A screen for small molecule inhibitors of soluble Abeta oligomer assembly

可溶性 Abeta 寡聚物组装的小分子抑制剂的筛选

• 批准号: 7273895
• 负责人: HARRY LEVINE
• 金额: $ 18.14万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7273895
• 关键词: Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Animal Model; Antibodies; Attention; Automation; Avidin; Binding; Binding Sites; Biological; Biological Assay; Biology; Biotin; Blocking Antibodies; Brain; Cathepsins B; Cellular Assay; Clinical; Collaborations; Collection; Compatible; Cultured Cells; Detection; Development; Effectiveness; Endopeptidases; Enzymes; Epitopes; Evaluation; Immunoassay; Impaired cognition; In Vitro; Insulinase; Label; Lead; Libraries; Liquid substance; Measures; Mediator of activation protein; Methods; Microbiology; Neprilysin; Numbers; Peptide Hydrolases; Peptides; Performance; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacologic Substance; Procedures; Process; Property; Protocols documentation; Publishing; Research Personnel; Resistance; Robotics; Screening Result; Screening procedure; Site; Specimen; Staging; Streptavidin; Structure; Structure-Activity Relationship; Symptoms; System; Testing; Therapeutic; Treatment Effectiveness; Trypsin; abeta oligomer; amyloid formation; base; experience; high throughput screening; in vivo; inhibitor/antagonist; innovation; interest; monomer; neurotoxic; novel; pharmacophore; prevent; programs; secretase; small molecule; therapeutic target; tool

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.

描述（由申请人提供）：阿尔茨海默病β-肽（Abeta(1-42)）的可溶性寡聚形式作为Abeta神经毒性活性的潜在介质以及作为阿尔茨海默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以及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探索受到了阻碍。该探索性提案采用了一种新颖、廉价的检测方式来筛选能够抑制可溶性 Abeta 寡聚物形成或破坏其结构稳定性、促进生物清除的化合物。将筛选一个小型药剂库以确定性能参数，然后评估更广泛的药效基团库以识别潜在感兴趣的结构。具体目标 1. 开发高通量检测系统来鉴定抑制 Abeta 寡聚物形成的化合物。在早期阶段防止 Abeta(1-42) 寡聚物组装将防止形成稳定的有毒物质。合成 N 末端生物素化 Abeta(1-42) 的寡聚体将通过新型生物素-亲和素单位点结合测定进行定量。这种方法甚至比寡聚物特异性抗体更适合初级筛选，因为它避免了大量的假阳性、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. 开发高通量检测系统来鉴定破坏 Abeta 寡聚物结构稳定的化合物。 Abeta 寡聚物具有高度蛋白酶抗性，因此很难从大脑中清除。使寡聚物不稳定并允许Abeta代谢蛋白酶降解的化合物可用于降低寡聚物水平并具有潜在的治疗价值。在化合物存在下形成的合成Abeta寡聚物，或预先形成然后用化合物处理的合成Abeta寡聚物将用胰蛋白酶筛选，如果呈阳性，则用胰岛素降解酶和脑啡肽酶消化，蛋白酶相信在体内降解Abeta肽。还将使用为 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 寡聚物的蛋白酶敏感性的影响。该方法有望检测生物体液（包括动物模型和临床标本）中的寡聚 Abeta，以确定抗寡聚体治疗的有效性。具体目标 3. 确定具体目标 1 和 2 中开发的测定方法在筛选 LOPAC 化合物库和更广泛的 Hit-Finder(tm) 药效基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 中优化的协议进行故障排除。该筛选的结果将用于预测 16,000 种 Maybridge Hit-Finder(tm) 化合物集合中的哪些化合物可能具有阻断低聚物形成的活性。尽管这一探索性/开发提案的目的主要是验证检测方法，但由于构成 LOPAC 的药剂与细胞系统兼容，因此活性化合物将提供可用于探索 Abeta 寡聚物的生物效应的工具。抗寡聚物化合物可能会阻止寡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。

项目成果

Clioquinol and other hydroxyquinoline derivatives inhibit Abeta(1-42) oligomer assembly.

氯碘羟喹和其他羟基喹啉衍生物抑制 Abeta(1-42) 寡聚物组装。

• 发表时间: 2009-11-06
• 影响因子: 2.5
• 作者: LeVine H 3rd;Ding Q;Walker JA;Voss RS;Augelli-Szafran CE
• 通讯作者: Augelli-Szafran CE

Dihydroxybenzoic acid isomers differentially dissociate soluble biotinyl-Aβ(1-42) oligomers.

二羟基苯甲酸异构体差异解离可溶性生物素-Aβ(1-42) 低聚物。

• DOI: 10.1021/bi201288x
• 发表时间: 2012-01-10
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: LeVine H 3rd;Lampe L;Abdelmoti L;Augelli-Szafran CE
• 通讯作者: Augelli-Szafran CE

Synthesis and application of β-carbolines as novel multi-functional anti-Alzheimer's disease agents.

β-咔啉作为新型多功能抗阿尔茨海默病药物的合成和应用。

• DOI: 10.1016/j.bmcl.2016.11.067
• 发表时间: 2017-01-15
• 期刊: Bioorganic & medicinal chemistry letters
• 影响因子: 2.7
• 作者: Horton W;Sood A;Peerannawar S;Kugyela N;Kulkarni A;Tulsan R;Tran CD;Soule J;LeVine H 3rd;Török B;Török M
• 通讯作者: Török M