Small chemical modulators of SMN biology as candidate therapeutics for SMA

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DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA)-the most common genetic cause of death in infancy-is a neurodegenerative disease caused by reduced expression of the survival motor neuron (SMN) protein. SMA patients have homozygous loss of the SMN1 gene and retain at least one copy of the nearly identical SMN2 gene. The SMN2 gene produces low levels of SMN leading to motor neuron degeneration and skeletal muscle wasting. No therapy for SMA is currently available. Therefore, there is an urgent need to identify treatments that can restore SMN levels or can correct the deficits downstream of SMN depletion. Since higher copy numbers of the SMN2 gene reduce SMA disease severity, to date most drug screening efforts have focused on increasing expression of SMN from the SMN2 gene. However, most of these have used reporter assays that do not directly assess SMN function. Thus, compounds acting on the disease pathway rather than on the disease trigger are not screened for. To address these shortcomings, we established cell model systems that use proliferation defects triggered by SMN deficiency in mammalian cells as phenotypic readout of functional SMN levels. In particular, we developed a NIH3T3 cell line with regulated knockdown of endogenous mouse SMN in which cell proliferation is dependent on SMN levels produced by the human SMN2 gene. This cell line was used to establish a cell-based proliferation assay in 96-well format, and the suitability of the assay to high- throughput screening applications was subsequently validated through small pilot screens. In this project, we propose to use this newly developed platform to screen a large library of chemical compounds in order to identify small molecule modulators of SMN biology as candidate SMA therapeutics. A unique advantage of the system we developed is its capacity to capture compounds that act through multiple mechanisms of action. In the primary screen, hits will be defined as compounds that promote proliferation of SMN-deficient NIH3T3 fibroblasts that contain the SMN2 gene. For his confirmation and selection, active compounds will be analyzed by repeat testing in multiple replicates and in counter screen assays specifically designed to reveal whether they act non-specifically. Confirmed hits will then be moved to the subsequent phase in our validation pipeline, which will employ a panel of orthogonal cell-based assays designed to capture different mechanisms of action. This will determine whether compounds increase SMN transcription or splicing, enhance SMN function or influence SMN-dependent downstream events. Compounds will also be tested for their ability to promote survival of SMA motor neurons differentiated from mouse embryonic stem cells. Together, these studies will provide a first insight into mechanism(s) of action and priority ranking of compounds for future in-depth analysis. We anticipate that this project will lead to the identification of diverse small molecule as candidate SMA therapeutic compounds to advance to preclinical studies. It may also lead to the discovery of compounds as new research tools to study SMN biology and disease mechanisms.