A screen for small molecule inhibitors of soluble Abeta oligomer assembly

描述(由申请人提供)：阿尔茨海默氏症β-肽(Abeta(1-42))的可溶性低聚形式作为Abeta神经毒性活动的潜在介体和阿尔茨海默病的可能致病因素正受到越来越多的关注。由于缺乏高通量的筛选试验和化合物与抗体的人工相互作用，对潜在治疗性抗低聚物化合物的寻找一直受到阻碍。这一探索性的建议应用了一种新颖、廉价的分析格式来筛选抑制可溶性Abeta寡聚体的形成或破坏其结构的化合物，从而促进生物清除。将筛选一个小的药剂库，以建立性能参数，然后评估更广泛的药效团库，以确定潜在的感兴趣的结构。具体目的1.建立一种高通量检测系统，用于鉴定抑制β-寡聚体形成的化合物。在早期阶段阻止Abeta(1-42)齐聚物的组装将防止形成稳定的有毒物种。合成的N-末端生物素化的Abeta(1-42)的寡聚物种将用一种新的生物素-亲和素单点结合分析来定量。这种方法甚至比寡聚体特异性抗体更适合于初步筛选，因为它避免了大量的假阳性，即干扰抗体表位和抗体的非活性化合物。该程序将在英国的Tecan Genesis LiHa/Temo机器人系统上实施。具体目的2.建立一种高通量检测系统来鉴定破坏Aβ齐聚物结构稳定的化合物。Aβ寡聚体对蛋白酶的抗性很强，因此很难从大脑中清除出去。使低聚物不稳定的化合物可以被Abeta代谢酶降解，这将有助于降低低聚物的水平，并具有潜在的治疗价值。在化合物存在下形成的合成的Abeta寡聚体，或预先形成然后用化合物处理的低聚物，将用胰酶筛选，如果呈阳性，则用胰岛素降解酶和Neprilysin消化，被认为是体内降解Abeta多肽的蛋白酶。选定的阳性化合物还将使用为Luminex-100(R)Beadlyte(Tm)系统配置的新的高灵敏单点免疫分析来测试它们对从AD脑中提取的可溶性Abeta寡聚体的酶敏感性的影响。这种方法有望检测生物液中的寡聚体Abeta，包括动物模型和临床标本，以确定抗低聚物疗法的有效性。具体目标3.确定在具体目标1和2中开发的分析在筛选Lopac化合物文库和对更广泛的Hit-Finder(Tm)药效团集合进行定向筛选方面的性能。LOPAC库将用于对在筛选格式中应用的特定目标1和2中优化的协议进行故障排除。这一筛选的结果将用于预测Maybridge Hit-Finder(Tm)收集的16,000种化合物中哪些化合物可能有效地阻止低聚物的形成。尽管这一探索性/开发性建议的目的主要是验证分析，但活性化合物将提供有用的工具，用于探索Abeta寡聚体的生物效应，因为组成Lopac的药剂与细胞系统兼容。反寡聚体化合物可能会阻止寡聚体在动物模型中引发的认知障碍症状，并干扰阿尔茨海默病的进展。

DESCRIPTION (provided by applicant): Soluble oligomeric forms of the Alzheimer's beta-peptide (Abeta(1-42)) are receiving increasing attention as potential mediators of the neurotoxic activity of Abeta and as a possible causative agent for Alzheimer's disease. The search for potentially therapeutic anti-oligomer compounds has been hampered by the lack of a high-throughput screening assay and artifactual interactions of compounds with antibodies. This exploratory proposal applies a novel, inexpensive assay format to screen for compounds that inhibit the formation of soluble Abeta oligomers or destabilize their structure, facilitating biological clearance. A small library of pharmaceutical agents will be screened to establish performance parameters followed by evaluation of a more extensive pharmacophore library to identify structures of potential interest. Specific Aim 1. To develop a high-throughput assay system to identify compounds to inhibit Abeta oligomer formation. Preventing Abeta(1-42) oligomer assembly at an early stage will prevent stable toxic species from being formed. Oligomeric species of synthetic N-terminally biotinylated Abeta(1-42) will be quantified with a novel biotin-avidin single site binding assay. This method is preferable for primary screening over even oligomer-specific antibodies because it avoids a significant number of false positives, inactive compounds that interfere with antibody epitopes and antibodies. The procedure will be implemented on a Tecan Genesis LiHa/TeMo robotics system at UK. Specific Aim 2. To develop a high-throughput assay system to identify compounds to destabilize Abeta oligomer structure. Abeta oligomers are highly protease-resistant and thus poorly cleared from the brain. Compounds that destabilize oligomers allowing degradation by Abeta-metabolizing proteases would be useful in reducing oligomer levels and be of potential therapeutic value. Synthetic Abeta oligomers formed in the presence of compound, or preformed and then treated with compound, will be screened with trypsin, and if positive, digested with insulin-degrading enzyme and neprilysin, proteases believed to degrade Abeta peptides in vivo. Selected positive compounds will be also tested for their effect on the protease sensitivity of soluble Abeta oligomers extracted from AD brain employing a new highly sensitive single site immunoassay configured for the Luminex-100(r) Beadlyte(tm) system. This method shows promise for the detection of oligomeric Abeta in biological fluids, including animal models and clinical specimens to determine the effectiveness of anti-oligomer therapeutics. Specific Aim 3. To determine the performance of the assays developed in Specific Aims 1 and 2 on screening the LOPAC compound library and a targeted screen of the more extensive Hit-Finder(tm) pharmacophore collection. The LOPAC library will be used to troubleshoot the protocols optimized in Specific Aims 1 and 2 applied in a screening format. Results from this screen will be used to predict which compounds in the 16,000 compound Maybridge Hit-Finder(tm) collection may be active in blocking oligomer formation. Although the intent of this exploratory/developmental proposal is primarily to validate the assays, active compounds will provide tools that will be useful in probing the biological effects of Abeta oligomers since the pharmaceutical agents that make up LOPAC are compatible with cellular systems. Anti-oligomer compounds could potentially block the cognitive impairment symptoms that oligomers elicit in animal models and interfere with the progression of Alzheimer's disease.