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

高内涵筛选鉴定用于重折叠 SOD1 突变体的小分子

• 批准号: 9310821
• 负责人: Shengyun Fang
• 金额: $ 30.32万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9310821
• 关键词: Aftercare; Alpha Cell; Amyloidosis; Amyotrophic Lateral Sclerosis; Antibodies; Binding; Biological Assay; Cell Nucleus; Cells; Cessation of life; Chemicals; Collaborations; Collection; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasm; Development; Disease; FDA approved; Familial Amyotrophic Lateral Sclerosis; Fluorescence; Future; Goals; Hela Cells; Human; Image; Immunofluorescence Immunologic; Lead; Libraries; Link; Mediating; Molecular Chaperones; Molecular Conformation; Monitor; Muscular Atrophy; Neurodegenerative Disorders; Nuclear; Nuclear Export; Paralysed; Pathogenesis; Pathway interactions; Patients; Peptide antibodies; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Pharmacotherapy; Prealbumin; Quality of life; Reporting; Research; Riluzole; Signal Transduction; Staining method; Stains; Structure; Superoxide Dismutase; Symptoms; Treatment Efficacy; Validation; base; clinical application; clinically relevant; copper zinc superoxide dismutase; counterscreen; cytotoxic; cytotoxicity; improved; inhibitor/antagonist; misfolded protein; motor neuron degeneration; mutant; oxidative DNA damage; pre-clinical; protein aminoacid sequence; public health relevance; restoration; screening; seal; small molecule; stable cell line

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.

肌萎缩侧索硬化症（Amyotrophic Lateral Sclerosis，ALS）是一种以运动神经元进行性变性为特征的致死性神经退行性疾病，从症状出现起3-5年内可导致肌肉萎缩、瘫痪和死亡。FDA批准的唯一一种治疗ALS的药物阿舒唑只有中等效果，平均延长生存期三个月。因此，迫切需要新的策略来开发可以显着改善ALS患者生存和生活质量的药物。令人信服的证据表明，铜和锌超氧化物歧化酶（SOD 1）的错误折叠成有毒构象的发病机制的基础上约20%的熟悉ALS，也可能是一些散发性ALS。因此，稳定SOD 1结构并恢复其天然构象应该是治愈SOD 1相关ALS的理想方法。小分子化学分子伴侣已被FDA批准用于治疗由错误折叠蛋白引起的其他疾病，如囊性纤维化和甲状腺素运载蛋白淀粉样变性。然而，没有这样的化学分子伴侣已经报道了错误折叠的SOD 1的重折叠，甚至在实验阶段。 在这项提案中，我们将开发一种高内涵筛选（HCS）的方法，以确定小分子，促进SOD 1突变体的重折叠到其野生型（wt）构象。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，通常隐藏在天然野生型SOD 1，但暴露在ALS连接的SOD 1突变体和野生型SOD 1的错误折叠形式。重要的是，暴露的肽序列类似于核输出信号（内斯），并且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 SOD 1和SOD 1突变体从核中清除，而天然的wt SOD 1定位于核和细胞质中。我们提出了一种基于成像的测定，以确定小分子，恢复核定位的SOD 1突变体作为一个指标的重折叠天然野生型SOD 1构象。我们已经产生了针对NLP的抗体，其特异性识别细胞中的SOD 1突变体和错误折叠的wt SOD 1，但不识别天然wt SOD 1。因此，NLP抗体将用于区分SOD 1的错误折叠构象与天然形式，从而可以验证化合物处理后SOD 1突变体的折叠状态。这些初步研究使我们追求三个具体目标：1）开发用于初级和反向筛选的基于细胞的双链平台，以鉴定将突变体SOD 1重折叠为其天然野生型构象的小分子伴侣，2）对NCGC Pharmaceutical Collection化合物文库进行自动化中试筛选，以鉴定突变体SOD 1重折叠伴侣，以及3）命中验证和表征。完成后，拟议的研究将建立第一个HCS检测，并为重新折叠SOD 1的化学分子伴侣建立验证管道。未来的研究将包括与NCATS的Mark亨德森博士合作进行的大规模筛查。此外，本研究中确定的候选分子伴侣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SOD 1错误折叠来治疗SOD 1相关ALS的临床相关药物。