Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis

SUMMARY Tuberous sclerosis complex (TSC) is a rare genetic disorder caused by mutations in the mTOR pathway genes TSC1 or TSC2 . TSC can affect multiple organs including the brain, and most patients (75-90%) present with seizures during early childhood and intractable epilepsy throughout life. mTOR inhibitors, part of the current standard of care, lack the optimal characteristics to fully address patient phenotypes. Here, we report on the application of our all-optical electrophysiology platform for phenotypic screening in a human neuronal model of TSC. We used CRISPR/Cas9-isogenic TSC2 −/− iPS cell lines to identify disease-associated changes to neuronal morphology, transcript expression and neuronal excitability. We established a robust multiparametric electrophysiological phenotype which we then validated in TSC patient-derived neurons. We used this phenotype to conduct a screen of ∼30,000 small molecule compounds in human iPS cell-derived neurons and identified chemical scaffolds that rescued the functional TSC disease parameters. Confirmed hits may act via different mechanisms than direct mTOR pathway inhibition. This strategy provides molecular starting points for therapeutic development in TSC and a framework for phenotype discovery and drug screening in other neurological disorders.