Dual targeting of the cancer antioxidant network with 1,4-naphthoquinone fused Gold(i) N-heterocyclic carbene complexes

Herein, we report a simple and rational approach to the design of a targeted therapy (i.e., complex 1) whose mechanism of action involves targeting a single cancer relevant pathway via two independent mechanisms.


Introduction
A paradigm shi in recent years has given rise to the eld of Systems/Network Pharmacology whose focus is identifying drug candidates that act via modulation of multiple networked targets. 1,2This focus is predicated on the thought that drugs possessing target promiscuity may result in enhanced efficacy.It is leading to a rethinking of the "magic bullet" approach involving drugs that bind and interact preferentially with a single disease target. 3,4This latter approach, while time-honored, is characterized by high drug attrition rates in clinical trials. 5,6he emerging appeal of systems-based therapeutic approaches has prompted efforts to identify viable targets within biological networks (Fig. 1).Unfortunately, to date, random deletions or inhibition of specic proteins have typically led to poor phenotypic outputs due to the scale-free nature of biological networks. 7As a consequence, the targeting of single proteins or nodes within a biological system oen does not lead to viable drug candidates (Fig. 1b).On the other hand, dual knockout yeast model studies have lent support to the suggestion that the simultaneous deletion of two genes can result in a phenotypic alteration under conditions where the targeting of a single gene will not. 8,9][12] With such considerations in mind, we have developed a new approach that involves the dual targeting of antioxidant response mechanisms.9][20][21] To achieve a systems-based approach to targeting the antioxidant pathway, we suggest that it would be benecial to develop an agent that both reduces ROS tolerance (by inhibiting reducing metabolites) while increasing ROS production (Fig. 2).This would lead to antioxidant homeostasis being perturbed from both ends, thus overwhelming the network and promoting cell death (Fig. 1c and 2).Here, we present the results of a rst study along these lines.Specically, we present the synthesis, in vitro, and preliminary in vivo testing of a series of redox active, quinone-annulated gold(I) Nheterocyclic carbene complexes that both promote singlet oxygen generation and inhibit thioredoxin reductase (TrxR).
Thioredoxin reductase (TrxR) is a selenoenzyme that plays a central role within the antioxidant system.It regenerates thioredoxin (Trx) through an NADPH-dependent reduction of the active site disulde bond (Cys32 & Cys35) present in oxidized Trx. 22,23The reduced form of Trx reacts with ROS and thus helps overcome oxidative stress.This has made inhibition of Trx/TrxR an attractive strategy for patients undergoing radiation therapy. 24onsistent with other types of cancer, TrxR is overexpressed in human lung carcinoma models (e.g., the A549 cell line), providing a relevant model for antioxidant network targeting. 25,26pecic knockdown of TrxR by 90% (via siRNA), however, provided little to no phenotypic change in cell proliferation. 26In addition, treatment with auranon, a Au(I) complex that targets TrxR, resulted in no difference in cell proliferation between TrxR knockdown A549 and A549 cells treated with mock siRNA.This robustness of TrxR is consistent with a highly networked endogenous antioxidant system that would require multiple modes of drug targeting to be suppressed in a therapeutically useful manner (Fig. 1b and c).
The overexpression and robustness of TrxR reported in several cancer models makes it a unique challenge within the context of network pharmacological drug development.Specic small molecule inhibition of TrxR yielded non-signicant changes in cell growth, suggesting that a combined system approach is necessary to bypass the inherent redundancy. 26To explore this possibility we sought to develop a single molecular entity capable of both TrxR inhibition and redox cycling.In principle, this would both allow an increase in ROS production (through redox cycling) and a reduced ability to decrease the effects of ROS-based oxidative stress (through TrxR inhibition).
Quinones are venerable redox cycling agents.Under biological conditions, many quinones can accentuate ROS production beyond the buffering capacity of the cell.This is a feature that has long been appreciated in the context of cancer therapy, [27][28][29][30] and one that is potentially useful in targeting the antioxidant pathway.Separate seminal work by Berners-Price and Filipovska led to an appreciation that appropriately designed gold(I) Nheterocyclic carbene (NHC) complexes can inhibit TrxR.2][33][34] This has encouraged us and others to explore the utility of mono-NHC and bis-NHC gold(I) complexes as potential anticancer therapeutic agents  with recent examples being efficacious in mammalian xenogra bearing models. 10, We nw suggest that using a quinonebearing Au-NHC complex will allow a two-fold interruption of the antioxidant pathway via both overproduction of ROS and a decrease in TrxR-based ROS mediation.
To assign the molecular structure unambiguously, X-ray diffraction quality single crystals of 1 and 3 were grown by slowly diffusing hexanes into a concentrated 1,2-dichloroethane solution (see ESI † for structure of 3).Thermal ellipsoid plot of the resulting structure is presented in Fig. 4. In the case of 1, a trans geometry was seen for the core [(4) 2 Au] + cation with a C-Au-C bond angle of 172.8(2) being observed.The Au-C carbene bond distances of 2.012(5) Å and 2.009(4) Å are in agreement with those for other reported NHC-Au-NHC complexes. 52,63,66,68,69As inferred from the molecular structure of 1 (Fig. 4), the two carbene units are rotated around the gold atom with a torsion angle of 62.6(3) .Presumably this twisting minimizes steric crowding.

