Dicobalt(ii) helices kill colon cancer cells via enantiomer-specific mechanisms; DNA damage or microtubule disruption

Highly diastereoselective self-assembly reactions give both enantiomers (Λ and Δ) of anti-parallel triple-stranded bimetallic Co(ii) and Co(iii) cationic helices, without the need for resolution; the first such reaction for Co. The complexes are water soluble and stable, even in the case of Co(ii). Studies in a range of cancer and healthy cell lines indicate high activity and selectivity, and substantial differences between enantiomers. The oxidation state has little effect, and correspondingly, Co(iii) compounds are reduced to Co(ii) e.g. by glutathione. In HCT116 colon cancer cells the Λ enantiomer induces dose-dependent G2-M arrest in the cell cycle and disrupts microtubule architectures. This Co(ii) Λ enantiomer is ca. five times more potent than the isostructural Fe(ii) compound. Since the measured cellular uptakes are similar this implies a higher affinity of the Co system for the intracellular target(s); while the two systems are isostructural they have substantially different charge distributions as shown by calculated hydrophobicity maps. In contrast to the Λ enantiomer, Δ-Co(ii) induces G1 arrest in HCT116 cells, efficiently inhibits the topoisomerase I-catalyzed relaxation of supercoiled plasmid DNA, and, unlike the isostructural Fe(ii) system, causes DNA damage. It thus seems very likely that redox chemistry plays a role in the latter.


Synthesis
All solvents and chemicals purchased from commercial sources (Sigma-Aldrich, Acros, Fisher Scientific or Alfa Aesar) were used without further purification unless otherwise stated.Sodium hydride dispersions in mineral oil were placed in a Schlenk vessel under an inert atmosphere and washed three times with diethyl ether to remove the oil, then dried and stored under argon in an MBraun dry box.Where appropriate, reactions were carried out under argon using a dual manifold argon/vacuum line and standard Schlenk techniques or in an MBraun dry box.Necessary solvents were dried by heating to reflux for 3 d under dinitrogen over the appropriate drying agents (potassium for tetrahydrofuran and sodium/potassium alloy for diethyl ether) and degassed before use.Tetrahydrofuran and diethyl ether were additionally pre-dried over sodium wire.Dried solvents were stored in glass ampoules under argon.All glassware and cannulae were stored in an oven at > 375 K. Deuterated solvents were purchased from Sigma-Aldrich and Cambridge Isotope Laboratories.
A precipitate was observed immediately, and the suspension was stirred for 1 h, then filtered, washed with CH3CN and dissolved in water (15 ml).The addition of NH4PF6 (0.598 g, 3.67 mmol) into the solution led to the formation of an orange precipitate which was filtered, washed with H2O and dissolved in CH3CN before the solvent was removed under vacuum to give the product as an orange crystallite.

Geometry optimisation
All calculations were carried out using ORCA program. 2 Initial monometallic sub-structures for each metallohelices were created from existing crystallographic fragments and optimised without symmetrical restrictions.The ground state geometry optimisation were performed using density functional theory (DFT) with B3LYP functional, employing a triple-zeta def2-TVZP basis set for metal atoms and def2-SVP basis set for other atoms in the gas phase. 3spersion was also addressed in the optimisation using Grimme's D3BJ dispersion correction. 4,5 he local minima of optimised structures were confirmed by the vibrational frequency analysis.
The starting geometries of bimetallic metallohelices were assembled from the corresponding optimised monometallic fragments, and further optimised using the same basis set as before.
Solvation energy correction was performed in water using SMD continuum solvation model.
The (U)BP86-D3(BJ) functional with a mixed basis set of def2-TVZP for metal ions and def2-SVP for other atoms was used for geometry optimisation in solution phase and single point energy calculations.

Electrostatic potential (ESP)
Quantitative calculation of electrostatic potential surfaces of metallohelices were obtained using Multiwfn program 6 using B3LYP/G-31G** level and corresponding plots were generated using the VMD program.

Computational Procedure for Spatial Analysis of hydrophobicity
The hydrophobicity calculations were achieved using ORCA program.The hydrophobicity index was defined by the principle that the solvent molecule was found near the hydrophilic region of the solute and far from the hydrophobic region of the solute.The previously optimised structures of metallohelices were used as basis to perform the calculation.The coordinate system was defined with one of the metal atoms serving as the origin, the primary axis formed by the two metal atoms, and a coordinating bipyridyl nitrogen atom (Npy) acting as a reference.
ii. Angular and Radial Variation: The spatial coordinates of the water oxygen atom were systematically defined through a fixed azimuthal angle relative to nitrogen atom Npy (varying from 0° to 359° in 5° increments, 72 azimuthal angles) and a fixed polar angle (ranging from 179° to 59° in 10° increments, 13 polar angles).The radial distance from the origin (initially set at 10 Å) was allowed to vary freely, encapsulating the degrees of freedom within the system.
iii.Energy Minimization An energy minimization was conducted using the PM7 basis set.The minimization process involved the variation of the water oxygen position, azimuthal and radial angles, and the positions of water hydrogen atoms.All other atoms within the metallohelicate were frozen at fixed positions.

