cold methanol for 15 min at RT. The fixed cells were washed twice with ice cold 1 x PBS. Coverslips were then mounted with a drop of vectashield mounting medium to microscope slides. Coverslips were sealed in place with nail polish. Trans light confocal images were obtained with a Zeiss LSM 710 microscope. Fluorescence from MM41 was collected with a 633 nm HeNe laser. Cells were imaged using a 63 x oil immersion lens.

Pharmacokinetics studies

A single dose of 20 mg/Kg was administered intravenously to mice and 25 μl blood was drawn from the tail vein at time-points 10, 20, 30, 60, 120, 240, 360 and 1440 mins. These samples were mixed immediately with 225 μl phosphate buffered saline and centrifuged for 5 min at 21,000 × g. 200 μl supernatant was transferred to a cryovial and frozen at –70 °C until analysis.

Plasma samples were thawed and the MM41 was extracted using SOLA HRP 10 mg/ml cartridges according to manufacturer’s instructions. Samples were dried under nitrogen and reconstituted in 200 μl mobile phase . High performance liquid chromatography was performed using a C18 reversed phase column with a water (0.1% TFA)/ acetonitrile (0.1% TFA) gradient. The flow rate was 1 ml/min and the column oven was set to 40°C. MM41 was detected by its fluorescence, with an excitation wavelength 280nm and an emission wavelength 660 nm. The standard curve range was 100–10,000 nM.