4[H]
[Cl], obtained from DPV measurements, are summarized in Table 1.In the CV measurements (scan rate ¼ 100 mV s À1 ), all four compounds (1-3 and 4[H][Cl]) displayed cathodic waves that occur in two sequential steps in which the rst wave is completely reversible and the second wave is quasireversible at a 0.1 mV s À1 scan rate; these are labeled as a and b in Table 1. 70,71These electrochemical features were attributed to the reduction of the quinone moiety to rst produce the semiquinone radical (NQ À ) and then produce the quinone dianion (NQ 2À ) forms of compounds 1-3 and 4[H][Cl]. 70,71The quinone reduction potential in 4[H][Cl] occurs at À0.38 V, the lowest of all the molecules studied, indicative of a positively charged imidazolium ring.The quinone couple at À0.42 V observed for compound 1 is ascribed to the presence of bis(NHC).The same wave in compound 3 appears at a more negative potential (À0.47 V), presumably due to greater trans effects excreted by quinone annulated NHC ligand than the metal-bound chloride.This analysis agrees well with the differences in the observed d 13 C (Au-C carbene ) resonances for compounds 1 and 3.
Having studied the electronic properties, we sought to probe the stability and electronic nature of 1 upon reduction by means of UV-vis spectroelectrochemistry. Upon bulk electrolysis of compound 2 at a potential of À1.5 V using a special electrochemical cell, reduced quinone species were generated and simultaneously probed using UV-vis spectroscopy.Characteristic absorbance features ascribable to reduced quinone moieties were observed. 64The original UV-Vis spectral trace of compound 2 can be obtained aer reduction (NQ / NQ 2À ) followed by subsequent oxidation (NQ 2À / NQ) (see ESI †).These ndings provide support for the reduced species being stable under the conditions of electrochemical analysis.

