Inhibitors of beta-Amyloid Production in Alzheimer's

描述(申请人提供)：阿尔茨海默病(AD)是一种进行性疾病，导致严重的痴呆症，目前无法阻止。细胞外β-淀粉样蛋白的异常积聚被认为是导致AD的原因，而阻止β-淀粉样蛋白产生的化合物被预测为治疗AD的有效药物。β-淀粉样蛋白是由一种较大的蛋白质--淀粉样前体蛋白(APR)--被称为β-分泌酶和伽马-分泌酶的蛋白酶裂解而产生的。抑制这两种分泌酶的化合物会减少β-淀粉样蛋白的产生，因此被认为是潜在有效的AD药物。脑神经元通过调节和构成的分泌途径分泌β-淀粉样蛋白。我们发现，神经元通过调节的分泌途径分泌绝大多数的β-淀粉样蛋白，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。我们发现的证据基于广泛的实验数据。我们从分离的受调控的分泌囊泡中纯化了β-分泌酶活性，并通过显微测序确定其活性来源于组织蛋白酶B和组织蛋白酶L。免疫电子显微镜观察发现，组织蛋白酶B和组织蛋白酶L与APP和β-淀粉样蛋白共同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抑制剂阻断了分离的受调节分泌小泡的内源性β-分泌酶活性，并阻止了在分离受调节的分泌小泡后继续自然产生的β-淀粉样蛋白。此外，我们发现组织蛋白酶B和组织蛋白酶L以极高的效率切割野生型APP中的β-分泌酶位点，野生型APP存在于99%的AD患者中。此外，我们发现组织蛋白酶B的特异性抑制剂CA-074显著减少神经元嗜铬细胞分泌抗体，但不减少抗体的结构性分泌。值得注意的是，我们在这项应用中表明，半胱氨酸蛋白酶抑制剂e64d在豚鼠AD动物模型中可以减少体内总脑组织和突触体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的肽类药物，以及我们发现的也抑制调节分泌囊泡中的β-分泌酶活性的肽类药物。具体地说，我们将评估这些肽类药物对原代神经元培养和体内β淀粉样蛋白分泌的影响。

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.