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

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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.