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群视力受损和丧失的主要原因。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，由于每年有200多万例白内障手术，36亿美元的医疗费用对医疗体系来说是一个巨大的负担。频繁地需要第二次手术，加上偶尔出现的其他并发症，增加了医疗保健成本和负担。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。推迟白内障的发病或减缓其增长--即使是几年--将极大地降低医疗保健成本，并提高老年人的生活质量。这表明了投资于预防的价值，包括开发抑制白内障形成的小分子疗法。寻找蛋白质聚集的抑制剂是治疗其他蛋白质聚集疾病的主要工作，如阿尔茨海默病、帕金森病和亨廷顿病。在上一个项目阶段，人们开发了体外系统，在pH为7时，部分解开的人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。体外聚集反应捕捉到了晶状体内形成白内障的一些特性。该项目将利用这些实验系统，高通量筛选抑制晶体蛋白聚集反应的体外启动或繁殖的化合物。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。靶反应使用携带氨基酸修饰的人类3D-和3C-晶体蛋白，代表在白内障中发现的各种形式的氧化和突变损伤。此外，来自外科手术的人类白内障聚集体的碎片将被用作体外反应的种子，并筛选出抑制晶体蛋白链添加到种子上的化合物。最初的HITS的特征是它们是否抑制聚集过程的启动或传播，以及它们对晶状体伴侣1-晶体蛋白与底物结合的影响。为了更好地寻找潜在的靶结合位点和可能的HITS，将利用核磁共振和低温EM资源来表征晶体蛋白底物/抑制剂的相互作用和底物/伴侣结合位点的构象。这将使在电子计算中搜索目标位置和新化合物的强大功能得以利用。初步的HITS将通过化学修饰和进一步的体外表征而得到改善。然后，这些假定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药物。 与公共健康相关：白内障是导致美国老年人失明的主要原因。拟议中的识别一组干扰受损或扰动的人类晶状体晶体蛋白在试管中聚集的小分子将为低成本的抗白内障治疗提供候选。如果这类药物的一部分能够将白内障的发病推迟几年，或者延缓白内障的生长，它们将显著减缓许多老年人的视力丧失和昂贵的白内障手术的需要。

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.