Cell proliferation assays
To gauge the ability of each complex to inhibit cancer cell growth, A549 lung cancer cells were treated with 1-3, 4[H][Cl], doxorubicin, and auranon in a dose responsive manner.Cellular vitality (i.e., mitochondrial reductase activity) was then quantied colorimetrically post treatment using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) (Table 2, Fig. 5).Dose responsive treatment of A549 cells  with doxorubicin and auranon provided growth inhibition curves and IC 50 values of 0.103 AE 0.023 mM and 1.67 AE 0.05 mM, respectively.These values were similar to those previously reported. 10,61In the case of the gold(I) NHC quinone complex 1, the corresponding IC 50 value was determined to be 0.073 AE 0.016 mM.A similar value was recorded in the case of complex 2 (see ESI †).Complex 3 was essentially inactive (i.e., >150Â less potent than 1).
To determine the relative contribution of the individual components present in 1 (i.e., the quinone moiety vs. the Au(I)-NHC subunit), positively charged complexes containing a naphthoquinone (i.e., 4[H][Cl]) and the [(NHC) 2 Au] + core (i.e., 5) were also studied; they gave IC 50 values of 0.99 AE 0.12 mM and 0.71 AE 0.06 mM, respectively.Improved antiproliferative activity (IC 50 ¼ 0.197 AE 0.057 mM) was observed when A549 cells were exposed to a combination of 4[H][Cl] and 5 in a 2 : 1 molar ratio that matches their stoichiometric ratio in 1.However, this combination was not as effective as complex 1 (by a factor of 2.7).
Further anti-proliferation studies were carried out with complex 1 and its naphthoquinone component 4[H][Cl] using the following cell lines: A2780 ovarian (a wt-p53 cell line sensitive to platinum treatment), 2780CP ovarian (isogenic to A2780 but expressing multi-drug resistance (MDR)), and PC-3 prostate (p53 null) (Table 3).While both complexes reduced proliferation in all three cell lines, complex 1 was found to be statistically more potent in each cell line relative to 4[H][Cl].

Cellular uptake and interaction with serum proteins
To quantify the extent to which variations in cellular uptake might account for the differences in anti-proliferative efficacy seen for the various gold(I) complexes of this study, inductively coupled plasma mass spectrometry (ICP-MS) was used to detect intracellular Au levels (Fig. 6a).In brief, cell cultures of A549 were treated with varying doses of 1, 3, 5, and auranon, collected and digested, and quantitatively assessed for intracellular Au content.It was found that regardless of dose, a 2-5 fold increase in intracellular Au concentrations was seen in samples treated with auranon as compared to complex 1.In the case of 3, a neutral complex, no intracellular Au was detected under conditions identical to those used to test complex 1.The intracellular Au levels were found to be identical in the case of complexes 1 and 5 (see ESI †).
To assess potential drug protein interactions, samples of fetal bovine serum (FBS) were treated with 25 mM 1, 3, and auranon prior to incubating at 37 C. Aliquots were taken and the free Au (non-protein bound, methanolic extracts) content was analyzed by ICP-MS (Fig. 6b).As expected, the free Au content in the FBS samples treated with auranon decreased in a time dependent manner. 72,73A similar reduction in free Au was observed for FBS samples treated with complex 3.In contrast, minimal changes in the free Au levels were seen as a function of time in the samples containing complex 1.This result is consistent with the notion that Au(I)-NHC 1 enters the cell via different mechanism than auranon (Fig. 6a).In addition, the protein binding differences between 1 and 3 could explain the relatively reduced potency seen in the case of 3.

