High-content screening for modulators of lamin B1 as a therapeutic target in autosomal dominant leukodystrophy

高内涵筛选核纤层蛋白 B1 调节剂作为常染色体显性遗传性脑白质营养不良的治疗靶点

• 批准号: 9912870
• 负责人: Quasar S Padiath
• 金额: $ 39.13万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912870
• 关键词: Adult; Age; Age of Onset; Animal Model; Architecture; Biological Assay; Cell Nucleus; Cell physiology; Cells; Cessation of life; Chemicals; Chromatin; Clinical; Clinical Trials; DNA Sequence Alteration; Data; Defect; Demyelinating Diseases; Demyelinations; Development; Disease; Disease Progression; Dose; Down-Regulation; Embryo; Engineering; Ensure; Eukaryotic Cell; Evaluation; Event; Exhibits; Fibroblasts; Functional disorder; Funding; Future; Gene Expression; Genes; Genetic; Grant; Heterogeneity; Human; Institutes; LMNB1 gene; Lamin B1; Lead; Libraries; Library Collection Development; Life; Link; Lipids; Mediating; Methodology; Mission; Modeling; Molecular Bank; Morbidity - disease rate; Multivariate Analysis; Mus; Myelin; National Institute of Neurological Disorders and Stroke; Nerve; Neuraxis; Neurodegenerative Disorders; Nuclear; Nuclear Inner Membrane; Nuclear Lamina; Nuclear Structure; Oligodendroglia; Onset of illness; Outcome; Patients; Penetration; Performance; Pharmaceutical Chemistry; Phase; Phenotype; Plant Roots; Play; Positioning Attribute; Property; Proteins; Protocols documentation; RNA Interference; Reducing Agents; Reporting; Role; Series; Structure; Structure-Activity Relationship; Symptoms; Testing; Therapeutic; Toxic effect; Transgenic Mice; Translational Research; United States National Institutes of Health; Universities; Wild Type Mouse; base; combinatorial chemistry; disability; drug discovery; efficacy study; experience; falls; high throughput screening; human disease; illness length; improved; in vivo; indexing; innovation; lead optimization; leukodystrophy; member; mouse model; novel; overexpression; pre-clinical; preclinical trial; programs; protein expression; repository; response; screening; single cell analysis; small molecule; small molecule libraries; statistics; therapeutic target

Autosomal dominant leukodystrophy (ADLD) is a fatal, progressive adult-onset disease characterized by widespread CNS demyelination and significant morbidity. The late age of onset together with the relatively slow progression of the disease provides a large therapeutic window for the disorder. However, no treatment exits for ADLD, representing an urgent and unmet clinical need. We have previously shown that ADLD is caused by duplications of the lamin B1 gene causing increased expression of the lamin B1 protein. In eukaryotic cells, lamin B1 is a major constituent of the nuclear lamina, a fibrous meshwork adjacent to the inner nuclear membrane. The nuclear lamina maintains the structural integrity of the nucleus and has essential roles in multiple cellular processes. We have recently demonstrated that transgenic mice with oligodendrocyte-specific over-expression of lamin B1 exhibit temporal and histo-pathological features reminiscent of the human disease. ADLD patient fibroblasts and mouse embryonic fibroblasts (MEFs) that we have engineered to overexpress lamin B1, manifest distinct and quantifiable nuclear abnormalities. As an increased level of lamin B1 is the causative agent triggering ADLD, approaches aimed at reducing lamin B1 levels and associated functional consequences represent a potent and innovative strategy for discovery of small molecule ADLD therapeutics. We have developed high-content analysis to define, analyze, and quantify the abnormal nuclear phenotype caused by lamin B1 overexpression. Preliminary assay performance suggests that the assay can satisfy HTS criteria. In three specific aims we will 1) optimize the HCS assay to accepted screening criteria; 2) perform a large scale HTS using the NIH Molecular Libraries Small Molecule Repository (MLSMR) maintained by University of Pittsburgh Drug Discovery Institute (UPDDI); and 3) credential positives for downregulation of lamin B1 in ADLD patient fibroblasts and for effects on the expression of functionally relevant lipid modifying genes in oligodendrocytes from lamin B1 overexpressing mice. As the discovery paradigm is based on a defined genetic mutation, clinical observations, and disease-relevant models, we expect to identify bona fide correctors of lamin B1 pathophysiology as candidates for development into potential therapies for ADLD in preclinical mouse models and clinical trials in patients.

常染色体显性白质营养不良（ADLD）是一种致命的进行性成人发病疾病，以广泛的中枢神经系统脱髓鞘和显著的发病率为特征。较晚的发病年龄和相对缓慢的疾病进展为该疾病提供了一个很大的治疗窗口。然而，目前还没有针对ADLD的治疗方法，这是一个迫切且未得到满足的临床需求。我们之前已经证明，ADLD是由层蛋白B1基因的复制引起的，引起层蛋白B1蛋白的表达增加。在真核细胞中，核层蛋白B1是核层的主要组成部分，核层是靠近核膜的纤维网。核层维持细胞核的结构完整性，在多种细胞过程中发挥重要作用。我们最近证明，少突胶质细胞特异性过表达层蛋白B1的转基因小鼠表现出与人类疾病相似的时间和组织病理特征。ADLD患者成纤维细胞和小鼠胚胎成纤维细胞（mef），我们已经设计过表达层粘连蛋白B1，表现出明显的和可量化的核异常。由于层粘连蛋白B1水平的升高是引发ADLD的病原体，旨在降低层粘连蛋白B1水平和相关功能后果的方法代表了发现小分子ADLD治疗方法的有效和创新策略。我们开发了高含量的分析来定义、分析和量化由层粘连蛋白B1过表达引起的异常核表型。初步分析结果表明，该方法可以满足HTS标准。在三个具体目标中，我们将1)优化HCS检测以达到公认的筛选标准；2)利用匹兹堡大学药物发现研究所（UPDDI）维护的NIH分子文库小分子库（MLSMR）进行大规模HTS；3) ADLD患者成纤维细胞中lamin B1的下调以及对lamin B1过表达小鼠少突胶质细胞中功能相关脂质修饰基因表达的影响均呈阳性。由于发现范式是基于确定的基因突变、临床观察和疾病相关模型，我们希望在临床前小鼠模型和患者临床试验中确定层状蛋白B1病理生理学的真正校正者，作为开发潜在治疗ADLD的候选药物。