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

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

Author information

1
Department of Radiation Oncology , New York Presbyterian Brooklyn Methodist Hospital , Brooklyn , New York 11215 , United States.
2
Department of Pharmacology and the Lineberger Comprehensive Cancer Center , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.
3
Department of Microbiology and Immunology and the Lineberger Comprehensive Cancer Center , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.
4
Department of Chemistry and the Lineberger Comprehensive Cancer Center , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.
5
Madera Therapeutics LLC , Chapel Hill , North Carolina 27517 , United States.
6
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences , Pushchino 142292 , Russian Federation.

Abstract

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.

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