Ruthenium(II) Polypyridyl Complexes as FRET Donors: Structure- and Sequence-Selective DNA-Binding and Anticancer Properties

Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-“light switch” complexes [Ru(dppz)2(5,5′dmb)]2+ and [Ru(PIP)2(5,5′dmb)]2+ (dppz = dipyridophenazine, 5,5′dmb = 5,5′-dimethyl-2,2′-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor–acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)2(5,5′dmb)]2+ acts to block DNA replication fork progression.


Experimental Section
General All chemical reagents and solvents were purchased from commercial sources (Sigma-Aldrich, ThermoScientific) and used as supplied. 1 H and 13 C NMR spectra were obtained using a Bruker Advance III 500 MHz Nuclear Magnetic Resonance Spectrometer. HRMS (high resolution mass spectroscopy) samples were analysed at the EPSRC UK National Mass Spectrometry Facility at Swansea University using a ThermoScientific LTQ Orbitrap XL 1 Mass Spectrometer. Fourier Transform Infrared Spectra were run on a Perkin Elmer FT-IR Spectrometer Spectrum TWO. Elemental analysis was performed by the Elemental Analysis Service at London Metropolitan University.

Complex synthesis and characterisation
[Ru(N^N) 2 Cl 2 ] intermediate complexes were prepared using a modified method described by Sullivan et al. 1 Briefly, RuCl 3 .3H 2 O was added to a stirring solution of ligand (2 eq) in DMF. LiCl (7 eq) was added, and the mixture was sparged with N 2 for 20 minutes and protected from light. The mixture was then heated at reflux for 8 hours under nitrogen atmosphere. The products were precipitated from solution by the addition of acetone and collected by filtration and washed with distilled water and diethyl ether. The resultant brown intermediates were used in subsequent reactions without further purification or characterisation. [Ru(bpy) 2 (dppz)] 2+ (1) and [Ru(5,5'dmb) 2 (dppz)] 2+ (2) were prepared by previously-reported methods 2,3 from [Ru(bpy) 2 Cl 2 ] and [Ru(5,5'dmb) 2 Cl 2 ], respectively.
[Ru(bpy) 2 (dppz)] 2+ (1). 1 6 ] was added once the solution had cooled to room temperature, and the precipitate collected by filtration, before washing with distilled water and diethyl ether. The

FRET DNA binding assay
In a typical experiment, a concentration gradient of each compound (0.1 -20 M) was treated with 1 M of each DNA structure/sequence in 96 well plates (black, optical bottom, Thermo). 2x concentrations were prepared in advance and mixed 1:1 in each well to achieve the desired concentrations. After 30 mins, fluorescence spectra ( em = 550-850 nm) or intensity at a fixed wavelength (710 nm for FRET, 630 nm or 605 for MLCT emission of 1-4) were recorded by Tecan Infinite M Nano plate reader ( ex = 450 nm). The intensity of the FRET emission peak for each compound concentration was measured, background subtracted and normalized to the maximum FRET intensity. Binding curves were generated, fit to a sigmoidal binding model (Origin) and K d values extrapolated as the concentration required for 50 % binding, as described by Jarmoskaite et al. 12 Only fits with R-squared values of 0.9 or greater were used to derive K d values, otherwise it was concluded that binding saturation had not been reached and the K d value was greater than the maximum concentration employed.

Cytotoxicity and Olaparib synergy
Cells were seeded in 96 well plates and allowed to adhere for 24 h. Cells were then treated with a concentration gradient of each compound alone or in combination with Olaparib (10 M). After 72 h, solutions were removed and thiazolyl blue tetrazolium bromide (MTT) reagent dissolved in PBS was added (0.5 mg/mL). After 4 h, the solution was removed and the purple formazan crystals were then solubilized with 100 μL of DMSO and the absorbance at 570 nm (620 nm as reference wavelength) was measured using microplate reader. The average in percent reduction of cell viability was expressed relative to untreated control cells. Half inhibitory IC50 values were determined using GraphPad Prism software. Combination indices (CI) were calculated using CalcuSyn and CompuSyn software (Biosoft, Cambridge, UK) as established by Chou and Talalay. 13 CI < 1.0 indicates synergism, CI = 1.0 indicates additive, and CI > 1 indicates antagonism. GraphPad Prism Software was used to generate a 3-color scale based on CI values obtained, where synergism is represented by green, additive by yellow, and antagonism by red. The colors of each CI value were interpolated in between these constraints accordingly.

DNA fiber assay
MDA-MB-231 cells were treated with 20 M 3 or 4 for 1 h, complexes were removed, and pulselabeled with 25 μM CldU (Sigma-Aldrich) and 250 μM IdU (Sigma-Aldrich) for 20 min each. The DNA fiber assay, including image acquisition and data processing, was then performed as described within a recent publication. 14

Cell-cycle analysis
Cells were seeded at 3 x 10 5 cells/well 6-well plates and allowed to adhere for 24 h. Cells were then treated as stated in the main text, trypsinized and washed with PBS twice. Cells were then fixed with ice-cold 70% ethanol, centrifuged at 1,000 rpm for 5 min, and the resulting cell pellets were washed with PBS twice. Samples were resuspended in 500 µL PBS and treated with 5 µL of RNase A solution (10 mg/mL). After 15 min of incubation, the samples were stained with 2 µL propidium iodide (PI) (5 mg/mL) in the dark at room temperature. Samples were acquired and analysed with a NovoCyte flow cytometer (Agilent Technologies) and NovoExpress software. For each sample, a minimum of 10,000 cells were counted.

Annexin V binding assay
Cells were seeded at 3 x 10 5 cells/well 6-well plates and allowed to adhere for 24 h. Cells were then treated as stated in the main text, trypsinized and washed with PBS twice. Then, 500 µL 1X binding buffer and 5 µL Annexin V-FITC (Invitrogen) was added for 20 min at RT. 5 µL PI (20 µg/mL) was added prior to flow cytometric analysis using a NovoCyte flow cytometer and the results were analysed using NovoExpress software. For each sample, a minimum of 10,000 cells were counted. Tables   Table S1 Photophysical properties of 3