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））的可溶性寡聚体形式作为Abeta神经毒性活性的潜在介体和作为阿尔茨海默氏病的可能病原体正受到越来越多的关注。由于缺乏高通量筛选测定和化合物与抗体的人为相互作用，对潜在治疗性抗寡聚体化合物的研究受到阻碍。这一探索性的提议应用了一种新型的、廉价的测定形式来筛选抑制可溶性Abeta寡聚体形成或破坏其结构稳定的化合物，从而促进生物清除。将筛选一个小的药物库，以确定性能参数，然后评价更广泛的药效团库，以鉴定潜在的感兴趣结构。具体目标1。开发高通量检测系统，以鉴定抑制Abeta寡聚体形成的化合物。在早期阶段防止Abeta（1-42）寡聚体组装将防止形成稳定的有毒物质。将采用新型生物素-抗生物素蛋白单位点结合试验定量合成N-末端生物素化Abeta（1-42）的寡聚体物质。该方法对于甚至寡聚体特异性抗体的初级筛选是优选的，因为它避免了显著数量的假阳性、干扰抗体表位和抗体的无活性化合物。该程序将在英国的Tecan Genesis LiHa/TeMo机器人系统上实施。具体目标2。开发一种高通量检测系统，以鉴定使Abeta寡聚体结构不稳定的化合物。Abeta寡聚体具有高度的蛋白酶抗性，因此难以从大脑中清除。使寡聚体不稳定从而允许通过A β代谢蛋白酶降解的化合物将可用于降低寡聚体水平并且具有潜在的治疗价值。将用胰蛋白酶筛选在化合物存在下形成的合成Abeta寡聚体，或预先形成然后用化合物处理的合成Abeta寡聚体，并且如果阳性，则用胰岛素降解酶和脑啡肽酶（据信在体内降解Abeta肽的蛋白酶）消化。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，测试选定的阳性化合物对从AD脑中提取的可溶性Abeta寡聚体的蛋白酶敏感性的影响。该方法显示出用于检测生物流体（包括动物模型和临床标本）中的寡聚体Abeta以确定抗寡聚体治疗剂的有效性的前景。具体目标3。确定特定目标1和2中开发的试验在筛选LOPAC化合物文库和更广泛的Hit-Phase（tm）药效团集合的靶向筛选方面的性能。LOPAC库将用于对筛选格式中应用的特定目标1和2中优化的方案进行故障排除。该筛选的结果将用于预测Maybridge Hit-16,000化合物（TM）集合中的哪些化合物可能在阻断寡聚体形成中具有活性。尽管该探索性/开发性提案的目的主要是验证试验，但活性化合物将提供可用于探测Abeta寡聚体生物学效应的工具，因为构成LOPAC的药物与细胞系统相容。抗寡聚体化合物可以潜在地阻断寡聚体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.