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

ACS Chem Biol. 2019 May 17;14(5):1020-1029. doi: 10.1021/acschembio.9b00222. Epub 2019 May 1.


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 analogues (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 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- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Chromatography, Affinity
  • Endopeptidase Clp / antagonists & inhibitors*
  • Endopeptidase Clp / genetics
  • Endopeptidase Clp / metabolism
  • Enzyme Activation
  • Gene Knockdown Techniques
  • Heterocyclic Compounds, 4 or More Rings / chemistry
  • Heterocyclic Compounds, 4 or More Rings / pharmacology*
  • Humans
  • Imidazoles
  • Mitochondria / drug effects
  • Mitochondria / enzymology
  • Pyridines
  • Pyrimidines


  • Antineoplastic Agents
  • Heterocyclic Compounds, 4 or More Rings
  • Imidazoles
  • Pyridines
  • Pyrimidines
  • TIC10 compound
  • ClpP protein, human
  • Endopeptidase Clp