Development of Novel Calpain Protease Inhibitors Using Phage Peptide Display

Many researchers have shown that over activation of the calcium-dependent proteases, the calpains, likely contributes to the cytoskeletal disruption and cell death that occurs following a stroke or heart attack. Therefore, it is critical to develop therapeutic agents to stem this over activation and reduce or prevent the subsequent cellular and tissue destruction. In preliminary work, we have used phage peptide display to identify a class of peptides that bind to calpain in a calcium-dependent manner. Interestingly, the representative peptide, LSEAL, has homology to a conserved repetitive sequence found in calpastatin. Hence, we hypothesize that we have identified a novel class of calpastatin peptide mimetic. The goal of this proposal is to develop and test a novel family of calpain inhibitor for potential clinical use in the treatment of ischemic stroke or heart attack by carrying out the following specific aims: Specific Aim 1: Use phage peptide display to identify peptides that bind calpain in a calcium-dependent manner. Specific Aim 2: To test the hypothesis that phage peptide display-clerived peptides are novel modulators of calpain activity in vitro. In preliminary work, we have found that a representative peptide from the initial library (LSEAL) is a potent inhibitor of calpain's actions on proteolysis of the microtubule-associated protein tau, a known calpain substrate. Specific Aim 3: To evaluate the hypothesis that the novel peptide LSEAL is a peptide mimetic of calpastatin. BIAcore technology will be used in addition to Elisa based assays, to assay for calcium-dependent competitive binding between calpastatin and LSEAL. Specific Aim 4: To evaluate the specific of LSEAL and related peptides for calpain versus other calcium binding proteins and other proteases using Elisa based assays and BIAcore technology. Specific Aim 5: To evaluate the hypothesis that LSEAL and related peptides act as novel calpain inhibitors in a cellular context. In preliminary work, we have used cultured cortical neurons to demonstrate that cell death caused by increased cytosolic calcium mediated by either ionomycin or UV light is prevented by treatment with LSEAL and not by a scrambled sequence, negative control peptide, ELLAS. These studies will evaluate key aspects of LSEAL actions and provide important evidence to support the future development of this potentially new class of calpain inhibitor for use in the treatment of stroke and for heart attack.