Accentuation of reactive oxygen species (ROS)
To establish whether or not the complexes of this study would increase intracellular ROS levels, A549 cells were treated with each complex in a dose responsive manner.ROS uctuations were monitored post treatment via ow cytometry using the uorescein-based general ROS indicator (5-and-6)-chloromethyl-2 0 ,7 0 -dichlorodihydro-fuorescein diacetate, acetyl ester (CM-H 2 DCFDA).Following treatment with 2.5 mM 1, a 27-fold uorescence associated cell population shi was observed (Fig. 7a), a nding taken as indicative of a signicant increase in intracellular ROS in the case of this complex.A dose dependence was also seen (Fig. 7b).Upon treatment with the individual components of 1 (i.e.4[H][Cl] and 5), a more modest increase in ROS was observed ($11-fold increase at the 2.5 mM dose level in each case), while minimal or no ROS increase was observed in the case of 3, auranon, or doxorubicin (Fig. 7c).When A549 cells were exposed to a 2 : 1 molar ratio of 4[H][Cl] and 5 a dose-dependent increase in ROS was observed that statistically similar to that produced by 1.This is rationalized in terms of the ROS enhancement produced upon exposure to the individual components present in 1 being additive and not synergistic.2. Error bars represent the standard error of the mean.
To further elucidate the subcellular loci of ROS accentuation, confocal microscopy was employed to uorescently image A549 cancer cells treated with vehicle (DMSO) and 1.25 mM complex 1 (Fig. 7d).All cells were selectively stained for visualization of ROS accentuation (green, CM-H 2 DCFDA), mitochondria (red, Mitotracker Red), and nuclei (blue, Hoechst).No ROS accentuation was observed in cells treated with DMSO.A549 cells treated with complex 1 resulted in a general green uorescence increase with localized areas of higher green uorescence (Fig. 7d, image F).Once merged, evident overlap of localized ROS accentuation with mitochondria (red) suggests that ROS accentuation is arising from mitochondria (Fig. 7d, image H).

Inhibition of thioredoxin reductase
To assess whether any or all of the present gold complexes could serve as TrxR inhibitors, standard tests involving the reduction of the oxidized form of the cell-permeable cofactor lipoate to its corresponding reduced form, dihydrolipoate, were carried out.Briey, plateau phase A549 cells were exposed to variable doses of complexes 1, 3, We thus propose that complex 1 will be able to act as both a TrxR inhibitor and a general agonist of oxidative stress.

Induction of apoptosis
To determine whether complex 1 also promotes apoptosis, ow cytometry studies in conjunction with annexin-V staining were carried out.In brief, plated exponential growth phase A549 cells were exposed to various concentrations of 1 and incubated for 24 h.At that point, all cells (adhered and oating) were collected, washed, and stained with uorescein-labeled annexin-V and propidium iodide (PI) and subjected to ow cytometry (Fig. 9).At low doses, evidence of early stage apoptosis was seen, as inferred from the binding of annexin-V to the still-intact and impermeable cell membrane (resulting in FITC-only uorescence).As the dose escalation progressed, a larger percentage of late stage apoptosis/necrotic (FITC positive and PI positive from staining of nuclear material) cells became evident.Treatment of A549 cells with doxorubicin (a known inducer of apoptosis) provided similar results in both the early and late stage apoptotic quadrants (see ESI †).On this basis, we conclude that complex 1 induces controlled cell death via an apoptotic mechanism.

Toxicity and efficacy studies in zebrash
The anticancer activity of the complex 1 was tested using a qualitative high throughput zebrash tumor xenogra model (IACUC # I13009). 74First, zebrash embryos were divided into 7 groups at an average of 65 embryos per group.Each group was treated with vehicle (DMSO) or complex 1 at variable concentrations to identify the maximum tolerable dose (MTD) (Fig. S29 †).A dosing of 0.5 mM was found to induce no observable toxic effect relative to vehicle (p-value > 0.1) and was deemed to be the MTD for zebrash embryos.6][77] Briey, live human lung cancer cells (A549) were labeled with CM-DiI (red) and only live cells were transplanted via injection into the perivitelline space of 30 zebrash embryos 24 hours post fertilization (hpf). 74,78,79Tumor inoculated zebrash embryos were allowed to grow for one day till 48 hpf.This allows for establishment of the cancer cells in the host zebrash embryos.At 48 hpf, the xenogra bearing zebrash embryos were split into 2 groups (15 embryos per group) and treated with vehicle (DMSO) or complex 1 at 0.5 mM for one additional day (72 hpf), and cancer cell death was observed using acridine orange staining (green).Live zebrash-A549 tumor xenogras treated with DMSO display features consistent with the presence of tumor cells (red, white arrows in Fig. 10A).On the other hand, few, if any, tumors and little evidence of host cell apoptosis was seen with acridine orange staining (green, arrowhead in Fig. 10B-D).Finally, live zebrash-A549 tumor xenogras (red, white arrows in Fig. 10D) treated with complex 1 showed evidence of apoptosis for the majority of tumor cells under conditions of acridine orange staining (green, arrowhead in Fig. 10E and yellow or orange cells in Fig. 10F and G).

