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

描述（由申请人提供）：成熟型白内障是老年人视力受损和丧失的主要原因。白内障手术和透镜植入术的成功改善了许多成年人的这种情况。然而，每年有超过200万例白内障手术，36亿美元的医疗保健费用是医疗保健系统的重大负担。经常需要第二次手术，加上偶尔的其他并发症，增加了额外的医疗保健费用和负担。另一个不幸的问题是，由于经济、社会或心理因素，数十万美国人仍然无法接受白内障手术。如果能延迟白内障的发病时间或减慢其生长速度，即使只延迟数年，也会大大减低长者的医疗费用，并改善他们的生活质素。这表明投资于预防的价值，包括开发抑制白内障形成的小分子疗法。鉴定蛋白质聚集的抑制剂是用于其它蛋白质聚集疾病如阿尔茨海默氏症、帕金森氏症和亨廷顿氏症的主要努力。在最后一个项目期间，在体外系统中，部分未折叠的人晶状体蛋白聚集成光散射复合物在pH 7和淀粉样蛋白纤维在低pH值。体外聚集反应捕获白内障形成的透镜内的一些属性。本项目将利用这些实验系统进行高通量筛选的化合物，抑制在体外启动或传播的晶体蛋白聚集反应。最初的努力将利用哈佛医学院朗伍德设施提供的25万种化合物，以及麻省理工学院化学系合成的一小批活性淀粉样蛋白抑制剂。靶反应使用携带代表在白内障中发现的各种形式的氧化和突变损伤的氨基酸修饰的人3D-和3C-晶状体蛋白。此外，将来自外科手术的人白内障聚集体的片段用作体外反应中的种子，并筛选抑制晶体蛋白链添加到种子中的化合物。初始命中将表征为它们是否抑制聚集过程的起始或传播，以及它们对透镜伴侣1-晶状体蛋白的底物结合的影响。为了改善对潜在靶结合位点和推定命中的搜索，将利用NMR和cryoEM资源来表征晶状体蛋白底物/抑制剂相互作用和底物/伴侣结合位点的构象。这将使利用强大的计算机计算搜索的目标网站和新的化合物。将通过化学修饰和进一步体外表征来改善初始命中。这样的推定的抗白内障剂然后将是用于在白内障动物模型中测试的候选物。 公共卫生相关性：白内障是美国老年人视力丧失的主要原因。提出的鉴定一组干扰试管中受损或扰动的人透镜晶体蛋白聚集的小分子将为低成本抗白内障治疗提供候选物。如果这些药物的一个子集能够将白内障的发病延迟几年，或延缓白内障的生长，它们将显着减缓视力丧失和许多老年人对昂贵的白内障手术的需求。

DESCRIPTION (provided by applicant): 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 3D- and 3C-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 1-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. PUBLIC HEALTH RELEVANCE: Cataracts are the leading cause of vision loss among older Americans. The proposed identification of a set of small molecules that interfere with aggregation of damaged or perturbed human lens crystallins in the test tube will provide candidates for low-cost anti-cataract therapy. If a subset of such agents were able to delay the onset of cataract by a few years, or retard cataract growth, they would significantly slow vision loss and the need for expensive cataract surgery for many older adults.