High-Content Screening to identify small molecules for refolding SOD1 mutants

肌萎缩性侧索硬化症（ALS）是一种致命的神经退行性疾病，其特征是运动神经元进行性变性，在出现症状后3-5年内导致肌肉萎缩、瘫痪和死亡。唯一被fda批准用于治疗ALS的药物利鲁唑（Riluzole）只有中等效果，平均可延长3个月的生存期。因此，迫切需要新的策略来开发能够显著提高ALS患者生存和生活质量的药物。令人信服的证据表明，铜锌超氧化物歧化酶（SOD1）错误折叠成毒性构象是约20%常见ALS和一些散发性ALS发病机制的基础。因此，稳定SOD1结构和恢复其天然构象应该是治疗SOD1相关ALS的理想途径。小分子化学伴侣已被FDA批准用于治疗由错误折叠蛋白质引起的其他疾病，如囊性纤维化和甲状腺转视蛋白淀粉样变性。然而，即使在实验阶段，也没有这样的化学伴侣蛋白用于错误折叠的SOD1的再折叠。在本提案中，我们将开发一种高含量筛选（HCS）方法来识别促进SOD1突变体重折叠到其野生型（wt）构象的小分子。我们的初步研究有力地支持了这种筛选策略的成功发展。我们已经确定了一个肽序列，该序列通常隐藏在原生wt SOD1中，但暴露在als连锁SOD1突变体和错误折叠形式的wt SOD1中。重要的是，暴露的肽序列类似于核输出信号（NES），确实具有核输出活性。因此，由于nes样肽（NLP）介导的核输出，错误折叠的wt SOD1和SOD1突变体被从细胞核中清除，而原生wt SOD1则定位于细胞核和细胞质中。我们提出了一种基于成像的检测方法，以识别能够恢复SOD1突变体核定位的小分子，并将其作为重折叠到原生wt SOD1构象的指标。我们已经产生了一种针对NLP的抗体，它可以特异性识别SOD1突变体和错误折叠的wt SOD1，但不能识别细胞中的原生wt SOD1。因此，NLP抗体将用于区分错误折叠构象与SOD1的天然形式，从而验证SOD1突变体在复合处理后的折叠状态。这些初步的研究引导我们追求三个特定的目标：1)开发一个基于细胞的双链平台，用于鉴定将突变SOD1重新折叠成其原生wt构象的小分子伴侣蛋白；2)对NCGC Pharmaceutical Collection化合物库进行自动先导筛选，以鉴定突变SOD1重新折叠伴侣蛋白；3)命中验证和表征。完成后，拟议的研究将建立第一个HCS分析和重复SOD1的化学伴侣的验证管道。未来的研究将包括与NCATS的Mark Henderson博士合作进行大规模筛查。此外，本研究确定的候选伴侣蛋白将与相关领域的专家合作进行临床前评估和优化，最终目标是通过直接纠正SOD1错误折叠，开发出临床相关的一类药物来治疗SOD1相关的ALS。

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons, leading to muscular atrophy, paralysis and death in 3-5 years from the onset of symptoms. The only FDA-approved drug for the treatment of ALS, Riluzole, has only moderate effects, prolonging survival by three months on average. Therefore, new strategies are urgently needed to develop drugs that can significantly improve survival and quality of life of ALS patients. Compelling evidence indicates that the misfolding of copper and zinc superoxide dismutase (SOD1) into toxic conformation underlies the pathogenesis of about 20% of familiar ALS and maybe, also some sporadic ALS. Therefore, stabilization of SOD1 structure and restoration of its native conformation should be a desirable approach to cure SOD1-related ALS. Small molecule chemical chaperones have been approved by FDA for treating other diseases caused by misfolded proteins, such as cystic fibrosis and transthyretin amyloidoses. However, no such chemical chaperone has been reported for refolding of misfolded SOD1, even at experimental stage. In this proposal, we will develop a high-content screening (HCS) approach to identify small molecules that promote refolding of SOD1 mutants to their wild type (wt) conformation. Successful development of such a screening strategy is strongly supported by our preliminary studies. We have identified a peptide sequence that is normally concealed in native wt SOD1 but exposed in ALS-linked SOD1 mutants and a misfolded form of wt SOD1. Importantly the exposed peptide sequence resembles nuclear export signal (NES) and indeed, has nuclear export activity. Consequently, misfolded wt SOD1 and SOD1 mutants are cleared from the nucleus due to the NES-like peptide(NLP)-mediated nuclear export, while native wt SOD1 is localized in both the nucleus and the cytoplasm. We propose an imaging-based assay to identify small molecules that restore nuclear localization of SOD1 mutants as an indicator of refolding to native wt SOD1 conformation. We have generated an antibody against NLP that specifically recognizes SOD1 mutants and misfolded wt SOD1, but not native wt SOD1 in cells. Accordingly, the NLP antibody will be used to distinguish the misfolded conformation from the native form of SOD1, and thereby can validate the folding status of SOD1 mutants after compound treatment. These preliminary studies lead us to pursue three specific aims: 1) develop a cell-based biplex platform for primary and counterscreens to identify small molecule chaperones that refold mutant SOD1 to its native wt conformation, 2) perform an automated pilot screening of NCGC Pharmaceutical Collection compound library to identify mutant SOD1 refolding chaperones, and 3) hit validation and characterization. Upon completion, the proposed research will establish the first HCS assay and hit validation pipeline for chemical chaperones that refold SOD1. Future studies will include large-scale screen in collaboration with Dr. Mark Henderson at NCATS. Furthermore, candidate chaperones identified in this study will undergo preclinical assessment and optimization in collaboration with experts in related fields, with the ultimate goal being a clinically relevant class of drugs to treat SOD1-linked ALS through direct correction of SOD1 misfolding.