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β肽（Abeta(1-42)）的可溶性寡聚物形式作为Abeta神经毒性活性的潜在介质和阿尔茨海默氏病的可能病原体而受到越来越多的关注。由于缺乏高通量筛选试验和化合物与抗体的人为相互作用，对潜在治疗性抗低聚物化合物的研究一直受到阻碍。这一探索性的建议采用了一种新颖的、廉价的检测格式来筛选抑制可溶性β低聚物形成或破坏其结构稳定的化合物，促进生物清除。一个小的药物制剂库将被筛选，以建立性能参数，随后评估一个更广泛的药效团库，以确定潜在的兴趣结构。具体目标开发一种高通量测定系统来鉴定抑制β寡聚物形成的化合物。在早期阶段阻止β(1-42)寡聚物的组装将阻止稳定的有毒物质的形成。人工合成的n端生物素化Abeta（1-42）的寡聚物种将通过一种新的生物素-亲和素单位点结合试验进行定量。这种方法是优选的初级筛选，甚至寡聚特异性抗体，因为它避免了大量的假阳性，无活性的化合物，干扰抗体表位和抗体。该程序将在英国的Tecan Genesis LiHa/TeMo机器人系统上实施。具体目标2。开发一种高通量分析系统，以鉴定破坏β低聚物结构的化合物。-低聚物具有高度的蛋白酶抗性，因此很难从大脑中清除。使低聚物不稳定的化合物允许β代谢蛋白酶降解，将有助于降低低聚物水平，并具有潜在的治疗价值。在存在化合物的情况下形成的合成的Abeta低聚物，或者预先形成然后用化合物处理，将用胰蛋白酶筛选，如果阳性，则用胰岛素降解酶和neprilysin消化，这些蛋白酶被认为可以在体内降解Abeta肽。选定的阳性化合物还将采用为Luminex-100(r) Beadlyte（tm）系统配置的新型高灵敏度单位点免疫测定法，测试其对从AD脑中提取的可溶性Abeta低聚物的蛋白酶敏感性的影响。该方法有望检测生物体液中的低聚β，包括动物模型和临床标本，以确定抗低聚物治疗的有效性。具体目标3。确定在Specific Aims 1和2中开发的筛选LOPAC化合物库和更广泛的Hit-Finder（tm）药效团集合的靶向筛选的测定方法的性能。LOPAC库将用于对特定目标1和2中以筛选格式应用的优化协议进行故障排除。该筛选结果将用于预测Maybridge Hit-Finder（tm）收集的16,000种化合物中哪些化合物可能具有阻断低聚物形成的活性。虽然这一探索性/开发性提案的目的主要是验证分析，但活性化合物将为探测β低聚物的生物效应提供有用的工具，因为组成LOPAC的药物制剂与细胞系统兼容。抗低聚物化合物可能潜在地阻断低聚物在动物模型中引起的认知障碍症状，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.