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

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.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development

“…Compared to ADEP1, a known activator of bacterial ClpP and human CLPP (Brötz-Oesterhelt et al 2005;Wong et al 2018), the imipridones were at least 20-fold more potent in these in vitro assays ( Figure S1D). These results demonstrate that ONC201 and ONC212 inhibit human cell proliferation by inducing unregulated CLPP proteolytic activity, as recently shown in other studies (Graves et al 2019;Ishizawa et al 2019).…”

“…CLPX also performs chaperone functions independently of CLPP, as it promotes heme biosynthesis and enhances the DNA-binding activity of TFAM, a mitochondrial transcription factor (Kasashima et al 2012;Yien et al 2017). TFAM itself was also depleted in response to ONC212, in agreement with previous studies in other cell lines (Greer et al 2018;Graves et al 2019). Consistent with previous reports of mitochondrial defects caused by ONC201 treatment in various cell lines (Greer et al 2018;Ishizawa et al 2019), we found that both ONC201 and ONC212, but not an inactive isomer of ONC201, caused a collapse of mitochondrial membrane potential and an increase in mitochondrial reactive oxygen species levels in NALM-6 cells ( Figure S3).…”

“…HRP-conjugated goat anti-rabbit or goat anti-mouse (Jackson ImmunoResearch Laboratories) secondary antibodies were detected with Ultrascence Western Substrate (FroggaBio) and imaged on a ChemiDoc MP (Bio-Rad, Hercules, CA). In vitro degradation assays were performed with purified recombinant human CLPP (Profoldin) or E. coli ClpP (Li et al 2010) at 37°in 384-well plates in triplicate using fluorescein isothiocyanate (FITC)-labeled casein (FITC-casein) or N-acetyl-Trp-Leu-Ala-7-amino-4-methylcoumarin (Ac-WLA-AMC) substrates (Graves et al 2019). Fluorescence intensity was measured at l ex /l em of 485/528 or 350/460 nm for FITC-casein and Ac-WLA-AMC, respectively.…”

“…Cell biological characterization suggests that ONC201 disrupts mitochondrial function and that cancer cells that do not depend on mitochondrial respiration are ONC201-insensitive (Greer et al 2018). It has recently been shown that ONC201 and other imipridones directly bind and activate CLPP in the absence of CLPX, resulting in the degradation of selected mitochondrial proteins and loss of mitochondrial integrity (Graves et al 2019;Ishizawa et al 2019).…”

Impact of pharmacological agents on mitochondrial function: a growing opportunity?

“…Follow-up studies discovered that ONC201 did not directly activate TRAIL in all of the cancer cells in which it was effective but was impairing mitochondrial function [75]. Recent data from two studies identified the target of ONC201 as being the mitochondrial CLPP protease which is activated by ONC201 and results in increased cancer cell killing [76,77].…”