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Novel calpain inhibitors based on phage display

基于噬菌体展示的新型钙蛋白酶抑制剂

基本信息

批准号：
6893402
负责人：
RODNEY P GUTTMANN
金额：
$ 17.03万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-05-15 至 2007-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6893402
关键词：
biomimetics biotechnology brain injury calpain drug discovery /isolation enzyme inhibitors enzyme linked immunosorbent assay fluorescence microscopy laboratory rat peptide chemical synthesis phage display protein binding statistics /biometry stroke therapy tissue /cell culture

项目摘要

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.

描述(由申请人提供)：许多研究人员已经证明，钙依赖蛋白水解酶的过度激活很可能是脑损伤后细胞死亡的原因。因此，开发治疗药物来阻止这种过度激活并减少或防止随后的细胞和组织破坏是至关重要的。在前期工作中，我们使用噬菌体多肽展示来鉴定一类以钙依赖的方式与钙蛋白结合的多肽。有趣的是，代表性多肽LSEAL与Calastatin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调蛋白多肽模拟物。这项建议的目的是证实和扩大我们最初的观察结果，即我们已经通过实现以下特定目的来确定一种新的钙蛋白酶抑制剂家族，用于脑损伤的潜在临床治疗：特定目的1“使用噬菌体展示技术以钙依赖的方式鉴定与钙蛋白酶结合的多肽。我们已经确定了一个候选肽，LSEAL。将使用更严格的额外文库来寻找潜在更有效的序列。具体目的2：验证噬菌体多肽展示衍生的多肽是新型和特异的钙蛋白酶活性抑制剂的假说，其作用机制类似于钙调蛋白。在初步工作中，我们发现来自初始文库的一个有代表性的肽(LSEAL)是Calain对微管相关蛋白tau的蛋白分解作用的有效抑制剂，微管相关蛋白tau是一种已知的calain底物。除了基于酶联免疫吸附试验的检测外，还将使用Biacore技术来检测钙调蛋白、tau和LSEAL之间的钙依赖竞争结合。我们还将测试额外的钙蛋白酶底物。具体目标3：评估LSEAL和相关多肽在细胞环境中作为新的钙蛋白酶抑制剂的假设。在初步工作中，我们使用培养的皮质神经元来证明，由离子霉素或紫外线介导的胞浆钙增加引起的细胞死亡可以通过LSEAL处理而不是通过扰乱序列、负控制肽Ellas来防止。这些研究将评估LSEAL作用的关键方面，并提供重要证据，以支持这种潜在的新型钙蛋白酶抑制剂用于脑损伤治疗的未来发展。