Supramolecular delivery of dinuclear ruthenium and osmium MCU inhibitors

The transmembrane protein known as the mitochondrial calcium uniporter (MCU) mediates the influx of calcium ions (Ca2+) into the mitochondrial matrix. An overload of mitochondrial Ca2+ (mCa2+) is directly linked to damaging effects in pathological conditions. Therefore, inhibitors of the MCU are important chemical biology tools and therapeutic agents. Here, two new analogues of previously reported Ru- and Os-based MCU inhibitors Ru265 and Os245, of the general formula [(C10H15CO2)M(NH3)4(μ-N)M(NH3)4(O2CC10H15)](CF3SO3)3, where M = Ru (1) or Os (2), are reported. These analogues bear adamantane functional groups, which were installed to act as guests for the host molecule cucurbit-[7]-uril (CB[7]). These complexes were characterized and analyzed for their efficiency as guests for CB[7]. As shown through a variety of spectroscopic techniques, each adamantane ligand is encapsulated into one CB[7], affording a supramolecular complex of 1 : 2 stoichiometry. The biological effects of these compounds in the presence and absence of two equiv. CB[7] were assessed. Both complexes 1 and 2 exhibit enhanced cellular uptake compared to the parent compounds Ru265 and Os245, and their uptake is increased further in the presence of CB[7]. Compared to Ru265 and Os245, 1 and 2 are less potent as mCa2+ uptake inhibitors in permeabilized cell models. However, in intact cell systems, 1 and 2 inhibit the MCU at concentrations as low as 1 μM, marking an advantage over Ru265 and Os245 which require an order of magnitude higher doses for similar biological effects. The presence of CB[7] did not affect the inhibitory properties of 1 and 2. Experiments in primary cortical neurons showed that 1 and 2 can elicit protective effects against oxygen-glucose deprivation at lower doses than those required for Ru265 or Os245. At low concentrations, the protective effects of 1 were modulated by CB[7], suggesting that supramolecular complex formation can play a role in these biological conditions. The in vivo biocompatibility of 1 was investigated in mice. The intraperitoneal administration of these compounds and their CB[7] complexes led to time-dependent induction of seizures with no protective effects elicited by CB[7]. This work demonstrates the potential for supramolecular interactions in the development of MCU inhibitors.


Synthesis of 2⋅2H2O⋅NaCF3SO3
The triflate (CF3SO3 -) salt of Os245′ (80 mg, 0.060 mmol) was dissolved in 10 mL methanol with stirring.A solution of NaOAd (24.4 mg, 0.120 mmol) in 5 mL methanol was added to the yellow Os245′ solution dropwise with stirring.The clear, yellow solution was heated at 50 °C for 48 h.The solution was then concentrated to dryness and redissolved in ~3 mL THF.This solution was placed in a vial and was slowly evaporated at 4 °C over a period of ~5 d until yellowish brown crystals formed.These crystals were collected, washed with 10 mL ethanol, 10 mL acetone, and 10 mL diethyl ether, followed by drying in vacuo for 12 h.Yield: 78.

Synthesis of Cucurbit-[7]-uril (CB[7])
This procedure was adapted from a literature procedure. 5Glycoluril (3.00 g, 21 mmol) was added to 3 mL concentrated HCl and stirred at room temperature.Slowly, paraformaldehyde (1.35 g, 45 mmol) was added, and the cloudy, white mixture was heated at 100 °C for 12 h.The solution, now orange and clear, was added to 25 mL H2O and stirred at room temperature for 1 h.A white precipitate, presumably a mixture of CB [6]  and CB [8], formed and was removed by filtration.The remaining clear, orange filtrate was collected, and 25 mL methanol was added.A white precipitate formed immediately, which was filtered and washed with an additional 15 mL methanol.This white solid, a mixture of CB [5] and CB [7], was next added to 32 mL 20% (v/v) glycerol in H2O and heated at 100 °C for 4 h.The remaining white precipitate was filtered and dried as crude CB [5].The filtrate, containing the desired CB [7] product, was heated at 110 °C for 2 h or until the mixture became viscous.The thick mixture was added to 50 mL stirring methanol and continued stirring for 12 h at room temperature.The final white precipitate in this mixture was filtered, washed with 25 mL methanol and 25 mL diethyl ether, and dried.Yield: 257 mg (7.6%, 0.221 mmol). 1 H NMR (500 MHz, D2O + 0.1% DCl) δ (ppm) = 5.78 (d, 14 H), 5.53 (s, 14 H), 4.24 (d, 14 H). 13

