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

常染色体显性遗传性白质营养不良(ADLD)是一种以广泛的中枢神经系统脱髓鞘和显著发病率为特征的致死性、进展性成人疾病。发病年龄较晚，加上疾病进展相对缓慢，为这种疾病提供了一个很大的治疗窗口。然而，ADLD目前还没有治疗方法，这代表着一种紧急的和未得到满足的临床需求。我们之前已经证明，ADLD是由于Lamin B1基因的复制导致Lamin B1蛋白表达增加所致。在真核细胞中，层蛋白B1是核层的主要组成部分，核层是靠近内核膜的纤维网络。核膜维持细胞核的结构完整性，在多种细胞过程中起着重要作用。我们最近已经证明，具有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学特征。我们设计的ADLD患者成纤维细胞和小鼠胚胎成纤维细胞(MEF)过表达Lamin B1，表现出明显的和可量化的核异常。由于层蛋白B1水平的升高是引发ADLD的原因，旨在降低层蛋白B1水平和相关功能后果的方法代表了一种发现小分子ADLD疗法的有效和创新的策略。我们已经开发了高含量分析来定义、分析和量化由lamin B1过度表达引起的异常核表型。初步检测结果表明，该检测方法能够满足HTS标准。在三个特定目标中，我们将1)根据公认的筛选标准优化HCS分析；2)使用匹兹堡大学药物发现研究所(UPDDI)维护的NIH分子文库小分子仓库(MLSMR)进行大规模HTS；以及3)证实ADLD患者成纤维细胞中层蛋白B1的下调以及对层蛋白B1高表达小鼠的少突胶质细胞中功能相关脂修饰基因表达的影响。由于发现范式是基于明确的基因突变、临床观察和疾病相关模型，我们希望确定Lamin B1病理生理学的真正矫正者作为候选，在临床前小鼠模型和患者临床试验中开发出潜在的ADLD治疗方法。

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.