In this report, we utilized a yeast model to decipher the cytotoxic action of binaphthoquinones. We showed that binaphthoquinones are capable of depolarizing the mitochondrial membrane and that interference with cellular respiration increases the sensitivity of the cells to binaphthoquinones. Furthermore, we demonstrated that binaphthoquinones generate ROS and that the toxic effects of many binaphthoquinones is dependent on NAD(P)H:quinone oxidoreductase type II, NDE1.
Our screen results underscore the importance of intact cellular respiration in diminishing the negative effects of binaphthoquinones on cellular growth. Among the most sensitive strains, several are involved in ubiquinone synthesis. Ubiquinone is a monobenzoquinone present in the mitochondria of most eukaryotic cells as a part of the electron transport chain which participates in aerobic cellular respiration 
. Ubiquinone possesses antioxidant properties and disruption of ubiquinone synthesis leads to increased ROS production as seen in our binaphthoquinones sensitive mutants 
. Disruption of electron flow, such as would occur in ubiquinone deficient cells, would be expected to result in a backlog of electrons along the NADH utilizing electron transport pathway and this likely synergizes with increased ROS production by biquinones to cause cytotoxicity. Accordingly we observed substantial increases in ROS production compared to wild type when the ubiquinone synthesis mutant coq5
Δ was treated with binaphthoquinones.
To determine whether binaphthoquinones affect the mitochondrial membrane potential, we studied the ability of BiQs to depolarize mitochondrial membrane potential in both dextrose and glycerol-containing media. All tested BiQs were capable of depolarizing mitochondrial membrane in both media, though the effect was stronger in nonfermentable media. Attenuation of the effect of BiQs on depolarizing mitochondrial membrane potential by dextrose is possibly due to the partial repression of the activity of respiration enzymes, including NDE1
, by glucose 
. Interestingly, at IC50
concentrations of binaphthoquinones the inhibition of respiratory chain is most noticeable in BiQ11. This might be due to the presence of the pyran ring in this molecule which increases its lipophilicity.
Furthermore, binaphthoquinones were found to be capable of inducing oxidative stress in yeast cells by increasing ROS production. Additionally, binaphthoquinone cytotoxicity was abolished in mitochondrial DNA deficient yeast strains which lack critical respiratory chain catalytic subunits and have diminished ROS production 
A screen for deletion mutants resistant to binaphthoquinones identified NDE1
, a mitochondrial external NADH dehydrogenase (a type II NAD(P)H:quinone oxidoreductase) that catalyzes the oxidation of cytosolic NADH. Nde1p and Nde2p function primarily to provide cytosolic NADH to the mitochondrial respiratory chain 
. Our findings suggest NDE1
is involved in bioactivation of BiQs. Deletion of NDE1
increased resistance of yeast to binaphthoquinone action, while overexpression of NDE1
has an opposite effect. Interestingly, and in contrast to nde1
Δ, the ndi1
Δ strain demonstrated sensitivity to BiQs and this was not reproduced in relationship to other quinone containing drugs (doxorubicin, mitomycin C and menadione). It is possible that binaphthoquinones, due to their larger size and varied hydrophilicity are less accessible to the internal side of the mitochondrion. In addition, NDE1
may play a unique role in the bioactivation of BiQs. Indeed, another oxidoreductase, LOT6
, has been previously described as having the ability to detoxify quinones in yeast but in our studies, growth of the lot6
Δ mutant did not differ significantly from the wild type control when exposed to a range of binaphthoquinone concentrations (data not shown) 
In contrast to the mitochondria of fungi and plants, mammalian mitochondria do not harbor external NADH dehydrogenases and instead depend on redox shuttle mechanisms to couple the oxidation of cytosolic NADH to internal NADH dehydrogenases 
. Despite this, the main enzymes involved in quinone metabolism in human cells belong to the NAD(P)H:quinone acceptor oxidoreductase (NQO) gene family and, with respect to binaphthoquinone metabolism, may function similarly to NDE1
. For example, in last two decades, attention has been given to beta-lapachone, an ortho-naphtoquinone antineoplastic drug, and other quinone based drugs which take advantage of NQO1 overexpression in human cancers and selectively target them 
. NQO1 reduces beta-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to fully oxidized parent molecule, perpetuating a futile redox cycle. Deficiency or inhibition of NQO1 renders cells resistant to beta-lapachone 
. Additionally, our preliminary data in human cells overexpressing NQO1 show increased sensitivity of these cells to both beta-lapachone and binaphthoquinones (unpublished results). Based on these findings, we propose that fully oxidized binaphthoquinones undergo enzymatic reduction by NAD(P)H:quinone oxidoreductases to fully reduced form of bi-hydronaphthoquinones. Subsequently, through a series of oxidation steps, hydronaphthoquinones convert to semiquinones and ultimately back to oxidized binaphthoquinones. These stepwise oxidations culminates in ROS generation ().
Proposed mechanism of binaphthoquinones redox cycling.
Compared to other binaphthoquinones and quinones tested in this study, BiQ7 shows the most potent cytotoxic activity. Several observations might explain this phenomenon. Because BiQ7 possesses a hydroxyl group at the 5-position, it is able to form an intramolecular hydrogen bond with the carbonyl group's oxygen. Interestingly, previous studies of monomeric naphthoquinones with a hydroxyl group at position 5 on the aromatic ring have also shown an increase in toxicity toward rat hepatocytes compared to other naphthoquinones 
. There are several other potential factors that might have contributed to the higher potency of BiQ7 and quinone drugs with hydroxyl group at 5-position: 1) increased efficiency of redox cycling in BiQ7, 2) increased stability of the semiquinone derived from 5-hydroxy-1,4-naphthoquinone as compared to 1,4-naphthoquinone, which may lead to a higher semiquinone concentration and thereby a higher rate of autoxidation, 3) stabilization of other BiQs after deprotonation of their core hydroxyl groups, which BiQ7 lacks, and donation of the electrons from the deprotonized oxygen to the quinone ring to form tautomers, and 4) better utilization of oxidoreductases 
In conclusion, here we use high throughput screens in yeast to elucidate the basic cellular mechanisms mediating binaphthoquinone cytotoxicity. We find that treatment with binaphthoquinones depolarizes mitochondrial membranes and results in the generation of ROS. Accordingly, binaphthoquinone cytotoxicity can be abrogated in yeast mitochondrial DNA deficient mutants. Furthermore, we demonstrate the dependency of binaphthoquinone cytotoxicity on NDE1 and the ability to sensitize yeast to binaphthoquinones by overexpression of this enzyme. These mechanisms are likely paralleled in mammalian cells and manipulation of these pathways may allow for enhanced use of these drugs as therapeutic agents.