X-Ray Crystallography
Single crystals of 1 were obtained via the vapor diffusion of n-hexane into a solution of the compound in THF at room temperature.Single crystals of 2 were grown through the evaporation of a THF solution of the compound at 4 °C.Low-temperature X-ray diffraction data for 1 and 2 were collected on a Rigaku XtaLAB Synergy diffractometer coupled to a Rigaku Hypix detector with Cu Kα radiation (λ = 1.54184Å) from PhotonJet micro-focus X-ray sources at 100 K.The diffraction images were processed and scaled using the CrysAlisPro software (Rigaku Oxford Diffraction, The Woodlands, TX).The structures were solved through intrinsic phasing using SHELXT 6 and refined against F 2 on all data by full matrix least squares with SHELXL 7 following established refinement strategies. 8All non-hydrogen atoms were refined anisotropically.Hydrogens atoms on the complex cations of 1 and 2 were added with a riding model (HFIX 137, HFIX 23, or HFIX 13).The hydrogen atoms on the solvent water molecules could not be located on the difference map and were therefore not included in the model.This omission led to B-level CheckCIF alerts.However, based on charge balance considerations and putative hydrogen-bonding interactions within the crystal structure, the hydrogen atoms are present to afford neutral water molecules.Both structures were isomorphic, and their asymmetric unit comprised half of the complex cations of 1 and 2, which resided on crystallographic inversion centers, a THF molecule, two triflate anions, and residual electron density peaks.Charge balance considerations required the presence of an additional cation within the unit cell.Accordingly, residual electron density could be satisfactorily modeled as a half-occupancy sodium atom with site position disorder with a lattice water molecule with occupancy factors fixed at 0.50 for each component.Within 1, both of the outer-sphere triflate ions exhibited conformational disorder, which was modeled using appropriate similarity restraints.In addition, the adamantyl moiety of this complex was also disordered over two rotational orientations, which were modeled with similarity restraints.For 2, one of the triflate ions had elongated ellipsoids, indicating unresolved disorder, which triggered B-level alerts in the CheckCIF.Based on our refinement model, the final formula unit within each unit cell was [(C10H15CO2)Os(NH3)4(µ-N)Os(NH3)4(O2CC10H15)](CF3SO3)3•NaCF3SO3•THF•3H2O.CCDC 2352958-2352959 contain the supplementary crystallographic data for this paper.b] 1.027 1.051 R1/wR 2 (all data) [c] 0.0622/0.15210.0604/0.1441R1/wR 2 (I > 2σ) [c] 0.0595/0.1505a] Cu Kα λ = 1.54184Å [b] GoF = {Σ(w(Fo 2 -Fc 2 ) 2 /(n -p)} 1/2 , where n = number of data and p is the number of refined parameters.

