12 June 2012. Taking the road less traveled may prove beneficial when it comes to certain therapeutic targets. In the June 10 Nature Chemical Biology, researchers published a new way to tackle a cell death enzyme that causes neurodegeneration and may lead to Alzheimer's, Huntington's, and other diseases. Instead of targeting the cleavage site of caspase-6, the group looked for inhibitors that bind elsewhere on the protease. They found a peptide that converts the caspase to its inactive state—the zymogen—and traps it there, a bit like a wheel clamp. "To our knowledge, no one has identified ligands that bind to the zymogen," said senior author Rami Hannoush, Genentech, South San Francisco, California.
Caspases are a family of proteases that induce programmed cell death, or apoptosis. Though caspase 6’s role in apoptosis is unclear (unlike that of caspase-3), Genentech researchers previously reported that the protease facilitates a death cascade sparked by the N-terminal of APP (see ARF related news story). It may also regulate formation of toxic proteins in HD (see ARF related news story on Graham et al., 2006) and early neurodegeneration in AD (see Klaiman et al., 2008).
Andrea LeBlanc, McGill University, Montreal, Canada, showed that the caspase is elevated in the brains of people with AD or mild cognitive impairment (see Albrecht et al., 2007). The same study showed raised caspase-6 levels in the entorhinal cortices—where AD pathology may begin—of older, cognitively normal people, though it is not known whether those people are at greater risk for the disease. "Inhibiting caspase-6 in the entorhinal cortex before it propagates to the areas of the hippocampus might prevent the progression of Alzheimer's disease," LeBlanc told Alzforum.
Most attempts at blocking caspases have focused on their catalytic sites, which are homologous. Therefore, chronically targeting one caspase may mean blocking the others. Indeed, side effects have plagued researchers who tried to develop caspase inhibitors as drugs, and liver toxicity has prevented caspase inhibitors from becoming widely used as therapeutics. "Given the pleiotropic effects of caspases ... it is perhaps not surprising that these broad inhibitors have not yet proven effective in treating human chronic diseases," Kevin Roth, University of Alabama at Birmingham, told Alzforum in an e-mail (see full comment below).
Instead of targeting the active site, first author Karen Stanger and colleagues approached the problem from a different angle. They screened a peptide phage display library to find molecules that would bind allosterically—i.e., outside of the active site—to the zymogen, or inactive form, of caspase-6. They found a peptide—pep419—that fit the bill. Pep419 selectively inhibited caspase-6, only weakly blocked caspase-7 and -9, and had no effect on the other seven caspases. In SK-N-AS neuroblastoma cells treated with staurosporine to activate caspase-3, -6, and -7, pep419 prevented cleavage of the caspase-6-specific substrate lamin A.
How does pep419 work? Using biophysical techniques, Stanger and colleagues found that active caspase-6 exists as a dimer, while the zymogen is made up of a tetramer. They found that pep419 stabilized the latter, and also bound active caspase-6 dimers and locked them into the tetrameric zymogen-like state.
The work "demonstrates very precisely a mechanism that caspase-6 would use to regulate itself, thereby revealing the Achilles' heel by which a small molecule could block the activity of the protease," said Guy Salvesen, Sanford Burnham Medical Research Institute, La Jolla, California. In future work, the researchers could both optimize the peptide and test it in animal models of HD or AD, and use the pep419 to design a small molecule inhibitor that would be more stable in the body, he suggested.
Alternatively, researchers could use this same approach to directly screen for small molecules that bind the zymogen of caspase-6, said Hannoush. This study provides a "proof of concept that compounds exist that selectively bind to allosteric sites on the zymogen, and opens the door to screen small molecules and identify drug leads," he said.
Whether this work may translate into an AD drug remains to be seen. LeBlanc is hopeful, though admits a drug is pretty far off. "I would love for them to be able to follow through with this," he said. "But that will take many more years of work."—Gwyneth Dickey Zakaib.
Stanger K, Steffek M, Zhou L, Pozniak CD, Quan C, Franke Y, Tom J, Tam C, Elliott JM, Lewcock JW, Zhang Y, Murray J, Hannoush RN. Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization. Nat Chem Biol 2012 June 10. Advance online publication. Abstract