Discussion
Naphthoquinone functionalized N-heterocyclic carbene supported gold(I) complexes (1-3) were designed to test whether the inhibition of TrxR in parallel with an increase of network stress (higher levels of ROS) would lead to an enhanced phenotypic response (reduction in cell growth).The Au(I)-NHC and naphthoquinone moieties of the present study were specically chosen to (1) inhibit TrxR via irreversible binding of an Au(I) center to the selenothiol-containing active site and (2) accentuate ROS via redox cycling of the naphthoquinone moieties.The goal was to achieve these complementary functions using a single molecule.The use of a single molecule that achieves two targeting functions concurrently is expected to allow for better control ultimately over such key design features as metabolism, uptake, localization, and clearance, to name a few.With such considerations in mind, two bis-carbene Au complexes with different counter anions were prepared (i.e., 1 and 2).][82][83][84] Initial side-by-side comparisons revealed no appreciable difference between 1 and 2 in their ability to inhibit cell proliferation, induce exogenous ROS, or inhibit TrxR activity.ICP-MS analysis of cells treated with 1 and 2 showed similar Au uptake between complexes.Considering the intracellular uptake of the [AgCl 2 ] À counterion of 1, a 7 : 1 Au : Ag uptake ratio was observed via ICP-MS.This suggests that the [AgCl 2 ] À minimally enters the cell possibly due to ion exchange with salts within the cell culture medium (see ESI † for a complete comparison between 1 and 2).Detailed studies were thus carried out with 1 and various controls.As noted in the Results section above, in cell proliferation studies, this complex proved much more active than aur-anon (23-fold), 4[H][Cl] ($14-fold), 5 ($10-fold), or a 2 : 1 mixture of the latter species (Fig. 5 and Table 2).The stark (i.e., $10Â) increase in potency seen for 1 relative to its individual parts (i.e., 4[H][Cl] and 5) leads us to suggest that both the Au(I) NHC and quinone moieties contribute to the observed antiproliferative activity.Furthermore, the ability of 1 to inhibit cell proliferation was found to be 165-fold greater than the mon-oNHC-Au(I) complex 3. ICP-MS data provide support for the conclusion that complexes 1 and 5 enter cells more effectively than 3 (Fig. 6 and S20 †).Drug uptake is multifactorial, and the varying cellular uptake levels seen for the various Au(I) complexes could reect differences in lipophilicity, the presence of positive charges facilitating passive diffusion, and complex-serum protein interactions. 32Auranon is known to bind serum proteins, such as human serum albumin and bovine serum albumin, which are thought to provide a transport mechanism into the cell. 72,73,85,86On the other hand, the reduced uptake seen for 3 (in contrast to 1 and 5) is ascribed to irreversible sequestration by serum proteins, a conclusion that is consistent with recent structural work showing that mono-NHC ligated Au(I) complexes bind lysine residues within protein models. 87,88On the basis of the present work, we propose that such irreversible binding be avoided if the goal is to achieve cell uptake and targeting of the antioxidant network (Fig. 6a and b). 72,73,85,86ased on ICP-MS analysis, we conclude that complex 1 is less reactive towards serum proteins than auranon.This corresponds to an increase in complex stability that we ascribe to the differences in Au-ligation (i.e., NHC versus phosphine).Notwithstanding its increased stability relative to auranon, complex 1 was found to inhibit well the activity of TrxR.This nding is ascribed to complex 1 undergoing facile exchange with Se containing biomolecules (i.e., TrxR) with no appreciable reactivity towards other common biological nucleophiles, including protein thiols or amines.The combined benets of a decrease in serum protein reactivity (which prevents loss of active Au) while retaining effective TrxR inhibition provide for a potential increase in therapeutic benet and an enhanced safety window. 