Identification of New Hit Compounds Using a High-Throughput Phenotypic Screen with SMA Patient iPSC-Derived Motor Neurons

Spinal muscular atrophy (SMA) is an inheritable cause of infant mortality that is characterized by the loss of lower motor neurons and skeletal muscle atrophy. The degeneration of motor neurons is caused by insufficient levels of survival motor neuron (SMN) protein, which is encoded by two nearly identical genes SMN1 and SMN2. Most cases of SMA harbour homozygous deletions of the SMN1 gene and retain at least one copy of SMN2. Hence, a promising treatment strategy is to upregulate levels of the full-length SMN protein originating from the SMN2 gene. Drug discovery screening platforms typically use SMA fibroblasts or lymphocytes, yet the identified molecules often had limited efficacy in SMA mouse models, especially rescuing motor neuron (MN) degeneration. Therefore, MNs from SMA patients should be used early in drug discovery to increase the likelihood of identifying effective small molecule therapeutics. At BrainXell, we established new technologies to rapidly differentiate SMA patient induced pluripotent stem cells (iPSCs) into large quantities of neurons. We also used genome editing to endogenously fuse SMN2 with a nanoluciferase (NLuc) reporter, which enables high-throughput screening (HTS) that monitors the expression levels of SMN after 48h exposure to each compound. The assay was adapted to meet HTS requirements, including: large batch sizes, 1536-well format, minimal well-to-well variation, short-term culture, plating by automated dispenser, and low reagent volumes. Applying a quantitative HTS approach, we screened the LOPAC, NPC, and MIPE libraries (>6,000 compounds) in a dose dependent manner. After demonstrating feasibility, we expanded the screen to the larger Genesis library (~95,000 compounds) in order to identify novel hit molecules. Compounds that increased SMN2 expression by >20% were considered hits. Analysis of the combined ~100,000 compound qHTS identified 81 hit candidates, which were rescreened in triplicate. Ten compounds increased SMN2 expression by 20% with EC50 <10 μM. We then used an ELISA to validate the increased SMN2 expression after 48h treatment. This screening paradigm identified and validated at least one new hit compound that has promising efficacy, but the potency will require optimization.