Inhibitors of beta-Amyloid Production in Alzheimer's

描述（由申请人提供）：阿尔茨海默病（AD）是一种导致严重痴呆的进行性疾病，目前无法停止。细胞外脑β-淀粉样蛋白的异常积累被认为是导致AD的原因，并且阻断β-淀粉样蛋白产生的化合物被预测对治疗AD有效。β-淀粉样蛋白是由称为β-分泌酶和γ-分泌酶的蛋白酶从更大的蛋白质，淀粉样前体蛋白（APR）裂解而产生的。抑制这些分泌酶中的任一种的化合物减少β-淀粉样蛋白的产生，因此被认为是潜在有效的AD药物。脑神经元通过调节性和组成性分泌途径分泌β-淀粉样蛋白。我们发现神经元通过调节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负责产生大部分分泌的β-淀粉样蛋白。我们发现的证据是基于广泛的实验数据。我们从分离的调节分泌囊泡中纯化β-分泌酶活性，并通过微测序确定β-分泌酶活性是由于组织蛋白酶B和组织蛋白酶L。我们通过免疫电镜发现，组织蛋白酶B和组织蛋白酶L与APP和β-淀粉样蛋白共定位于分离的分泌囊泡中。我们观察到，组织蛋白酶B和/或组织蛋白酶L抑制剂阻断内源性β-分泌酶活性在分离的调节分泌囊泡和停止β-淀粉样蛋白的持续自然生产后发生的分离的调节分泌囊泡。此外，我们发现组织蛋白酶B和组织蛋白酶L以优异的效率切割在野生型APP中发现的β-分泌酶位点，在99%的AD患者中发现的APP形式。此外，我们发现，组织蛋白酶B特异性抑制剂CA-074，显着减少调节分泌的抗体，但不是其组成性分泌，由神经元嗜铬细胞。值得注意的是，我们在本申请中显示，半胱氨酸蛋白酶抑制剂e64 d在豚鼠AD动物模型中减少体内总脑和突触体β-淀粉样蛋白。在这个项目中，我们将评估特异性抑制组织蛋白酶B或组织蛋白酶L的肽模拟物，我们发现它们也抑制调节分泌囊泡中的β-分泌酶活性。具体来说，我们将评估这些肽模拟物对β-淀粉样蛋白分泌的影响，在原代神经元培养和体内。

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.