32 dose responsive increase in ROS was observed in A549 cells for several of the complexes, which culminated in a maximal 27fold increase at 2.5 mM in the case of 1.This ROS accentuation by 1 was found to be localized to the mitochondria as evidenced by confocal microscopy studies.In contrast to what was seen in the cell proliferation studies, 2 : 1 molar mixtures of 4[H][Cl] and 5 (a "cocktail" mimicking the stoichiometry of the subunits within 1) engendered statistically similar levels of ROS to that of complex 1.The difference between the growth and ROS phenotypes is consistent with the notion that two different modes of action (ROS generation and TrxR inhibition) are responsible for the observed biological activity.In fact, complex 1, in contrast to previous systems we have studied, 10 was found to inhibit TrxR activity strongly (i.e., at levels similar to aur-anon (Fig. 8)).This was also true for the 2 : 1 stoichiometric mixture of 4[H][Cl] + 5 (cocktail), but not for either of the components (4[H][Cl] or 5) when tested individually.This leads us to suggest that the increased TrxR inhibition by 1 is due to the presence of the naphthoquinone moieties and not due to potential differences in Au-carbene metal ligand interactions.The statistical indifference between 1 and the 2 : 1 stoichiometric mixture of 4[H][Cl] and 5 regarding ROS accentuation and TrxR inhibition is in stark contrast to the 2.7-fold difference in growth phenotypes.We ascribe the increase in biological potency relative to what one might expect based on a simple sum of the chemical and enzymatic inhibition effects provided by the individual components (i.e., 4[H][Cl] and 5) to the effect of conjugation.The tethered system 1 helps assure the concurrent subcellular localization of both active species, namely the naphthoquinone and the NHC-complexed Au centers.
Doxorubicin, a conjugated anthracycline possessing a quinone moiety, is thought to mediate its anticancer effect through inhibition of topoisomerase II via DNA intercalation.However, it has been established that doxorubicin localization to healthy cardiac tissue induces cellular stress and dose limiting toxicity via mitochondrial ROS accentuation. 29,60,62,63his duality of action led us to question whether the naphthoquinone complexes of the present study (e.g., 1 and 4[H][Cl]) would also interact with DNA, mediating an effect apart from their targeted ROS producing function.To test this possibility, thermal denaturation studies with short DNA duplexes were carried out.Signicant DNA stabilization was observed in the case of doxorubicin; however, no thermal stabilization of DNA by 1 or 4[H][Cl] was observed under the study conditions (see ESI †).This is consistent with complex 1 and doxorubicin operating via different mechanisms.However, both induce cell death via apoptosis as inferred from the formation of two positive annexin-V populations (+PI, ÀPI) (Fig. 9).
Complex 1 was also found to inhibit cell proliferation across several cancer cell lines displaying varying p53 status and drug resistance proles, namely PC3 prostate (p53 null), A2780 ovarian (wt-p53 platinum sensitive), and 2780CP (isogenic partner to A2780 displaying multidrug resistance (MDR)) (Table 3).The gold-free naphthoquinone 4[H][Cl] was also tested.Across all cell lines, complex 1 proved more potent than 4[H] [Cl].Of the four cell lines tested, it should be noted that complex 1 displayed higher potency in the A2780 cell line, as compared to the A549, 2780CP and PC-3 cell lines (where similar potency levels were observed).Regarding naphthoquinone 4[H][Cl], a stronger antiproliferative effect was observed in the A2780 and PC-3 cell lines relative to 2780CP and A549.These trends may reect differing pharmacological proles that warrant further exploration.
To assess the utility of complex 1 in vivo, a zebrash xeno-gra tumor model was used. 74Such zebrash models are attractive as they (a) provide a qualitative high throughput cancer drug screen in a system that maintains the complex physiology of the human tumor and (b) allow for the assessment of dose limiting toxicity through non tumor specic cell death.This efficacy/toxicity system presents a preliminary valuation for cancer death selectivity and therapeutic index.Using this kind of model, it was found that zebrash embryos tolerated well a 0.5 mM dose of complex 1 with no signicant host cell apoptosis.Moreover, at that dose, cancer specic cell death was seen in zebrash embryos bearing human A549 lung cancer xenogras.Based on this nding, the induced apoptosis is thought to be largely, if not completely, localized within the tumor xenogras.These preliminary studies validate the ability of complex 1 to selectively induce cancer cell death in vivo at levels that do not produce toxic effects and warrant further investigation in mammalian murine models.