iv. Calculations
Two sets of calculations were performed: the first with the origin at one metal centre, resulting in 936 distinct calculations, and the second with the origin at the other metal centre, thus covering the "sphere" of helicate with an additional 936 calculations.Prior to use the working electrode was mechanically polished first using a micropolish alumina (0.05 µm) paste (Buehler, Germany) on a microcloth pad (Buehler, Germany), and then polished on a wetted (with ultra-pure water) alumina-free pad and thoroughly rinsed.The experiment was performed using a 1 mM aqueous solution of Λ-7 complex containing 0.1 M KCl under argon, to remove dissolved oxygen from solution.The pH of the solution was 6.42.
CVs were recorded from -0.For the mixture of Co(III) complex and GSH, the peaks of Co(III) complex were reduced by factor of 2.4 and there were new peaks (in pale green region) observed.
All cell lines were routinely maintained as monolayer cultures and sub-cultured or harvested for chemosensitivity studies when approximately 70-80% confluent.Cells were seeded into 96-well tissue culture plates at a density of 2×10 3 cells/well for HCT116, and ARPE-19 cells/well.Plates containing cells were incubated for 24 h at 37°C in an atmosphere of 5% CO2 prior to drug exposure.Cell media (200 µl) was added to the control cells and differing concentrations (0 to 50 μM) of drug solution (200 µl) were added to the remaining wells.All complexes were directly dissolved in cell media.The plates were incubated for a further 72 or 96 h at 37°C in an atmosphere of 5% CO2.Media was removed and replaced with fresh media prior to the assay.3-(4,5-Dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/ml, 20µl per well) was added to each well and incubated for a further 4 h at 37°C in an atmosphere of 5% CO2.Upon completion, all solutions were removed from the wells and dimethyl sulfoxide (150 µl,) was added to each well to dissolve the purple formazan crystals.A Thermo Scientific Multiskan EX microplate photometer was used to measure the absorbance at 540 nm.Lanes containing 100% cell media and untreated cells were used as a blank and 100% cell survival respectively.Cell survival was determined as the true absorbance of treated cells divided by the true absorbance of untreated controls; this value was expressed as a percentage.The IC50 values were determined from a plot of percentage cell survival against drug concentration (µM).All assays were conducted in triplicate and the mean IC50 ± standard deviation was determined.

Cellular uptake
HCT116 cells (as in manuscript Table 2) were seeded at a density of 2x10 6 cells/Petri dish and grown overnight.Culture medium was replaced with fresh medium containing 5 µM metallohelices (5 mL) and the cells were incubated for 8 hours.The cells were then harvested (trypsinization), washed (PBS), counted and pelleted by centrifugation.It was verified that at the time of harvest, at least 95% of cells was viable as determined by the trypan blue exclusion test.Cell pellets were digested using the microwave acid digestion system (CEM Mars®).
Cobalt amounts were determined with ICP-MS.

Cell death
To investigate the mode of cell death induced in HCT116 cells by the tested compounds annexin-V/propidium iodide assay was employed.The cells were seeded at a density of 3x10

Nuclear uptake
HCT116 cells were seeded in Petri dishes at a density of 2x10 6 cells/dish and allowed to attach overnight.The cells were then exposed to 5 µM compounds for 8 hours.The cells were then harvested by trypsinization and processed with Nuclei EZ Prep Kit according to the recommended protocol.The nuclei were then counted and pelleted.The pellets were digested with the microwave acid (HCl) digestion system (CEM Mars®).Cobalt amounts were determined with ICP-MS.

DNA transcriptional activity
This assay is based on the use of a fluorescent analog of UTP (UTP-gamma-AmNS; Jena Bioscience, Jena, Germany) as one of the nucleotide substrates. 9Incorporation of UMP in RNA strand by RNA polymerase leads to the release of gamma-AmNS, which exhibits higher intrinsic fluorescence than UTP-gamma-AmNS.Circular pBR322 plasmid DNA was used as RT. 1 unit of DNA topoisomerase I (Takara Bio Inc., Shiga, Japan) was then added into each tubulin polymerization buffer (80 mM PIPES pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA and 15% glycerol) and variable concentrations of metallohelices were added in 96-well plate (white, flat bottom).GTP was added to final concentration of 1 mM immediately before inserting the plate in fluorimeter.SPARK reader (Tecan) was set to 37 °C; Ex/Em=360/420 nm.Tubulin polymerization was recorded as increase of fluorescence.

Microtubule imaging
HCT116 cells were seeded on coverslips in 6-well plates at a density of 1.5x10 5 cells/well and grown overnight.The cells were treated with the compounds at concentrations corresponding to 5x-and 10xIC50 for 6 hours.The cells were then washed with PBS, fixed with 4% paraformaldehyde, permeabilized with PBST (0.1% Triton X-100 in PBS), blocked with 5% FBS and immunostained with primary (anti-α-Tubulin antibody; abcam; ab176560) and secondary (Goat Anti-Rabbit IgG-Alexa Fluor® 488; abcam; ab150077) antibodies and mounted with ProLong Diamond Antifade with DAPI (Invitrogen).The imaging was performed with Leica TCS SP8 SMD (Leica microsystems GmbH, Wetzlar, Germany).The images were sequentially scanned with first scan 405 nm (diode laser)/emission window 420 to 550 nm, second scan 488 nm (white laser)/emission window 520 to 600 nm).Pinhole was set to 1 AU.

