Abstracts

Rationale: Malignant migrating partial seizures of infancy is a rare, devastating form of pediatric epilepsy most commonly associated with mutations in the potassium channel Slack. However, the lack of selective molecular probes and absence of an animal model of the disorder leaves many unanswered questions about the role Slack plays in childhood epilepsies. In order to address these concerns we have begun develop highly selective molecular probes, while exploring the neurobehavioral effects of a specific epilepsy-associated Slack mutation in a knock-in murine model. Methods: To discover selective Slack modulators, we screened >50,000 members of a structurally diverse library of drug-like small molecules using a HTS-compatible Tl+ flux assay on two engineered HEK293 cell lines that express either wildtype (WT) Slack, or a Slack mutant channel, A934T in a 384-well format. This particular mutation was chosen because it is associated with the most severe human MMSPI phenotype and was used to generate our genetically modified mouse. Using CRISPR-Cas9 we were able to generate a knockin mouse expressing an MMPSI-associated Slack mutation. Male and female mice representing WT, heterozygous and homozygous A913T Slack point mutation were analyzed in a series of neurobehavioral assays to assess motor activity, anxiety-related behaviors, and sensorimotor function. Studies are currently underway to evaluate the effects of the Slack A913T mutant on electrical activity in the brain. Results: We have identified inhibitors of mutant Slack channels with a 200% greater efficacy and 300-fold increase in potency than that of quinidine, the current add-on therapy for the treatment of MMPSI. The inhibitors we have discovered also showed improved selectivity across a variety of structurally related potassium channels. Furthermore, preliminary behavioral assays indicate a significant increase in the A913T homozygous mice anxiety-related behavior, indicated by an increase in the percentage of time spent in the closed chambers of elevated zero maze and light-dark transition tests (n=3-6, p<0.05). Conclusions: We have identified novel Slack inhibitors giving us a starting point to develop the potent and selective proves needed to help us assess the Slack channels' role in epilepsy as well as provide a mechanism to assess the therapeutic potential of selective Slack inhibition. Additionally, we have generated a mutant Slack mouse expressing an MMPSI-associated mutation. Preliminary behavioral studies show a trend of Slack knockin animals to be more anxious. Together these two approaches allow for the investigation of Slack's role in neuronal excitability and the future treatment of MMPSI. Funding: PhRMA Foundation Predoc Fellowship AwardR21 NS109521-0