Encapsulation Studies and Binding Constant Determination
Encapsulation Studies by 1 H and DOSY NMR Solutions of 1 (1 mM), 2 (1 mM), or CB [7] (2 mM) were prepared in D2O (pD = 7). 1 H and DOSY NMR experiments were carried out for 1 or 2 ± CB [7].The diffusion-ordered experiments were analyzed via the pulse gradient spin-echo experiment.The signal amplitude of each resulting 1 H NMR spectra following the spin echo pulse sequence was fit to Eqs 1 and 2. By plotting the natural logarithm of the normalized signal intensities versus the gradient field strength, the slope of the linear fit determines the diffusion constant (D).Results are presented as the average D of three independent experiments ± SD.    [7], and c) 2 ± CB [7].Integrations of the proton resonances of each species were plotted against the relative gradient field strength (s•cm -2 ) to measure the diffusion constant (D) as the slope of the resulting line.On each plot is shown the plot of H2O, which consistently measured to be 1.8 × 10 5 cm 2 s -1

Binding Constant Determination by ITC
In these experiments, 50 mM MOPS-buffered solutions (pH 7.4) were used.Prior to buffer preparation, glassware was washed with 10% HNO3 to remove any trace metals.A 50 mM MOPS solution was prepared and adjusted to pH 7.4 using HCl or NaOH.The buffer was then treated with Chelex 100 resin for at least 1 h while stirring.Prior to storing at room temperature, the buffer was filtered through a 0.22 micron filter.Aliquots for CB [7], NaOAd, 1, and 2 were prepared by creating a stock solution in this buffer.All aliquots were stored at -20 °C until needed for experimentation.During these experiments, evolution of heat is monitored by measuring the differential power, or heat flow, required to maintain constant temperature within both cells for each injection of the titrant. 9,10The resulting thermogram from graphing heat flow (µJ s -1 ) over time (s) is integrated and normalized for concentration to quantify the stoichiometry (n), binding constant (KITC), and change in enthalpy (∆HITC). 10Each ITC experiment consisted of 20 injections of 2.5 µL and were analyzed using NanoAnalyze software.Experiments were carried out in triplicate, and the post hoc analysis to determine the Ka for each species was carried out according to literature.[7] added to 500 µM NaOAd (left).Concentrations of CB [7] range from 125 µM to 1.5 mM.The percent of encapsulated species is plotted against the concentration of CB [7], demonstrating the 1:1 stoichiometry (right).

Aquation Kinetic Studies by UV-vis
Solutions of 1 or 2 (25 µM) with or without CB [7] (0 or 50 µM) in 50 mM MOPS buffered solution (pH 7.4) in a quartz cuvette were placed in a water-jacketed cuvette holder and incubated at 37 °C.The absorbance spectrum of the solution was recorded every 20 min over a period of 16 h for 1 and every 24 h for 120 h for 2. The pseudo-first order overall rate constant kobs (s -1 ) was determined by fitting the decrease in absorbance at 261 nm for 1 and 242 nm for 2 as a single exponential decay.

Cytotoxicity
HeLa cells were seeded in 96-well plates with ~4000 cells per well and incubated overnight at 37 °C.On the following day, the culture media was removed, and cells were treated with media containing varying concentrations of the test complex and further incubated for 72 h.The media was removed, and cells were incubated in DMEM containing 1 mg mL -1 MTT without FBS for 3 h.Following incubation, the media was removed, and the purple formazan crystals were dissolved in 200 µL of an 8:1 DMSO/glycine buffer (pH 10) mixture.The absorbance of 570 nm of each well was measured using a BioTek Synergy HT plate reader.The average absorbance of control cells was set to 100% viability, and the average absorbances of treated cells were normalized to the control absorbance.Data were plotted as percent viability versus the log[concentration].The Hill Equation was applied to the data to determine the IC50.Data are reported as the average of three independent biological replicates ± SD.

Mitochondrial Membrane Potential via JC-1 Assay
Approximately 10 5 HeLa cells were seeded in 35 mm glass-bottomed dishes (MatTek Life Sciences, Ashland, MA) and incubated overnight at 37 °C.On the next day, cells were treated with 50 µM of the desired complex and incubated for an additional 24 h at 37 °C.The culture media was then removed and replaced with fresh media supplemented with 10 µM JC-1 dye followed by incubation in the dark for 30 min at 37 °C.The dye-containing media was removed, and the cells were washed with 2 × 1 mL phosphate-buffered saline (PBS, Corning Life Sciences).The cells were imaged in 1 mL PBS.Control dishes were handled identically to treated dishes.For the positive control dishes, 50 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) in PBS was used, and the images were collected without the removal of CCCP.Cells were imaged using an EVOS M5000 fluorescence microscope (ThermoFisher, Waltham, MA) with a green fluorescence protein (GFP) filter cube (ex.457-487/em.502-538) for the green monomer fluorescence and a Texas red filter cube (ex.542-583/em.604-644) for the red Jaggregate fluorescence.The cellular images were analyzed using ImageJ (NIH) and the corrected total cellular fluorescence (CTCF) was calculated using the following formula:

