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.

Reference:
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

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Comments on News and Primary Papers

  1. A variety of caspases have been implicated over the last 15 years in regulating the extensive neuron degeneration that occurs in Alzheimer's disease. Originally, most investigators focused on the effector caspase, caspase-3, whose activity in many experimental systems was found to be a critical trigger for neuron apoptosis. Since neuron death is now widely recognized as a late event in the course of Alzheimer's disease pathogenesis, occurring well after synapse loss and neuronal cytoskeletal changes have occurred, the potential therapeutic utility of developing inhibitors of effector (executioner) caspases would at first glance appear limited. However, a series of studies performed over the last five to 10 years have consistently drawn attention to a potentially unique role of a second effector caspase, caspase-6, in cell death-independent neurodegeneration. Caspase-6 exists at baseline in neurons as an inactive precursor (zymogen) that is cleaved by initiator caspases into large and small subunits which together form the active caspase-6 enzyme. Unlike caspase-3, which triggers neuron death and destruction of the neuronal cell body, activated caspase-6 has been implicated in degradation of synapses and neuronal processes. Elevated levels of activated caspase-6 have been found in Alzheimer's disease brain and in the brains of patients with mild cognitive impairment prior to extensive neuron loss, suggesting that caspase-6 may play an important role in regulating the earliest pathological events in Alzheimer's disease progression. Thus, inhibition of caspase-6 activity may represent a new and important therapeutic target for attenuating the neuropathological changes observed in Alzheimer's disease, and potentially delaying or preventing neurocognitive decline.

    The article by Stanger et al. is potentially an important first step in developing just such an inhibitor. To date, there has been very limited success in developing specific inhibitors of individual caspases, since the caspases represent a relatively large family with extensive sequence and structural homology. Broad spectrum caspase inhibitors that target the active site of multiple caspases have not yet been proven therapeutically useful due to a variety of side effects. Given the pleiotropic effects of caspases and the important homeostatic function of regulated cell death in a variety of organs, it is perhaps not surprising that these broad caspase inhibitors have not yet proven effective in treating human chronic diseases.

    In the current article, Stanger et al. have taken a different approach to identifying an effective and specific caspase-6 inhibitor. Rather than targeting the active site of the "processed" active caspase-6 enzyme, they used phage display to discover peptides that bind to the caspase-6 zymogen and screened such compounds for their effects on caspase-6 enzymatic activity. They report the identification of pep419 as a selective allosteric inhibitor of caspase-6 function with minimal inhibitory activity on other caspases. Pep419 was found to selectively inhibit the degradation of a caspase-6 substrate in a cell culture model of neuronal apoptosis, and did not inhibit the degradation of caspase-3 or caspase-7 substrates in the same experimental system. Although it is unclear if pep419 can selectively inhibit caspase-6 activity in vivo, the article describes several important conceptual advances. First, selective caspase inhibitors can be developed by targeting caspase zymogens rather than the active enzymes themselves; second, the molecular mechanism by which pep419 inhibits caspase-6 activity is distinct from previously developed small molecule caspase inhibitors; and third, phage display screening against caspase zymogens may lead to the development of small molecule allosteric ligands that selectively inhibit the activity of specific caspases in vivo. Further research and drug development is essential to move these findings into clinical settings, but if effective and safe caspase-6 specific inhibitors can be developed, they would hold significant promise as Alzheimer's disease-altering agents, since increased caspase-6 activity may play an early and relatively selective role in Alzheimer's disease-related neurodegeneration prior to irreversible neuron loss.

    View all comments by Kevin Roth

References

News Citations

  1. Copper Mountain: Death and Trophin Receptors—New Insight, New Drugs?
  2. The Unkindest Cut: Caspase-6 Cleavage of Huntingtin Produces Killer Fragment

Paper Citations

  1. . Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin. Cell. 2006 Jun 16;125(6):1179-91. PubMed.
  2. . Targets of caspase-6 activity in human neurons and Alzheimer disease. Mol Cell Proteomics. 2008 Aug;7(8):1541-55. PubMed.
  3. . Activation of caspase-6 in aging and mild cognitive impairment. Am J Pathol. 2007 Apr;170(4):1200-9. PubMed.
  4. . Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization. Nat Chem Biol. 2012 Jul;8(7):655-60. PubMed.

External Citations

  1. SK-N-AS

Further Reading

Papers

  1. . Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization. Nat Chem Biol. 2012 Jul;8(7):655-60. PubMed.
  2. . Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays. J Biol Chem. 2012 May 4;287(19):15371-9. PubMed.
  3. . Rescue from excitotoxicity and axonal degeneration accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice. Hum Mol Genet. 2012 May 1;21(9):1954-67. PubMed.

Primary Papers

  1. . Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization. Nat Chem Biol. 2012 Jul;8(7):655-60. PubMed.