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MT-OXC MitoTox™ Complete OXPHOS Activity Assay
Overview
Drugs can bind to or alter key metabolic enzymes, thus inhibiting their activity, and among the most critical metabolic enzymes are the complexes involved in the electron transport chain and ATP synthesis in oxidative phophorylation (OXPHOS).
Assay Details
The assays measure the effects of drugs added directly to the 5 OXPHOS complexes. Complexes I,IV and V are each immunocaptured from bovine heart mitochondria in 96-well plates, and their activities measured by simple spectrophotometric assays, made possible because other enzymes competing for substrate and product are now absent. Complexes II+III, which utilize a unique substrate (succinate), are measured spectrophotometrically in a 96-well plate in provided mitochondrial membranes.
Positive controls are provided for Complex I (rotenone), Complexes II+III (antimycin), and Complex V (oligomycin). The recommended positive control for Complex IV is potassium cyanide, and this compound must be provided by the customer.
Figure 1. Some drugs have inhibitory effects on a single OXPHOS complex but many affect mutliple complexes. This is evident with tamoxifen which inhibits complexes II+III, IV and V with different IC50 values. The fact that complex I is not inhibited significantly rules out that the drug is acting by inhibiting ubiquinone binding. Note that complex V, the ATP synthase, is not fully inhibited. The approx 20% residual activity is characteristic of a number of inhibitors of this enzyme that block cooperativity between the 3 active sites e.g. aurovertin.
Product References
Nadanaciva, S. et al., Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays, Toxicology in Vitro, Volume 21, Issue 5, August 2007: 902-911.
Nadanaciva, S. et al., Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration, Toxicology and Applied Pharmacology, Volume 223, Issue 3, 15 September 2007: 277-287.
Dykens, J., et al., Strategies to reduce late-stage drug attrition due to mitochondrial toxicity. Expert Rev Mol Diagn. 2007 Mar;7(2):161-75.
Frequently Asked Questions
Q1. Why use bovine heart mitochondria for the MT-OXC assays?
A1. Bovine heart tissue is readily available and can be obtained very fresh. It is a rich source of mitochondria which shows reproducible properties from one batch of tissue to another. Mitochondria from human tissues are more difficult to obtain and are subject to degradation post mortem. Rodent mitochondria are obtained in much smaller yields and differ from batch to batch, possibly related to diet, etc.
Many different types of study have shown the strong correlation between observations made on bovine material and human. The similarity in stoichiometry of the OXPHOS components and their >95% sequence similarity add additional support for using bovine heart mitochondria as a model for drug effects on OXPHOS.
Q2. Can the MT-OXC assays be used to detect a compound's toxicity in vivo after animal dosing?
A2. Sometimes. The major issue is the binding affinity of the compound with the OXPHOS complexes. Most compounds that enter the cell will enter mitochondria, and some (in particular weak, hydrophobic acids) can be concentrated in the organelle for reaction with the OXPHOS machinery. In evaluating such toxicity, the problem is that the tissue of interest must be disrupted and solubilized for analysis, and this dilutes the cell extract and thereby affects the drug binding. We have been able to detect rotenone inhibition of Complex I in animals does with the drug, but the IC50 for this complex is in the nanomolar range. The inhibition of Complex I by nefazodone, which is evident in cell culture experiments (IC50 in the micromolar range), is not seen in animal dosing experiments. We are currently working to define conditions of minimal dilution of samples for the assays.
Q3. Are there secondary consequences of the inhibition of one or more of the OXPHOS complexes that can contribute to the overall effect of my drug on cells or tissues?
A3. Yes. Reduced activity of Complexes I and III has been shown to result in increased release of damaging oxidative and nitrative free radicals, which can be detected through post-translational modification of mitochondrial and cytosolic proteins using the MitoTox™ OxStress™ assay. Also, at a critical threshold of inhibition of energy production, apoptosis is induced in many cell types, which is detectable using the MitoTox™ ApoTox™ assay. Important to note: when the drug alters the amount of an enzyme rather than or in addition to an effect on activity, this can be detected in both in vitro and in vivo dosing studies using the MitoTox™ MitoBiogenesis&trade and MitoTox™ Protein Expression assays.
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