Conclusions
Herein we report that targeting a highly networked antioxidant regulator (TrxR) results in a greater phenotypic alteration (cell proliferation) when combined with a network stress inducer (i.e., accentuation of oxidative stress via redox cycling).The incorporation of redox cycling naphthoquinone subunits within an Au(I)-NHC core to give complexes such as 1 and 2 leads to an enhancement in the anti-proliferative activity.This enhancement is ascribed to the combination of ROS accentuation and TrxR inhibition provided by the individual components and to the fact that the species are tethered to one another, thus controlling co-localization (e.g., uptake and clearance).The anticancer activity and low toxicity seen in the zebrash-A549 tumor xenogra provides support for the notion that complex 1 warrants further study as a potential anticancer agent.We view these preliminary ndings as promising considering clinical drugs such as anthracyclines and platinums induce organ specic toxicities that are dose limiting.More broadly, the success of 1 relative to various controls, including auranon and doxorubicin, provides "proof-of-principle" support for the suggestion that targeting key cancer-related pathways via multiple modes of action may have utility as a therapeutic paradigm.Further tests of this hypothesis are ongoing in our laboratories.

Fig. 1
Fig. 1 Schematic view of bimodal network targeting.(a) Normal (green) signal transduction within a generic biological network.(b) In the presence of a small molecule protein inhibitor, the pathway is shut down (black); however, no change in response is observed due to redirection of the signal transduction.(c) It is hypothesized that biochemical targeting with the same protein inhibitor in conjunction with a small molecule capable of inducing general pathway stress (e.g., a redox cycler) will shut down the network, resulting in a greater alteration in the phenotypic response.

Fig. 2
Fig. 2 Mechanism based rationale regarding a dual targeting approach in drug design.(A) Elevated endogenous ROS that is tolerated by cancer cells.(B) Accentuating exogenous ROS by a single mechanism may not reach the cell death threshold.(C) Antioxidant inhibitors reduce the concentrations of reducing metabolites, thus lowering the cell death threshold.(D)The dual targeting approach involves the use of a redox cycler to accentuate exogenous ROS in combination with a reducing metabolite inhibitor to lower the cell death threshold.This combination is expected to overwhelm the system and drive it towards death.

Fig. 5
Fig. 5 Cell proliferation profiles of A549 lung cancer cells treated with 1, 3, 4[H][Cl], 5, and a 2 : 1 molar concentration of 4[H][Cl] and 5 (cocktail), respectively.Data for doxorubicin and auranofin are not shown for clarity purposes but are provided in the ESI † as well as in Table2.Error bars represent the standard error of the mean.
4[H][Cl], a 2 : 1 molar ratio of 4[H][Cl] and 5 (cocktail), auranon, and doxorubicin for 6 h.Post treatment, the live cells were monitored colorimetrically over 180 min for their ability to reduce lipoate (Fig. 8a).Depending on the incubation concentration distinct differences in the time dependent inhibition of TrxR are evident.At low concentrations (0.1-0.6 mM), inhibition of TrxR was apparent in A549 cells exposed to 1, auranon, and 4[H][Cl] + 5, while little to no inhibition was seen in the case of 3-5 or doxorubicin.At higher concentrations (1.25-5.0 mM), inhibition of TrxR by 4[H][Cl] and 5 became evident, while 3 or doxorubicin remained inactive over the full concentration range used in the study (see ESI †).

