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Differentiating Mitochondrial Toxicity Drug Discovery New Assay Chemistry Can Identify the Type of Mechanistic Toxicity and Help Reduce Drug Failures Michele Arduengo Mitochondria are responsible for the pro- duction of >90% of mammalian cell energy and are intimately linked to the apoptosis signaling cascade. They have critical func- tions specific to certain tissues including in- volvement in hemoglobin synthesis, estrogen and testosterone production, and cholesterol metabolism. There are several mechanisms by which chemical compounds can adversely affect mitochondria. Mitochondrial DNA (mtD- NA) replication can be disrupted, as with the case of drugs like AZT that inhibit re- verse transcriptase of retroviruses. Some compounds can directly inhibit the electron transport chain or uncouple ATP synthesis from electron transport. Other compounds can inhibit the Krebs cycle, affect mitochon- drial membrane permeability or inhibit mi- tochondrial transporters. In spite of the critical roles that mitochon- dria play in all cells, mitochondrial toxicity is difficult to identify. Many of the cell lines used in high-throughput drug discovery screens are highly proliferative, immortal- ized cell lines that, in the presence of glucose, use glycolysis for energy production, despite abundant oxygen and functional mitochon- dria, a phenomenon known as the Crabtree effect. As a result these cells tend to be re- sistant to compounds that disrupt mitochon- drial oxidative respiration. Almost all cells can tolerate diminished mitochondrial membrane potential as long as minimal capacity is maintained; however, when that minimal capacity is lost, cells die rapidly via apoptosis or necrosis, and mi- tochondrial toxicity is not often identified as the underlying cause of cell death. Often with mitochondrial toxicity there is a lack of correlation between drug dose and toxic- ity, and toxicity can be missed in clinical tri- ASSAY TUTORIAL als because it is often highly dependent on individual genetics and organ history. Drug Failures Mitochondrial toxicity is implicated in the recent post-market withdrawal of several pharmaceuticals: tolcapone, tro- glitazone, amiodarone, and cerivastatin. Such post-market withdrawals are costly in terms of loss of life, detriment to patient health, as well as in financial resources. Identifying potential mitochondrial toxins as early as possible in the drug discovery process is critical. Methods to measure mitochondrial func- tion, such as monitoring oxygen consump- tion using electrodes, do not lend themselves easily to automation, and are not particu- larly well suited for drug screening activi- ties, although modifications using soluble electrodes can be used with 96-well plates, increasing throughput. However, these as- says still require isolated mitochondria. Isotope-based assays that monitor mi- tochondrial function by looking at mtD- NA replication and protein synthesis are expensive and carry with them the issues of disposal of hazardous waste. Assays us- ing phosphorescent probes to detect mito- chondrial respiration have been developed but are used most often on isolated mito- chondria. High-throughput activity assays for oxi- dative phosphorylation exist, but they rely on immunocapture. While giving a complete profile of the precise mode of action of the "mitotoxicant", these assays are used with isolated mitochondria rather than intact cells and therefore may not be the best option for early-stage drug screening activities. Oxidative Phosphorylation One way to facilitate the detection of mi- tochondrial toxicity is to conduct the toxicity assays on cells grown under conditions that shift the metabolic balance toward oxidative phosphorylation. HepG2 cells, which have high glycolytic activity, are resistant to mito- chondrial toxins under typical high-glucose culture conditions. Galactose medium renders the cells sensitive to drugs known to disrupt mitochondrial function. Indeed growing cell lines on galactose- and glucose-containing media in parallel for toxicity screens to detect mitochondrial toxins is becoming more com- mon in drug screening activities (Figure 1). For assays that measure ATP output, comparing results for the cells in the two dif- ferent growth conditions allows researchers to see decreases in ATP output that are only associated with growth in galactose-contain- ing medium, and therefore probably a result of mitochondrial dysfunction. Biologically Relevant Mitochondrial Toxicity Assay Creating a cell-based assay to monitor mi- tochondrial toxicity that overcomes the dif- ficulties in specifically detecting compounds that have deleterious effects on mitochondria 24 | October 1, 2012 | genengnews.com | Genetic Engineering & Biotechnology News