ABOVE: A model of ONC201 (red) binding to ClpP (gray) based on the crystal structureMICHAEL ANDREEFF

Scientists have known that the cancer drug ONC201 blocks cells from proliferating and kills tumors in cell and animal models—but they haven’t known exactly how it works, or what its molecular target is. Nevertheless, multiple clinical trials of the drug, in various cancer types, are underway. In 2018, ONC201, made by the Philadelphia-based company Oncoceutics, received a fast track designation from the Food and Drug Administration, meaning it gets expedited review, for the treatment of certain gliomas in adults. Now, two studies published independently this month reveal the drug’s mechanism of action: ONC201 works by activating ClpP, an enzyme that chews up misfolded proteins in mitochondria. 

“It’s important to understand how a drug works to understand how best to use it in patients, and in these two studies, they identified ClpP, a mitochondrial protease, as an important target of ONC201, which seems to account for some if not most of its clinical activity,” says Stanley Lipkowitz, chief of the Women’s Malignancy Branch of the National Cancer Institute’s Center for Cancer Research, who was not involved in either study but is currently collaborating with Lee Graves, who led one of the studies. 

ONC201 was originally identified as a potential cancer drug in a screen for molecules that induce the transcription of TRAIL, a gene that leads to apoptosis in tumors. Yet ONC201 doesn’t activate TRAIL in all of the cancer cells against which it is effective: In a 2018 paper, Yoshimi Greer and colleagues in the Lipkowitz lab reported that ONC201 worked against several lines of breast cancer cells—without upregulating TRAIL. Instead, they found, ONC201 was hindering the cancers’ mitochondrial function. But just how ONC201 was doing its mitochondrial damage was unclear. 

To find out, Graves, a University of North Carolina School of Medicine pharmacologist, and colleagues studied ONC201 as well as very similar molecules, called ONC201 analogs, generated by the Chapel Hill–based company Madera Therapeutics, of which one of Graves’s coauthors is president. They attached the ONC201 analog TR-80 to agarose beads to construct a column and ran the innards of HeLa cells, the immortal cervical cancer–derived cell line, through it to see what would stick. 

“We came at it from an old-fashioned affinity-chromatography approach—you know, ‘let’s make some bait and go fishing and see what we catch,’” Graves tells The Scientist. Mass spectrometry identified the protein they caught as ClpP. They repeated the experiments using cell lysates from other cancers, including breast, pancreatic, and lung, and in every case, they found that the ONC201 analogs bound ClpP, they report in ACS Chemical Biology. 

For all these new insights into how the drug works molecularly, one big mystery remains: how it targets cancer cells without harming healthy ones.



Graves’s work hasn’t demonstrated that ONC201 kills cancer cells, but it does prevent their growth. All of the compounds his team tested increased ClpP’s ability to cleave peptides, with the analogs being even more potent than ONC201. Knocking down ClpP production in breast cancer cells cancelled out the effect of the drugs. 

The other study, by Michael Andreeff, a researcher at the University of Texas MD Anderson Cancer Center, and colleagues, examined ONC201 and ONC212, another Oncoceutics drug, which is chemically identical to one of the analogs from Graves’s study, and reported their findings in Cancer Cell. Oncoceutics, in which Andreeff holds stock, provided the compounds for this study and one author is affiliated with the company. 

Andreeff’s study confirmed many of the findings from Graves’s team: ONC201 and ONC212 activate and bind to ClpP and are able to kill cancer cells—a stronger anti-tumor effect than Graves found—but only if they contain ClpP. “The more ClpP we have in cells, the more active the drug is,” says Andreeff.

Andreeff’s group also described the crystal structures of ONC201 and ONC212 bound to ClpP, which they compared to the crystal structure of ClpP alone. They found that when the drugs bind the protease, which is shaped like a barrel, the pore through which proteins enter widens and vent-like openings appear on the structure’s sides, presumably to release fragments of digested proteins. “So suddenly this whole thing becomes active by opening up its mouth as a channel, but also opening up the efflux side channels,” says Andreeff.  

He and colleagues also found that ONC201 treatment results in the degradation of certain proteins involved in the respiratory chain—part of the process by which cells convert glucose into energy—and impairs mitochondrial function in cancer cells. 

For all these new insights into how the drug works molecularly, one big mystery remains: how it targets cancer cells without harming healthy ones. “We don’t know,” says Andreeff, who adds that it may have to do with an overproduction of ClpP in cancer cells. 

“I think it comes back to the possibility that the tumor cells have a metabolic vulnerability,” says Graves.  He believes that the mitochondria of cancer cells are altered in such a way that they are more susceptible to ONC201 than the mitochondria of healthy cells.

Now that scientists know ONC201 acts on ClpP, they can look for other molecules that do the same thing and could be potential cancer drugs, says Graves. They could also look for drugs targeting other mitochondrial proteases, Andreeff adds. 

The knowledge that ONC201 targets ClpP and is more effective against tumors with higher ClpP levels could help scientists identify patients whose tumors would be most likely to respond to the drug, says Andreeff. It would also let scientists evaluate whether or not the drug is hitting its target—activating ClpP, in this case—in patients, Lipkowitz adds. 

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P.R. Graves et al., “Mitochondrial protease ClpP is a target for the anticancer compounds ONC201 and related analogues,” ACS Chemical Biology, doi:10.1021/acschembio.9b00222, 2019. 

J. Ishizawa et al., “Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality,” Cancer Cell, doi:10.1016/j.ccell.2019.03.014, 2019.

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