Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogs

ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogs (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ~50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response (ISR) protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ~10 fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- an...