Fig. 6
Fig. 6 (a) ICP-MS detection of intracellular Au levels as an indicator of complex uptake into A549 lung cancer cells.Students t-test (unpaired) of 1 (2.5 mM) compared to auranofin (2.5 mM) provided p-value <0.05, indicating statistical significance.A comparison of 1 to 3 (p-value >0.2) revealed no statistical significance.(b) Percent of free Au (non-protein bound) within samples of fetal bovine serum treated with 25 mM 1, 3, and auranofin.Error bars represent the standard error from the mean.

Fig. 7
Fig. 7 (a) A549 cell population shift (black ¼ vehicle treatment, red ¼ treatment with 2.5 mM 1) indicating accentuation of intracellular ROS via flow cytometry.(b) Dose responsive cell population shift of A549 cells treated with 1. (c) Dose responsive accentuation of intracellular ROS with various complexes post 6 h incubation.Error bars represent the standard error of the mean.Treatment of A549 cells with 100 mM H 2 O 2 was used as a positive control (see ESI †).Students t-test (unpaired) of 1 compared to the cocktail provided p-values >0.05 suggesting no statistical significance.Comparison of 1 to either 4[H][Cl] or 5 individually (p-values < 0.005) revealed a statistically significant difference in both cases.(d) Confocal microscopy studies illustrating mitochondria specific ROS generation in A549 cells treated with 1.25 mM complex 1.

Fig. 9
Fig. 9 Cell death via apoptosis as detected using flow cytometry.Study is suggestive of the activation of apoptosis by 1 due to the presence of two separate annexin-V positive populations representing early stage (bottom right) and late stage (top right) apoptosis.

Fig. 10
Fig. 10 Complex 1 induces tumor specific cell death in Zebrafish tumor xenografts.A-D are lateral view of 3 day old zebrafish tumor xenografts treated with 0.5 mM DMSO and E-H are lateral views of 3 day old zebrafish tumor xenografts treated with 0.5 mM complex 1.A and E shows the DiI labeled (Red, white arrow) A549 lung cancer cells in the DMSO and complex 1 treated xenografts, respectively.B and F shows Acridine Orange labeled (Green, white arrowheads) dead A549 cells within the DMSO and complex 1 xenografts, respectively.Whereas DMSO treated xenografts display very few dead cells (B), complex 1 treated xenografts display cell death of majority of tumor cells (F).C and G are the merge of DiI and Acridine orange staining of xenografts where yellow/orange indicate dead cells.D and H are the bright field images of DMSO and complex 1 treated xenografts showing no non-specific cell death in the developing zebrafish larvae.

Table 1
Electrochemical analysis of compounds 1-3 and 4[H][Cl].The potentials were obtained from differential pulse voltammetry measurements in DMSO using 0.1 M [N(nBu) 4 ] + [PF 6 ] À as the supporting electrolyte, 0.1 mM analyte, and referenced vs. SCE.See the ESI for the corresponding cyclic voltammograms and differential pulse voltammograms

Table 2
Cell proliferation data in A549 lung cancer cells

Table 3
IC 50 values of the naphthoquinone Au(I)-NHC complex 1 and the naphthoquinone imidazolium salt 4[H][Cl] in various cancer cell lines a a Error is represented as standard error from the mean.b Students t-test (unpaired) revealed 1 was signicantly more potent than 4[H][Cl] in every cell line (p-value <0.005 for A549, A2780, 2780CP; p-value <0.05 for PC-3).c Students t-test (unpaired) revealed that the potency was different in A2780.d Students t-test (unpaired) revealed no difference in potency between the A2780 and PC3 cell lines.2780CP was signicantly different from A549, A2780, and PC-3 (p-value <0.0005).