Actin polymerization
Actin Polymerization Biochem Kit TM (Cytoskeleton, Inc.) was used to determine the effect of the tested compounds on actin polymerization in vitro.The assay uses pyrene conjugated Gactin and enhanced fluorescence is recorded when F-actin is formed.The assay was performed according to the recommended protocol.Pyrene actin (0.4 mg mL -1 ) was polymerized in actin polymerization buffer in microplate format at room temperature on SPARK TM multimode reader (Tecan) (Ex.360 nm and Em.410 nm).

Actin imaging
HCT116 cells were grown, treated and fixed as in the case of microtubule imaging and stained with Alexa FluorTM 488 Phalloidin (Thermofisher Scientific, 40 min).The cells were mounted with ProLongTM Diamond Antifade with DAPI (Invitrogen).The imaging was performed with Leica TCS SP8 SMD microscope at the same setting as for microtubule imaging.
Typical of high spin Co(II) there are large hyperfine shifts and moderate broadening, with short electronic relaxation times (ps) and large magnetic anisotropy.Several broader resonances [ca 200 Hz (FWHM), corresponding to T2* = 1.6 ms at 7 T] are assigned to the bipyridyl, pyridyl, and benzylic protons most proximate to the metal centers for the three chemically inequivalent ligand strands.

Figure S15 .
Figure S15. the molecular electrostatic potential (ESP) map of the optimised structures of the metallohelices.

Figure S16 .
Figure S16.Schematic diagram of the hydrophobicity calculation, where the polar coordinates describing the watering oxygen atom's spatial relationship with metallohelices v. Coordinate Conversion and Visualization:Polar coordinates of the water oxygen atoms from each of the remaining 1868 calculations were converted into Cartesian coordinate system.Subsequently, these coordinates were plotted in MATLAB2019 using the scatter3 function.The obtained plots were fused with the electron density map of the molecule thereby demonstrating the hydrophobicity of the molecule.The colour scale of the resulting plot represented the minimized energy levels, ranging from relative hydrophilic (blue) to hydrophobic (red).

CO2) and subcultured
twice or thrice a week.Antiproliferative activities of the metallohelices were determined using the assay based on the tetrazolium compound MTT [3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide].The cells were seeded at optimal pre-determined densities in 96-well plates, incubated overnight and treated with a range of concentrations of the tested compounds for 72 hours.MTT was added (20 µL of 1.25 mg/mL PBS solution) for the final 3 hours.The medium was then replaced with 100 µL DMSO to dissolve the formazan products of cellular metabolization of MTT.Absorbance was read at 570 nM (reference 620 nm) and IC50 values were calculated.

a
template at the concentration of 0.96 μg/100 μl.Transcription was performed in a total volume of 28 μl in 10 mM Tris-HCl buffer (pH 7.6), 100 mM KCl, 5 mM MgCl2, 0.1 mM ATP, CTP and GTP, 0.01 mM UTP-gamma-AmNS, 2 mM DTT, and increasing concentrations of metallohelices.After 10 min of pre-incubation at RT, one unit of E. coli RNA polymerase holoenzyme (New England Biolabs, Beverly, MA, USA) was added to each sample and incubated for 2 h at 37 ºC.RNA polymerization was stopped by addition of 42 μl of 50 mM EDTA and transcription products were detected by measuring the sample fluorescence intensity using Varian Cary Eclipse spectrofluorophotometer with the following parameters: excitation wavelength 330 nm, emission wavelength 463 nm, excitation and emission slit widths 10 nm, and integration time 3 s.Topoisomerase I-catalyzed relaxation of negatively supercoiled DNA Metellohelices at various concentrations were mixed with 0.2 µg of supercoiled pUC19 plasmid DNA (2686 bp; New England Biolabs, Beverly, MA, USA) in 35 mM Tris-HCl, pH 8.0, 72 mM KCl, 5 mM MgCl2, and 5 mM DTT in a total volume of 8 µl and left for 15 min at

Table S1 .
The CH Instruments) was used as the working electrode.The SCE was routinely monitored for any drift, relative to a master SCE reference electrode and the coiled Pt wire counter electrode was flame cleaned before use to remove any unwanted contaminants.
result of the hydrophobicity calculation of three metallohelices, which contains the hydrophobicity map (top image refers to front view, bottom image refers to back view, coloured according to the energy scale, hydrophilic (blue) to hydrophobic (red)), the upper and lower limits of the energy.

Cell culture and Chemosensitivity -manuscript Table 1
Cells of human cervical carcinoma (HeLa) were kindly provided by Professor B. Keppler,