CTCF = Integrated density -(area of cell × mean fluorescence of background reading)
For each replicate, the average red/green fluorescence was determined using at least 8 independent cells and was normalized to the untreated control cells ([red]/[green]control = 1).Data are reported as the average of three independent trials ± SD.

Whole Cell Uptake in HeLa Cells
Approximately 10 6 HeLa cells were seeded in 6-well plates.On the day of the experiment, the culture media was removed, and the cells were treated with fresh media supplemented with 0 or 50 µM complex and incubated for 3 h at 37 °C.The culture media was then removed, and the adherent cells were washed with 3 × 1 mL PBS and detached with 0.05% trypsin + 0.53 mM ethylenediaminetetraacetic acid (EDTA; Corning Life Sciences).The detached cells were pelleted by centrifugation (800 × g for 10 min), and the supernatant was removed.The cell pellet was resuspended in PBS and pelleted again by centrifugation.This process was repeated twice more, and the resulting washed cell pellet was resuspended in ice cold lysis buffer (1% w/v 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5 mM EDTA, 50 mM tris(hydroxymethyl)aminomethane (Tris) and 100 mM NaCl; pH 7.4).The suspension was vortexed for 30 s and incubated on ice for 45 min.The cell lysate was then centrifuged to remove any precipitated debris and the supernatant was transferred to a clean tube prior to analysis.For solutions treated with any Ru containing compounds, the Ru content was determined using GFAAS and was normalized to the protein content of the sample, which was determined using the bicinchoninic acid (BCA) assay kit following manufacturer instructions (ThermoFisher).For solutions treated with any Os containing compounds, the 200 µL of the cell lysate solutions were immediately diluted to a total of 5 mL in stabilizing solution (500 µM each of ascorbic acid, thiourea, and EDTA). 4Samples were kept at room temperature until analysis and were analyzed for Os content with ICP-MS within 7 days of preparation.The total Os content was normalized against the protein content of the sample using the BCA assay kit.Results are reported as the mean mass ratio of Ru or Os to protein content (pg µg -1 ) in each sample ± SD.

Whole Cell Uptake in Primary Cortical Neurons
Cortical neuron cultures were seeded at a density of 200,000 cells/well in 24-well plates for uptake studies.After 12 d in culture, neurons were treated with 1 (0, 3, or 10 µM) or 2 (0, 3, or 10 µM) in the presence and absence of two equiv CB [7] (0, 6, or 20 µM) for 24 h.The media was then removed, and cells were washed with 3 × 500 µL cold PBS before the addition of 75 µL RIPA lysis buffer to each well.Plates were then placed on an orbital shaker for 30 min.Cell lysates were collected and centrifuged at 13,000 rpm for 15 min.The protein concentration of the cell lysates was determined using the BCA assay as mentioned previously.Samples were then stored at -80 °C for no longer than one week before analysis of Ru or Os content via GFAAS or ICP-MS, respectively.Results are reported as the mean mass ratio of Ru or Os to protein content (pg µg -1 ) in each sample ± SD.

