Inhibitors of beta-Amyloid Production in Alzheimer's

阿尔茨海默病中β-淀粉样蛋白生成的抑制剂

• 批准号: 7142250
• 负责人: GREGORY R HOOK
• 金额: $ 16.32万
• 依托单位: AMERICAN LIFE SCIENCE PHARMACEUTICALS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142250
• 关键词: Alzheimer' s disease; amyloid proteins; aspartic endopeptidases; biomimetics; brain disorder chemotherapy; brain metabolism; cathepsin B; drug delivery systems; drug design /synthesis /production; drug screening /evaluation; guinea pigs; neurons; neuropharmacology; nonhuman therapy evaluation; peptide analog; phthalimides; protease inhibitor; secretion; synaptosomes; therapy design /development; tissue /cell culture

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive disorder resulting in severe dementia which currently cannot be stopped. Abnormal accumulation of extracellular brain beta-amyloid is thought to cause AD and compounds that block beta-amyloid production are predicted to be effective for treating AD. Beta-amyloid is generated by cleavage from a larger protein, amyloid precursor protein (APR), by proteases called beta-secretases and gamma-secretases. Compounds that inhibit either of these secretases reduce beta-amyloid production and, therefore, are thought to be potentially effective AD drugs. Brain neurons secrete beta-amyloid by regulated and constitutive secretory pathways. We find that neurons secrete the vast majority of beta-amyloid via the regulated secretory pathway and that the cysteine proteases, cathepsin B and cathepsin L, are the beta- secretases of that pathway. Consequently, we find cathepsin B and cathepsin L are responsible for producing the majority of secreted beta-amyloid. Evidence for our finding is based on a wide range of experimental data. We purified beta-secretase activity from isolated regulated secretory vesicles and determined by microsequencing that the beta-secretase activity is due cathepsin B and cathepsin L. We found by immunoelectron microscopy that cathepsin B and cathepsin L are co-localized with APP and beta-amyloid in isolated secretory vesicles. We observed that cathepsin B and/or cathepsin L inhibitors block endogenous beta-secretase activity in isolated regulated secretory vesicles and stop the continued natural production of beta-amyloid that occurs after isolation of regulated secretory vesicles. Moreover, we found that cathepsin B and cathepsin L cleave with excellent efficiency the beta-secretase site found in wild-type APP, the APP form found in 99% of AD patients. Further, we found that a cathepsin B specific inhibitor, CA-074, dramatically reduces regulated secretion of Ab, but not its constitutive secretion, by neuronal chromaffin cells. Significantly, we show in this application that the cysteine protease inhibitor, e64d, reduces in vivo total brain and synaptosomal beta-amyloid in the guinea pig AD animal model. In this project, we will evaluate peptidomimetics that specifically inhibit cathepsin B or cathepsin L and which we find also inhibit beta-secretase activity in regulated secretory vesicles. Specifically, we will assess the affects of these peptidomimetics on beta-amyloid secretion in primary neuronal cultures and in vivo.

描述（由申请人提供）：阿尔茨海默氏病（AD）是一种进行性疾病，导致严重的痴呆症，目前无法停止。据认为，细胞外脑β-淀粉样蛋白的异常积累会引起AD，并且可以预测阻断β-淀粉样蛋白产生的化合物可有效治疗AD。 β-淀粉样蛋白是由较大蛋白质淀粉样蛋白前体蛋白（APR）的切割产生的，该蛋白质是由称为β-分泌酶和γ-分泌酶的蛋白酶产生的。抑制这两种分泌酶中的任何一种的化合物减少了β-淀粉样蛋白的产生，因此被认为是潜在的有效AD药物。脑神经元通过受调节和构成分泌途径分泌β-淀粉样蛋白。我们发现，神经元通过受调节的分泌途径分泌绝大多数β-淀粉样蛋白，并且半胱氨酸蛋白酶，组织蛋白酶B和组织蛋白酶L是该途径的β-分泌酶。因此，我们发现组织蛋白酶B和组织蛋白酶L负责生产大多数分泌的β-淀粉样蛋白。我们发现的证据基于广泛的实验数据。我们从孤立的调节分泌囊泡中纯化了β-分泌酶活性，并通过微糖测序确定β-分泌酶活性是由于组织蛋白酶B和组织蛋白酶L。我们通过免疫电子显微镜发现，在分离的分离式小牛siles中，我们通过免疫电子显微镜与app和beta-amymyloid共同定位了calter蛋白蛋白酶B和canterpersin b和canterepsin l。我们观察到组织蛋白酶B和/或组织蛋白酶L抑制剂在分离的分泌囊泡中阻断内源性β-分泌酶活性，并停止在分离受调节分泌囊泡后发生的β-淀粉样蛋白的持续自然产生。此外，我们发现组织蛋白酶B和组织蛋白酶l裂解具有极高的效率，β-分泌酶位点在野生型应用程序中发现的β-分泌酶位点，该应用程序在99％的AD患者中发现。此外，我们发现组织蛋白酶B特异性抑制剂CA-074大大降低了神经元铬蛋白细胞的AB的调节分泌，而不是其本构分泌的调节。值得注意的是，我们在此应用中表明，半胱氨酸蛋白酶抑制剂E64D在豚鼠AD动物模型中降低体内总脑和突触体β-淀粉样蛋白。在该项目中，我们将评估特异性抑制组织蛋白酶B或组织蛋白酶L的肽仪，并发现在受调节分泌囊泡中抑制β-分泌酶活性。具体而言，我们将评估这些肽仪对原代神经元培养物和体内β-淀粉样蛋白分泌的影响。