These should be the best of times for drug companies targeting the cell's garbage disposal system. The 2004 chemistry Nobel Prize affirmed for the world the importance of the system, the ubiquitinproteasome pathway, in human biology and cancer. The proteasome inhibitor Velcade (bortezomib), approved by the U.S. Food and Drug Administration in May 2003 for treating multiple myeloma, heralded an entirely new class of cancer drugs. In particular, E3 ubiquitin ligases, which catalyze the transfer of ubiquitin chains to proteins, marking them for degradation in the proteasome, offered seemingly ideal drug targets.
"Because each E3 is responsible for the destruction of a small number of proteins, specific inhibitors of E3s should be highly specific drugs with few side effects," J. Wade Harper, Ph.D., of Harvard University, wrote in Scientific American 4 years ago.
That optimism was premature. No known E3 inhibitor has yet reached the clinic or even appears to be close. Millennium Pharmaceuticals, which markets Velcade, is focusing mainly on other, less specific elements of the pathway. Rigel Pharmaceuticals has put two key E3 programs on hold, and few other companies are actively pursuing ligase inhibitors (see box, p. 167). "It's probably going to take a real visionary group that's willing to take a huge risk ... to make a breakthrough," said Ray Deshaies, Ph.D., of the California Institute of Technology in Pasadena.
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Biological complexity, more than any human failing, accounts for the retreat. Until the late 1970s scientists gave little thought to how cells dispose of their trash. In 1978, Israeli scientists Avram Hershko, M.D., Ph.D., and Aaron Ciechanover, M.D., D.Sc., baked some cells for a few days and purified the remains, thus discoveringalmost by accidentthe protein ubiquitin. (Hershko and Ciechanover, along with Irwin Rose, Ph.D., were rewarded with the 2004 Nobel Prize.) Six years later, Massachusetts Institute of Technology's Alexander Varshavsky, Ph.D., showed that the pathway was fundamental for protein turnover and the cell cycle in mammals.
From these beginnings, a regulatory system of almost dismaying complexity has been gradually revealed. The E1 enzyme activates ubiquitin and connects it to an E2, and the E3 facilitates the transfer of the activated ubiquitin to the protein, which is eventually marked for degradation in the proteasome by a ubiquitin chain. Researchers have now identified about 50 E2s and more than 500 E3sastounding numbers that do not include the 70 deubiquitinating enzymes and hundreds of ubiquitin-like proteins. The process of sorting out their individual functions has barely begun.
Some E3 ubiquitin ligases emerged as tempting anticancer targets. The Skp2 ubiquitin ligase, for example, causes the degradation of the key tumor suppressor gene p27, so Skp2 inhibitors should have antitumor effects. The anaphase promoting complex (APC) is an E3 that leads to degradation of key cell cycle proteins and is responsible for completing the final steps of mitosis. Blocking APC, the theory goes, would halt cell division. And the Mdm2 ubiquitin ligase causes degradation of p53, a major tumor suppressor. Inhibiting Mdm2 should elevate p53 levels, driving damaged cancer cells into apoptosis. Skp2 and Mdm2 are overexpressed in various tumors. "Conventional wisdom is that ... ligases must even be better as targets than the proteasome," said Deshaies.
But obstacles abound. Each ligase degrades multiple proteins, so inhibition may have unexpected effects. Skp2, for example, degrades the Myc oncogene in addition to p27, so blocking Skp2 might make tumors more aggressive, not less. "It's very difficult to predict what [effect] any one perturbation would have until you actually do the perturbation," said Deshaies.
Taking p53 to New Levels
The most popular ligase target for cancer is Mdm2, which Allan Weissman, M.D., and Karen Vousden, Ph.D., of the National Cancer Institute, demonstrated in 2000 to be a ubiquitin ligase that degrades p53. Vousden, now director of the Beatson Institute for Cancer Research in Glasgow, Scotland, has been working with Weissman, chief of the NCI's Laboratory of Protein Dynamics and Signaling, and biotech company Meso Scale Discovery to screen for Mdm2 inhibitors, and recently found some promising compounds. The group will now optimize them for bioavailability and potency.
But Mdm2 inhibitors won't necessarily work in people. In theory, blocking Mdm2 will work only in tumor cells harboring wild-type p53, since it doesn't make sense to elevate mutant, deactivated p53. And since normal cells also express wild-type p53, Mdm2 inhibitors might drive normal cells into apoptosis. But that's unlikely, in Weissman's view. "Tumor cells tend to apoptose, whereas normal cells tend to growth arrest," he said. Another worry is that Mdm2 inhibitors will block Mdm2 autoubiquitination and self-degradation, paradoxically elevating Mdm2. Mdm2 binding to newly elevated p53 may then block p53's tumor suppressor function. "That is a real concern that we have," said Weissman, adding that their experiments in tumor cells so far are reassuring.