Mitochondrial Isolation
Isolation of mitochondria in HeLa cells was performed using protocols previously reported.Approximately 10 6 HeLa cells were seeded in 10 cm 2 culture dishes and allowed to adhere overnight.Each replicate consisted of cells combined from two 10 cm 2 dishes.The next day, cells were treated with 50 µM of 1 ± 100 µM CB [7] in normal culture media for 24 h at 37 °C.The media was then removed, cells were washed with 3 × 3 mL room temperature PBS and harvested with trypsin.The cells were collected and pelleted by centrifugation (800 × g) for 10 min and resuspended in 500 µL ice cold mitochondrial isolation buffer (pH 7.4) containing 200 mM mannitol, 68 mM sucrose, 50 mM piperazine-N,N ′ -bis(2-ethanesulfonic acid), 50 mM KCl, 5 mM ethylene glycol-bis-(βaminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 mM MgCl2, 1 mM dithiothreitol, and 1:500 v/v protease inhibitor cocktail.The cell suspension was then incubated on ice for 20 min before it was homogenized through a 25-gauge needle using a 1 mL syringe with 40 passes.The homogenized suspension was centrifuged (150 × g) for 5 min, and the supernatant was transferred to a clean tube and centrifuged (14,000 × g) for 10 min to pellet the mitochondrial fraction.The supernatant from this step was kept as the cytosolic fraction, while the mitochondrial pellet was suspended in 300 µL RIPA lysis buffer and vortexed at the highest setting for 30 s.The Ru content was determined as described previously and was normalized to the protein content using the BCA assay as described above.Results are reported as the mean mass ratio of Ru to protein content (pg µg -1 ) in each sample ± SD.

Mitochondrial Ca 2+ Uptake in Permeabilized Cells
HEK293T cells were grown to near confluency in 10 cm 2 dishes and harvested with trypsin.The cells were pelleted by centrifugation (800 × g) and resuspended in ice cold PBS supplemented with 5 mM EDTA (pH 7.4).This cell suspension was counted using trypan blue, and the cells were pelleted by centrifugation (800 × g) for 5 min and resuspended in ice cold high KCl solution (125 mM KCl, 20 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), 2 mM K2HPO4, 5 mM glutamate, 5 mM malate, 1 mM MgCl2; pH 7.2) supplemented with 80 µM digitonin and 1 µM thapsigargin.The final solution contained <0.1% DMSO from the original digitonin and thapsigargin stocks.The cells were incubated on ice for 15 min and centrifuged (200 × g) for 10 min at 4 °C.Finally, the permeabilized cells were resuspended in high KCl solution containing 1 µM Calcium Green 5N (ThermoFisher, Waltham, MA) and 2 mM succinate to a final density of 1 × 10 7 cells mL -1 .For each experiment, 100 µL of the cell suspension with the desired concentration of the test complex were placed in each well of a black-walled 96-well plate.The background fluorescence of each well was recorded for 60 s prior to the addition of 20 µM CaCl2.The change in fluorescence of the dye (ex.488/em.528) in response to Ca 2+ was recorded every 5 s for at least 120 s or until the fluorescence returned to baseline.The mCa 2+ uptake rate was calculated as the time constant of the exponential fit of the decay in the fluorescence response curve.The Ca 2+ uptake rate of treated cells was normalized to that of untreated cells (0% inhibition), and each replicate was performed using independently prepared cell suspensions to account for differences in cell count.A BCA assay was performed on each cell suspension for every experiment, giving an average protein content of 1200 µg mL -1 each time.The Hill Equation was used to determine the IC50 of MCU inhibition.Data are presented as the average of three independent biological replicates ± SD.

