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

描述（申请人提供）：成熟性白内障是老年人视力受损和丧失的主要原因。白内障手术和晶状体植入的成功改善了许多成年人的视力。然而，每年有超过200万例白内障手术，36亿美元的医疗费用对医疗保健系统来说是一个沉重的负担。经常需要第二次手术，加上偶尔出现的其他并发症，增加了额外的医疗费用和负担。另一个不幸的问题是，由于经济、社会或心理因素，成千上万的美国人仍然无法接受白内障手术。延缓白内障的发病或延缓其生长——即使是几年——将大大降低保健费用，提高老年人的生活质量。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。识别蛋白质聚集抑制剂是治疗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）的主要工作。在最后一个项目期间，开发了体外系统，其中部分未展开的人晶体蛋白在pH7下聚集成光散射复合物，在低ph下聚集成淀粉样纤维。体外聚集反应捕获了晶状体内白内障形成的许多特性。该项目将利用这些实验系统进行高通量筛选，以抑制晶体蛋白聚集反应的体外启动或传播。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组较小的活性淀粉样蛋白抑制剂。靶反应使用人类3D-和3c -晶体蛋白携带氨基酸修饰，代表各种形式的氧化和突变损伤，这些损伤已经在白内障中发现。此外，手术过程中人类白内障聚集物的碎片将用作体外反应的种子，并筛选抑制晶体蛋白链添加到种子中的化合物。最初的撞击将被表征为它们是否抑制聚集过程的启动或传播，以及它们对透镜伴侣1-结晶蛋白结合底物的影响。为了更好地寻找潜在的靶标结合位点和假定的靶点，将利用核磁共振和低温电镜资源来表征晶体蛋白底物/抑制剂的相互作用以及底物/伴侣结合位点的构象。这将使利用强大的计算机搜索目标位点和新化合物成为可能。通过化学修饰和进一步的体外表征，将提高初始命中值。这些假定的抗白内障药物将成为白内障动物模型试验的候选药物。

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.