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A Novel Method for Stabilizing Small Alpha-Helices Will Promote Development of Protease Inhibitors

By LabMedica International staff writers
Posted on 10 Jan 2013
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By establishing a new method for stabilizing the alpha-helix structure in a small peptide, researchers were able to design a highly specific inhibitor of the enzyme calpain.

Although the physiological role of calpains, a family of calcium-regulated enzymes, is only poorly understood, they have been shown to be active participants in processes such as cell mobility and cell cycle progression, as well as cell-type specific functions such as long-term potentiation in neurons and cell fusion in myoblasts. Other reported roles of calpains are in cell function, helping to regulate clotting and the diameter of blood vessels, and playing a role in memory. Calpains have been implicated in apoptotic cell death, and appear to be an essential component of necrosis. Calpain is also involved in skeletal muscle protein breakdown due to exercise and altered nutritional states. Overexpression of calpain has been implicated as a factor in muscular dystrophy, AIDS, Alzheimer's disease, multiple sclerosis, and cancer.

Investigators at the University of Pennsylvania (Philadelphia, USA) and collaborators at the University of California, San Francisco (USA) and Queen's University (Ontario, Canada) screened 24 commercially available cross-linking reagents before succeeding to stabilize the alpha-helix at the center of the binding site between calpain and its natural inhibitor calpastatin.

Calpastatin consists of an N-terminal domain and four repetitive calpain-inhibition domains and is involved in the proteolysis of amyloid precursor protein. The calpain/calpastatin system is involved in numerous membrane fusion events, such as neural vesicle exocytosis and platelet and red cell aggregation. The encoded protein is also thought to affect the expression levels of genes encoding structural or regulatory proteins.

The investigators examined the effects of cross-linking on the alpha-helicity of selected peptides by CD (circular dichroism) and NMR (nuclear magnetic resonance) spectroscopy and found that structurally rigid cross-linkers were best for stabilizing alpha-helices. They reported in the October 24, 2012, issue of the Journal of the American Chemical Society that they had applied this strategy to the design of inhibitors of calpain that were based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn alpha-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. They expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes, which retained the specificity of the stabilized helical inhibitor.

"We have an interest in this protein because it is important for Plasmodium development," said senior author Dr. Doron Greenbaum, assistant professor of pharmacology at the University of Pennsylvania. "We initially found that calpain played a role in parasites being able to get out of their host cell, so we became interested in inhibitor development for human calpains."

"Traditionally people thought that alpha-helices normally make horrible inhibitors because it was thought that proteases do not like to bind to them, preferring to bind motifs called a beta-sheet," said Dr. Greenbaum. "We decided to take a different tack on inhibitor development, which has traditionally been designing small peptide-like inhibitors that fit across an enzyme’s active site. We found that there was a small alpha-helix that fit into the active site of the calpain enzyme. It is the first example of an alpha-helical inhibitor of any protease. Previously no one has ever tried using an alpha-helical motif. It opens up a new way of inhibiting proteases."

Related Links:

Queen's University
University of Pennsylvania
University of California, San Francisco


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