Mitochondrial Ca 2+ Uptake in Intact HeLa Cells using Rhod2-AM
Approximately 5 × 10 4 HeLa cells were seeded in an 8-well µ-slide (Ibidi USA, Inc., Fitchburg, WI) and incubated overnight at 37 °C.The following day, cells were treated with the desired complex in DMEM supplemented with 10% FBS for 1 h at 37 °C.The culture media was removed, and the cells were washed with 100 µL PBS before the cells were incubated in the dark for 30 min at room temperature in extracellular medium (ECM; 135 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) supplemented with 10 mM glucose, 3.2 mg mL -1 bovine serum albumin (BSA), 0.003% Pluronic F127, and 2 µM Rhod2-AM (Molecular Probes).The dye-containing ECM was removed, cells were washed with 100 µL ECM, and cells were treated with fresh ECM supplemented with 10 mM glucose and 3.2 mg mL -1 BSA followed by further incubation in the dark for 30 min at room temperature.The buffer was then removed, cells were washed with 100 µL ECM, and cells were treated with fresh ECM supplemented with 10 mM glucose and 3.2 mg mL -1 BSA.The cells were then incubated for 15 min at 37 °C before imaging using a Zeiss LSM i710 confocal microscope with a 40× water objective at an excitation wavelength of 561 nm and an emission window of 568-712 nm.After ~30 s of baseline recording, histamine (final concentration of 100 µM) was added to the dish and fluorescence images were collected every 3 s to monitor mCa 2+ uptake.Images were analyzed and quantified using ImageJ and the CTCF was calculated.The average of at least six individual cells were used to determine the average CTCF for each replicate.Results are reported as the average of two independent replicates ± SD.

Cell Viability after OGD
Primary cortical neuron cultures were seeded at a density of 150,000 cells/well in 48-well plates for cell viability studies.After 10-12 d, neurons were left untreated (control) or treated with the desired complex in the presence or absence of two equiv CB [7] for 3 h.Cells were then subjected to 90 min OGD.For OGD, culture media was removed and 300 µL of glucose-free balanced salt-solution (GBSS) containing corresponding concentrations of compound was added to wells.Culture plates were then placed in an airtight chamber flushed with a gas mixture of 90% nitrogen, 5% carbon dioxide, and 5% hydrogen for 3 min to remove any oxygen from the chamber.The chamber was then placed in a 37 °C incubator for the remainder of the OGD treatment (90 min total).After the OGD treatment, the GBSS was removed, and 500 µL fresh culture media containing corresponding concentrations of compound was added back to the wells.Cell viability was measured 24 h after OGD treatment using the MTT assay.

Figure S3 .
Figure S3.IR (ATR) spectrum of 2. The asymmetric OsNOs stretch on the IR spectrum is indicated with an asterisk (*).

Figure S17 .
Figure S17.Pulse-gradient spin echo (PGSE) experiment calculations for a) CB[7], b) 1 ± CB[7], and c) 2 ± CB[7].Integrations of the proton resonances of each species were plotted against the relative gradient field strength (s•cm -2 ) to measure the diffusion constant (D) as the slope of the resulting line.On each plot is shown the plot of H2O, which consistently measured to be 1.8 × 10 5 cm 2 s -1

Figure S25 .
Figure S25.Aquation kinetic experiments of a) 1 and b) 1 + CB[7] in pH 7.4 MOPS buffered solution at 37 °C as measured by UV-vis spectroscopy over 16 h.Shown for each are the change in spectral absorbance at 258 nm over time as indicated by an arrow, and the exponential fit of the absorbance over time to calculate the rate constant of aquation (kobs).

Figure S26 .
Figure S26.Aquation kinetic experiments of a) 2 and b) 2 + CB[7] in pH 7.4 MOPS buffered solution at 37 °C as measured by UV-vis spectroscopy over 120 h.Shown for each are the change in spectral absorbance at 244 nm over time as indicated by an arrow, and the exponential fit of the absorbance over time to calculate the rate constant of aquation (kobs).

Figure S30 .
Figure S30.Comparison of the Ru concentration in the extramitochondrial (EM) and mitochondrial (M) fractions of HeLa cells treated with a) 50 µM 1 and b) 50 µM 1 + 100 µM CB[7].Cells were treated with the desired complex for 24 h at 37 °C.Ru concentration was normalized to protein content using the BCA assay.Data are the mean of three trials ± SD (n = 3), **p < 0.01.

Table S1 .
X-ray crystal data and structure refinement details for 1 and 2. 7