Novel calpain inhibitors based on phage display

描述（由申请人提供）： 许多研究人员已经表明，钙依赖性蛋白酶（calpains）的过度激活可能导致脑损伤后发生的细胞死亡。因此，开发治疗剂以阻止这种过度活化并减少或防止随后的细胞和组织破坏是至关重要的。在初步工作中，我们已经使用噬菌体肽展示来鉴定一类以钙依赖性方式与钙蛋白酶结合的肽。有趣的是，代表性肽LSEAL与钙蛋白酶抑制蛋白中发现的保守重复序列具有同源性。因此，我们假设，我们已经确定了一类新的钙蛋白酶抑制蛋白肽模拟物。本提案的目的是确认和扩展我们的初步观察，即我们已经鉴定了一个新的钙蛋白酶抑制剂家族，通过执行以下具体目标，可用于治疗脑损伤的潜在临床用途：具体目标1”使用噬菌体肽展示来鉴定以钙依赖性方式结合钙蛋白酶的肽。我们已经确定了一个候选肽，LSEAL。增加严格性的额外文库将用于发现潜在更有效的序列。具体目标二：验证噬菌体肽展示衍生肽是钙蛋白酶活性的新型特异性抑制剂的假设，其机制与钙蛋白酶抑制剂相似。在初步工作中，我们已经发现，来自初始库（LSEAL）的代表性肽是钙蛋白酶对微管相关蛋白tau（一种已知的钙蛋白酶底物）的蛋白水解作用的有效抑制剂。除了基于Elisa的测定外，还将使用BIAcore技术来测定钙蛋白酶抑制蛋白、tau和LSEAL之间的钙依赖性竞争性结合。我们还将测试其他钙蛋白酶底物。具体目标3：评估LSEAL和相关肽在细胞环境中作为新型钙蛋白酶抑制剂的假设。在初步工作中，我们已经使用培养的皮层神经元，以证明细胞死亡引起的细胞溶质钙离子或离子霉素或紫外线介导的增加是防止治疗与LSEAL，而不是由乱序，阴性对照肽，ELLAS。这些研究将评估LSEAL作用的关键方面，并提供重要证据支持这种潜在的新型钙蛋白酶抑制剂用于治疗脑损伤的未来发展。

DESCRIPTION (provided by applicant): Many researchers have shown that over activation of the calcium-dependent proteases, the calpains, likely contributes to the cell death that occurs following brain injury. 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 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 confirm and extend our initial observation that we have identified a novel family of calpain inhibitor for potential clinical use in the treatment of brain injury 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. We have already identified a candidate peptide, LSEAL. Additional libraries of increased stringency are to be used to find potentially more effective sequences. Specific Aim 2: To test the hypothesis that phage peptide display-derived peptides are novel and specific inhibitors of calpain activity with a mechanism similar to calpastatin. 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. BIAcore technology will be used in addition to Elisa based assays to assay for calcium-dependent competitive binding between calpastatin, tau and LSEAL. We will also test additional calpain substrates. Specific Aim 3: To evaluate the hypothesis that LSEAL and related peptides act as novel calpain inhibitors in a cellular setting. 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 brain injury.