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New Method Found To Trigger Cancer Cell Suicide
Paul Hergenrother, Ph.D., is a 34-year-old chemist whose University of Illinois lab in Urbana is working on solving antibiotic resistance, neurodegenerative disease, and cancer. These are among the toughest unsolved problems in medicine, but that's why Hergenrother is drawn to them. "If we're all going to spend a lot of time doing something, we might as well do something important," he said.
The common element in Hergenrother's work is the use of small molecules to define new targets and create potential new drugs. In the October issue of Nature Chemical Biology, his group described what appears to be the first small-molecule activator of procaspase-3, a key enzyme in the apoptotic cascade that leads cells to commit suicide. They showed that the compound can kill cancer cells in cell culture and in primary human tumors, while leaving normal cells intact. The compound, PAC-1, also arrests tumor growth in mice. "It's exciting," said apoptosis researcher John Reed, M.D., Ph.D., the president of the Burnham Institute for Medical Research in La Jolla, Calif. "It's something that many of us in the apoptosis community have thought about."
For almost 20 years, cancer researchers have been intrigued by the prospect of using a drug to induce apoptosis in cancer cells, killing them with minimal side effects. Apoptosis, or programmed cell death, is a natural process that rids the body of cells to prevent uncontrolled growth. This process is blocked in cancer. In recent years, at least nine drugs designed to induce apoptosis have made their way to clinical trials, although it's too early to judge their effectiveness. All of them act to indirectly activate the so-called executioner caspases, which cut open other cellular proteins vital for cell survival, leading the cell to commit suicide. But Hergenrother's compound is the first known to directly activate caspase-3, the key executioner caspase.
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More Target, Less Drug
Caspase-3 is an obvious target. The enzyme sits at the downstream junction of the so-called intrinsic and extrinsic apoptotic signaling pathways, which are triggered by mitochondrial proteins and cell surface death receptors, respectively. Activation of caspase-3 from procaspase-3, the inactive form of the enzyme, is "the point of no return," Hergenrother explained. "Once caspase-3 is activated, there [are] a hundred-plus substrates that it cleaves in the cell. And the cell's going to die."
Even if the upstream apoptotic pathways are disabled through mutations or other abnormalities, Hergenrother added, caspase-3 activators should kill the cells because the enzyme itself remains inactive but intact. Based on this premise, Hergenrother screened more than 20,000 compounds collected by his lab for the ability to activate procaspase-3. The laborious effort yielded one compound: PAC-1.
As hoped, the compound proved effective against cancer cells. PAC-1 killed tumor cell lines and tumor cells from primary tumor samples at concentrations much lower than those able to kill normal cells, and it blocked growth of tumors in mice—all without apparent side effects.
Given that most normal cells also express procaspase-3, why isn't PAC-1 more toxic? Hergenrother notes that many cancer cells contain elevated levels of procaspase-3, and more targets directly translate to greater sensitivity to the drug. "The more [caspase-3] you have, the less compound you need, basically," he explained. "That gives you the therapeutic window." Hergenrother found that it took up to 2,000 times as much drug to kill normal cells in culture than cancer cells, a ratio which, if it translates to actual human cancers, could provide a relatively nontoxic therapy.
Pondering the Risks
But cell culture experiments poorly predict a drug's effect in people, and activating a powerful cell killer like caspase-3 with a drug carries a strong theoretical risk. "The biggest concern with a strategy like that is safety and whether such compounds will also induce normal cells to kill themselves," Reed said. Most other antiapoptotic drugs, Reed added, are designed to remove blocks to apoptosis. "You're sort of removing the brakes and allowing the natural pathway to occur when it should occur, or to try to nudge it along with complementary chemotherapy," he said. "This [PAC-1 approach], though, is going in and triggering the program. Theoretically, any cell should be a potential target for this—any cell that expresses caspase-3, and most cells do."
One way to lessen the risk of side effects might be to give the drug only to patients whose tumors express high levels of procaspase-3, since they're most likely to benefit at nontoxic doses. Many breast, colon, and lung cancers, among others, show such elevated levels, for unknown reasons. In theory, biopsy samples could be easily tested to identify such patients for PAC-1 therapy. Reed said more work needs to be done to define the range of cancers that overexpress procaspase-3. But "there's plenty of reason to go forward just based on what we know," Hergenrother said.
Hergenrother's group saw no side effects in mice treated with PAC-1, but it is now dosing mice with higher PAC-1 concentrations to see what kind of toxicity emerges. He's also hoping to see their tumors disappear. (PAC-1, so far, has been able to only halt tumor growth, not eliminate the tumors.) The group is also modifying the PAC-1 molecule to increase its potency and improve its ability to reach tumors intact. Reed speculated that the drug may already be potent enough. "There's a decent argument to be made that there's sort of a ‘sweet spot’ in terms of potency with a target like this," he said. "Highly potent activators might actually be toxic, whereas weakly activating might be tolerated." One promising idea is to combine PAC-1 with inhibitors of X-linked inhibitor of apoptosis (XIAP), since XIAP inhibitors target a pool of already activated caspase-3, so the two drugs would activate the enzyme at different stages. At least two XIAP inhibitors are already in clinical trials.
Feeling Validated
Alone or in combination, PAC-1 has the potential to be used as cancer therapy, said Hergenrother, but he knows the odds are long against this compound's becoming a drug. Of cancer drugs, 95% fail when tried in humans, according to a recent review in Nature Reviews Cancer, and PAC-1 is still a long way from the clinic. As an academic investigator, though, his greater goal is to prove that caspase-3 activators in general are viable drug candidates. "Then others can jump on it," he said, and make even better compounds. Several drug companies have already expressed interest in PAC-1, he said.
And Hergenrother hopes that drug companies will start looking for activators of other inactive protein-cutting enzymes (proenzyme proteases) now that he has shown that it can be done. His own group is using the same screening strategy to look for specific small-molecule activators of at least two other executioner caspases, caspase-6 and caspase-7. And there are dozens of other proteases that, in theory, could be easily turned on with a small-molecule drug. These compounds, if nothing else, could evolve into great laboratory tools for understanding basic biology, since the function of many of these enzymes is poorly understood. "[RNA interference] is great. You can turn things off," Hergenrother observed. "But there's no equivalent tool to turn something on. ... Can all proenzymes be autoactivated with some small molecule? I don't know."
Until PAC-1, the question couldn't be asked. Now it's just another big problem that Hergenrother's lab is trying to solve.
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