Testing of compound toxicity takes place during lead selection, allowing safety liabilities to be identified early in the drug discovery process. AstraZeneca’s mitochondrial toxicity screening cascade utilises two cell based assays to detect drug induced mitochondrial toxicity: the high throughput HEP-G2 Mitotox assay (measures ATP depletion) and the low throughput Seahorse assay (measures oxygen consumption).

A number of alternate cell based assays: THP-1 Mitotox and Mitoglo were evaluated to ascertain if changing the cascade would result in the detection of more mitotoxic compounds. THP-1 cells were successfully adapted to the necessary media conditions required for mitochondrial toxicity screening and expected differential growth rates observed. 37 compounds were then screened (n=3) in the Mitotox assay, changing the cell line from HEP-G2 to THP-1 resulted in the detection of fewer mitotoxic compounds, thus HEP-G2 cells were more sensitive to mitochondrial insult. Data obtained for the same compound set in the Mitoglo assay evaluation indicated that introducing the assay into the cascade would not increase the capability to detect both mito and cytotoxins. An example of structure activity relationship is shown where structures of AstraZeneca internal compounds were altered; this resulted in the elimination of a safety liability in the mitotox assay but not the seahorse assay, supporting the view that the change in oxygen consumption is a more sensitive endpoint of mitotoxicity than ATP depletion. The structure study illustrates the need to introduce a higher throughput oxygen consumption assay into the screening cascade for routine screening.