Screening for APPNeo Inhibitors

描述（由申请人提供）：阿尔茨海默病（AD）目前在美国折磨着超过500万人。在美国，每年大约有36万新的AD病例被诊断出来。在目前的治疗环境下，预计到2050年新病例数将增加3倍以上，达到每年约100万新病例。因此，迫切需要开发新的药物来预防或延缓疾病的发展。目前有四种获批的药物可用于治疗迟发性或散发性阿尔茨海默病的症状，但这些治疗不能预防或延缓疾病的进展。许多疾病修饰疗法的新方法正在开发中，但其临床结果仍不确定。在许多基于淀粉样蛋白的临床候选结果令人失望之后，仅针对A¿的治疗方法可能无法解决该疾病的所有致病事件。因此，迫切需要开发基于D664切割等其他靶点的新方法来开发AD的最佳治疗方法。本提案的目标是确定这个新靶点的抑制剂：APP在Asp664 （D664）位点的胞浆内c端蛋白水解。我们之前已经证明，APP的c端裂解导致高细胞毒性肽APP- c31的产生。最近，我们已经证明，在用某些他汀类药物治疗或停用血清后，可以在细胞裂解物中检测到来自该裂解的整个n端APP片段- APPneo片段-，并且该n端片段的产生与细胞死亡相关，可以作为该裂解的生物标志物进行测量。我们的研究表明，D664切割事件发生在人类患者中，并且与年龄匹配的对照组相比，在疾病的早期阶段增加；该位点的突变阻止了c -末端片段（C99）和A -本身的细胞毒性。重要的是，使用转基因小鼠模型，如果我们通过点突变（Asp664 -> Ala）从基因上阻止c端APP蛋白水解，我们就阻断了阿尔茨海默病的特征：记忆丧失、突触丧失、脑萎缩（萎缩）、电生理异常和脑瘢痕（胶质瘤）。我们还发现，在D664A突变的转基因小鼠大脑中，sAPP /A¿42比率也有所增加。因此，我们的数据表明，D664切割确实代表了一种新的、潜在的重要的阿尔茨海默病治疗靶点。我们已经开发了一种检测方法来筛选这种APP蛋白水解的抑制剂，这种新的检测方法适用于高通量筛选（HTS）格式。一个以cns为中心的小文库的筛选已经导致了一种有前途的抑制剂的鉴定。在这一建议中，我们概述了一个循序渐进的计划，以确定和开发这一目标的有效抑制剂。我们计划和科斯福德医生合作

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) currently afflicts more than five million people in the US. Approximately 360,000 new cases of AD are diagnosed annually in the US. Under the current therapeutic environment the number of new cases is projected to rise more than 3-fold to about 1 million new cases per year by 2050. Thus, development of new drugs that prevent or delay the progression of the disease are desperately needed. Currently four approved drugs are available for the treatment of the symptoms of late-onset or sporadic AD, but these treatments do not prevent or delay the progression of the disease. Many new approaches to disease modifying therapies are under development, but their clinical outcome remain uncertain. After disappointing results from many amyloid-based clinical candidates, it is likely that therapeutics targeting just A¿ alone will not address all of the pathogenic events in te disease. Therefore development of new approaches based on other targets such as the D664 cleavage is urgently needed to develop an optimal treatment for AD. The goal of this proposal is to identify inhibitors for this new target: the intracytoplasmic C-terminal proteolysis of APP at Asp664 (D664) site. We have previously shown that this C-terminal cleavage of APP leads to the production of highly cytotoxic peptide APP-C31. Recently we have shown that the entire N-terminal APP fragment from this cleavage ¿ APPneo fragment ¿ can be detected in cell lysates after treatment with certain statins or after serum withdrawal, and production of this N-terminal fragment correlates with cell death and can be measured as a biomarker of this cleavage. Our studies show that the D664 cleavage event occurs in human patients, and is increased in early stages of the diseases in comparison to age-matched controls; mutation of this site prevents cytotoxicity of the ¿-C-terminal fragment (C99) as well as by A¿ itself. Importantly, using a transgenic mouse model, if we genetically prevented the C-terminal APP proteolysis through a point mutation (Asp664 -> Ala), we blocked the features of Alzheimer's disease: the memory loss, synapse loss, the brain atrophy (shrinkage), the electrophysiological abnormalities, and the brain scarring (gliosis). We also see an increase in the sAPP¿/A¿42 ratios in the transgenic mouse brain with the D664A mutation. Our data thus argue that the D664 cleavage does indeed represent a novel and potentially important new therapeutic target in AD. We have developed an assay to screen for inhibitors of this APP proteolysis, and this new assay is adaptable to a high-throughput screening (HTS) format. Screening of a small CNS-focused library has already led to the identification of a promising inhibitor. In this proposal we outline a stepwise plan to identify and develop potent inhibitors of this target. In collaboration with Dr. Cosford we plan to screen the diverse Sanford- Burnham small-molecule library. Based on our preliminary data, the chance of successfully attaining the key goals of this proposal is high. This project fulfills an important gap, and would lead to identification of inhibitors for this APP processing pathway, and possibly a new class of therapeutics for AD. PUBLIC HEALTH RELEVANCE: The approved drugs for the treatment of Alzheimer's disease (AD) only provide temporary, symptomatic relief without affecting disease progression. We have identified a critical C-terminal proteolytic cleavage of APP that leads to cytotoxic APP fragments (Jcasp and APP-C31); production of these fragments can be measured by levels of the N-terminal APPneo fragment, and along with A? they contribute to the pervasive neurotoxicity seen in AD. Our initial screening effort has led to a promising hit that we plan to develop in this proposal, in addition using a high-throughput screening (HTS) assay we plan to identify new inhibitors of this cleavage from the diverse Sanford-Burnham library and to evaluate them as candidates for further development in AD.