Chemical-genetic profiling in yeast is a robust technique for exploring the mechanism of action of biologically active compounds. Profiling of compound 13 and 15, a nitroso-armed imidazo-pyridine and imidazo-pyrimidine, respectively, suggested that despite the strong likelihood that they cause oxidative stress, they might act in a mechanistically distinct manner. We provide several lines of evidence that compound 13 causes mitochondrial dysfunction, whereas compound 15 causes damage to the nuclear DNA. These different modes of action were also apparent when human cells were treated with compounds 13 and 15, illustrating the utility of chemical-genetic profiling in yeast in predicting mode of action in higher eukaryotes.
Nitroso aromatic compounds are bioactive mainly because they are readily reduced to highly reactive nitro radical anions which activate oxygen 
. Although there is a possibility that compound 13 and 15 are toxic to cells via other mechanisms, several lines of evidence suggest that compound 13 and 15 are acting as oxidizing agents in vivo. We found that 2-phenylimidazo[1,2-a
]pyridin-3-amine (compound 151), which is the reduced form of compound 13, was not active on yeast cells (). Furthermore, the antifungal activity of compound 13 and 15 could be partially suppressed by pre-treating cells to induce intracellular accumulation of reduced glutathione (Figure S2
), a protective small molecule that is part of the cellular defense against oxidative damage 
. Finally, we also found that chemical reduction of compounds 13 and 15 in vitro resulted in their inactivation (data not shown).
The chemical-genetic profile of compound 13 was significantly enriched for biological processes such as mitochondrial organization and biogenesis, and oxidative phosphorylation. This profile is reminiscent of that of H2
in which a specific requirement for an intact respiratory chain 
and a broader requirement for mitochondrial function 
was observed. Our data also suggest that compound 13 causes mitochondrial dysfunction since cells treated with compound 13 lose their peroxide tolerance, much like those treated with ethidium bromide or the ionophore FCCP. Consistent with this, compound 13 caused dramatic changes in mitochondrial morphology, resulting in extensive mitochondrial fragmentation, a phenotype known to disrupt mitochondrial activity. Maintenance of proper mitochondrial morphology is critical, and fragmentation of mitochondria is an important step in the progression of apoptosis 
Compound 15 displayed none of the mitochondria-specific characteristics seen with compound 13. The chemical-genetic profile of compound 15 showed little overlap with that of compound 13, and its profile clearly differed from other oxidizing agents examined. Few DNA repair genes have been identified in genome-wide screens with the oxidants H2
, linoleic acid 13-hydroperoxide, menadione, cumene hydroperoxide, and diamide 
. It has therefore been speculated that loss of viability following treatment with these agents is due to damage to proteins rather than to DNA 
. Our evidence that compound 15 causes DNA damage suggests that it will be a useful compound in the study of the cellular response to oxidative damage in the nucleus. Importantly, although targeting of oxidative stress to the nucleus has been reported 
, we provide evidence of targeted oxidative stress specifically causing nuclear DNA damage. Although this can be readily inferred from the importance of three DNA repair pathways in compound 15 tolerance, we also demonstrated that compound 15 causes activation of the DNA damage checkpoint and induces mutations in nuclear DNA.
The anti-proliferative and DNA damaging properties of compound 15 are shared by a number of cancer therapeutics, indicating that compound 15 or derivatives of it might be useful in this regard. It is of particular interest that tolerance of compound 15 in mammalian cells required the same DNA repair pathway, nucleotide excision repair, as was found in yeast. DNA repair pathway-specific toxicity affords the possibility of rational therapeutic approaches based on targeting cells defective in a given pathway. Additionally, synergy might be obtained by targeting multiple DNA repair pathways independently. Mitochondria are also an attractive anti-cancer target, as pharmacological modulation of mitochondrial permeability can result in apoptotic cell death 
. The mitochondrial fragmentation caused by compound 13 might reflect induction of the permeability transition pore, as has been observed with other forms of oxidative stress 
. Ongoing studies to determine the precise mechanism of action of compound 13 will reveal its potential as a therapeutic.