There is a broader concern: no one knows how to target E3 ligases with drugs. Kinase inhibitors such as imatinib mesylate (Gleevec) work simply by blocking the kinase ATP binding site, a straightforward task for medicinal chemists. But an E3 ligase "is a difficult target," said Julian Adams, Ph.D., Velcade's inventor, now at Infinity Pharmaceuticals. "It's a protein interaction target ... Ubiquitination itself is not a classical enzymatic reaction with a discreet catalytic site." Proteinprotein interactions are difficult to block with small molecules.
Last February in Science, Roche revealed Mdm2 inhibitors called Nutlins. Nutlins do block a proteinprotein interactionan important precedentbut only at very high concentrations, with antitumor activity in animals that one observer calls "rancid." Whether Nutlins have a future is widely questioned, because drug companies seldom publish on new drugs before they enter the clinic. "Is [the Science paper] a birth announcementor a death announcement?" asked Deshaies. ("The program is still ongoing," said Roche public affairs director Darien Wilson.) A molecule called Rita, reported by Karolinska Institute scientists in the December issue of Nature Medicine, functions similarly. Neither drug targets the active site of the enzyme, the traditional point of attack, so neither offers any lessons for targeting ligases as a class.
"We don't really understand very well how these enzymes work right now," said Deshaies. One theory is that the E3 changes the E2's conformation upon docking, somehow enabling transfer of ubiquitin to the protein substrate. Don Payan M.D., Rigel's chief scientific officer, calls this "a reasonable guess," but no one knows for sure, and details are completely lacking. "Maybe one of the groups that's doing this will get lucky [and] get a molecule that actually interferes with a catalytic mechanism," said Deshaies. "That may actually teach us something about how these things work.
"Somebody's going to have to be a hero to maybe come up with a general way to inhibit ligases," he added. "It would really be a groundbreaking, pathbreaking development."
Easier Targets, Messier Drugs?
But most companies are turning away from ligases, focusing instead on elements of the protein destruction pathway viewed as more "druggable." Unfortunately, these targets are less specific than E3s and less likely to generate nontoxic therapies. Millennium, for example, is going after the Nedd8 activating enzyme (Nae1). Nedd8 is a small ubiquitin-like protein that is required for the function of cullins, proteins common to many ubiquitin ligases. "Because the Nedd8 activating enzyme requires ATP, we feel that that's a really attractive approach," said Mark Rolfe, Ph.D., Millennium's senior director of discovery research. ATP blockade also worked for kinases, the thinking goes. But there are hundreds of cullin-based ligases, so Nae1 inhibitors wouldn't be very specific, although they would be somewhat more specific than Velcade.
Even less specific than Nae1 inhibitors are inhibitors of Rpn11, the metalloprotease-like component of the proteasome lid. Rpn11 removes ubiquitins from proteins hurtling into the proteasome and is required for the proteasome to function properly. Metalloproteases are druggable, but Rpn11 inhibitors are likely to be scarcely more specific than Velcade, if at all. "You may get a slightly different effect," said Rolfe.
Another class of emerging targets are the deubiquitinating enzymes, or DUBs. These cysteine proteases are druggable, but with only 70 DUBs identified, they are vastly less specific than ubiquitin ligases, and much less understood: Whether any DUBs are overexpressed in tumors remains unknown. "But the likelihood that you could find drugs is probably quite high," said Rolfe. On the other hand, designing inhibitors capable of distinguishing between the DUBs will be hard.
Finally, companies like Millennium and Proteolix are looking for novel proteasome inhibitors in the hope that a different version will avoid some of Velcade's toxicity. Velcade does need improvementit doesn't work in solid tumors, and it has serious side effects, especially neuropathy. Comparing it to standard chemotherapy, "it's not the worst drug, but not the most benign one," said University of North Carolina hematologist Robert Orlowski, M.D., Ph.D. But if the side effects are the result of proteasome inhibition itself and not Velcade's chemistry, Orlowski adds, then novel inhibitors won't be any less toxic.
Ubiquitin ligase inhibitors remain the best hope for well-tolerated, targeted therapies. But with most companies on the sidelines, the future of ubiquitin ligase inhibitors is at risk. "Investors have gotten much more conservative," said Deshaies. "It's very difficult to develop a completely new class of drug." Rigel is one of the few companies committed to ligases, signing a collaborative ligase development agreement with Merck last October. "[Ligases] are today where kinases were 10 to 15 years ago," said Rigel's Payan. "We're sticking our neck way out there. And, you know, sometimes you've got to stand up and say, We're going to try something really new."
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