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

常染色体显性脑白质营养不良（ADLD）是一种致命的、进行性的成人发病疾病，其特征是广泛的CNS脱髓鞘和显著的发病率。发病年龄晚，疾病进展相对缓慢，为该疾病提供了一个大的治疗窗口。然而，ADLD没有治疗，这代表了紧急且未满足的临床需求。 我们以前已经表明，ADLD是由核纤层蛋白B1基因的重复引起的，导致核纤层蛋白B1蛋白表达增加。在真核细胞中，核纤层蛋白B1是核纤层的主要组成部分，核纤层是与内核膜相邻的纤维网。核板层维持细胞核的结构完整性，并在多种细胞过程中发挥重要作用。我们最近已经证明，少突胶质细胞特异性过度表达核纤层蛋白B1的转基因小鼠表现出时间和组织病理学特征，让人想起人类疾病。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核纤层蛋白B1，表现出独特的和可量化的核异常。 由于核纤层蛋白B1水平的增加是触发ADLD的病原体，因此旨在降低核纤层蛋白B1水平和相关功能后果的方法代表了发现小分子ADLD治疗剂的有效和创新策略。我们已经开发了高含量的分析来定义，分析和量化核纤层蛋白B1过表达引起的异常核表型。初步测定性能表明，该测定可满足HTS标准。在三个具体目标中，我们将1）优化HCS测定以达到可接受的筛选标准; 2）使用由匹兹堡大学药物发现研究所（University of Pittsburgh Drug Discovery Institute，UDI）维护的NIH分子库小分子库（NIH Molecular Libraries Small Molecule Repository，MLSMR）进行大规模HTS;和3）在ADLD患者成纤维细胞中核纤层蛋白B1的下调以及对ADLD患者成纤维细胞中功能相关脂质修饰基因表达的影响的证书阳性。来自核纤层蛋白B1过表达小鼠的少突胶质细胞。由于发现范例是基于定义的基因突变，临床观察和疾病相关模型，我们希望确定真正的核纤层蛋白B1病理生理学校正剂作为临床前小鼠模型和患者临床试验中ADLD潜在疗法的候选药物。

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.