Compounds blocking crystallin aggregation in vitro; path to anti-cataract agents

项目摘要/摘要 早发性白内障是老年人群视力受损和丧失的主要原因。这个 白内障手术和晶状体植入术的成功改善了许多成年人的这种状况。然而，有超过2个 每年有100万例白内障手术，36亿美元的医疗费用是一个巨大的负担 医疗保健系统。经常需要第二次手术，以及偶尔的其他 并发症，增加了额外的医疗费用和负担。另一个不幸的问题是 数十万美国人仍然无法获得白内障手术，原因是经济、社会或 心理因素。推迟白内障的发病或减缓其生长--即使是几年--将会 显著降低医疗保健成本并提高老年人的生活质量 公民们。这表明投资于预防的价值，包括小分子的发展。 抑制白内障形成的治疗方法。识别蛋白质聚集的抑制剂是一项主要的努力 其他蛋白质聚集性疾病，如阿尔茨海默氏症、帕金森氏症和亨廷顿氏症。 在最后一个项目期间，开发了体外系统，在其中部分地展示了人类 在pH值为7时，晶体蛋白聚集成光散射复合体，在低pH时聚集成淀粉样纤维。试管苗 聚集反应捕捉到了晶状体内形成白内障的许多特性。这个项目 将利用这些实验系统对化合物进行高通量筛选 抑制晶体蛋白聚集反应的体外启动或繁殖。最初的努力将利用 通过哈佛医学院朗伍德设施提供的250,000种化合物，以及一组较小的 麻省理工学院化学系合成的活性淀粉样蛋白抑制剂。目标反应使用人类 D-和C-晶体蛋白携带氨基酸修饰，代表各种形式的氧化和 已在白内障中发现的突变损害。此外，人类白内障的碎片 来自外科手术的聚集体将被用作体外反应的种子，并筛选出 抑制晶体蛋白链加到种子上的化合物。最初的热门歌曲将被描述为 它们是否抑制聚集过程的启动或传播，以及它们对 底物由晶状体伴侣？-晶状体蛋白结合。改进潜在目标结合位点的搜索 并将利用可能的HITS、核磁共振和低温EM资源来表征晶体蛋白底物/抑制剂 相互作用和底物/伴侣结合位点的构象。这将使能够利用 在电子计算机搜索目标位置和新化合物方面功能强大。最初的热门歌曲将是 通过化学修饰和进一步的体外表征进行了改进。这些所谓的抗白内障药物 将成为在白内障动物模型上进行测试的候选对象。

Project Summary/Abstract Mature-onset cataracts are a major cause of sight impairment and loss in aging populations. The success of cataract surgery and lens implants has ameliorated this for many adults. However, with over 2 million cataract surgeries a year, the $3.6 billion dollar health care costs are a significant burden on the healthcare system. The frequent need for a second procedure, together with occasional other complications, add additional health care costs and burdens. An unfortunate additional problem is that hundreds of thousands of Americans remain unable to access cataract surgery, due to economic, social or psychological factors. Delaying the onset of cataract or slowing their growth - even by a few years - would yield a very significant reduction in health care costs and improvement in the quality of life for senior citizens. This suggests the value of investing in prevention, including the development of small molecule therapies that inhibit cataract formation. Identifying inhibitors of protein aggregation is a major effort for other protein aggregation diseases such as Alzheimer's, Parkinson's and Huntington's. In the last project period, in vitro systems were developed in which partially unfolded human crystallins aggregated into light-scattering complexes at pH7 and amyloid fibers at low pH. The in vitro aggregation reactions capture a number of the properties of cataract formation within the lens. This project will take advantage of these experimental systems by performing high-throughput screening for compounds that inhibit the in vitro initiation or propagation of crystallin aggregation reactions. The initial effort will tap the 250,000 compounds available through the Harvard Medical School Longwood Facility, and a smaller set of active amyloid inhibitors synthesized in the MIT Chemistry Department. The target reactions employ human ¿D- and ¿C-crystallins carrying amino acid modifications representing various forms of oxidative and mutational damage that have been found in cataracts. In addition, fragments of human cataractous aggregates from surgical procedures will be used as seeds in the in vitro reaction, and screened for compounds that inhibit addition of crystallin chains to the seeds. The initial hits will be characterized as to whether they inhibit the initiation or propagation of the aggregation process, and also by their effects on substrate binding by the lens chaperone ¿-crystallin. To improve the search for potential target binding sites and putative hits, NMR and cryoEM resources will be tapped to characterize the crystallin substrate/inhibitor interactions and the conformation of substrate/chaperone binding sites. This will enable utilization of powerful in silico computational searches for target sites and new compounds. The Initial hits will be improved by chemical modification and further characterization in vitro. Such putative anti-cataract agents would then be candidates for testing in animal models of cataract.