PHARMACOLOGICAL AND GENETIC APPROACHES PROBING THE ROLE OF THE SLACK POTASSIUM CHANNEL IN CHILDHOOD EPILEPSIES

Abstract

Malignant migrating partial seizures of infancy is a rare, devastating form of epilepsy most commonly associated with gain-of-function mutations in the potassium channel, Slack. Not only is this condition almost completely pharmacoresistant, there are not even selective drug-like tools available to evaluate whether inhibition of these over-activated, mutant Slack channels may represent a viable path forward toward new anti-epileptic therapies. Therefore, we used a high-throughput thallium flux assay to screen a drug-like, 100,000-compound library in search of inhibitors of both wild-type and a disease-associated mutant Slack channel. Using this approach, we discovered VU0606170, a selective Slack channel inhibitor with low µM potency. Critically, VU0606170 also proved effective at significantly decreasing the firing rate in over-excited, spontaneously firing cortical neuron cultures. Additionally, we have generated a mouse model of this condition by replacing the wild-type gene with one encoding KCNT1 A913T, a cytoplasmic C-terminal mutation homologous to the human A934T variant that results in MMPSI. We compared behavior patterns and seizure activity in these mice with those of wild-type mice. Anxiety-related behaviors were found to be increased in KCNT1+/A913T and KCNT1A913T/A913T animals in a gene dose-dependent manner. Similarly, increased response to startle stimuli and decreased sensorimotor gating were altered in a gene dose-dependent manner. Video-EEG monitoring of KCNT1+/A913T and KCNT1A913T/A913T animals revealed persistent interictal spikes and spontaneous seizures. Taken together, our data provide two critical tools for advancing our understanding of the role of the Slack channel in normal physiology and disease, as well as its potential as a target for therapeutic intervention. We have generated a novel animal model of MMPSI, recapitulating several phenotypes seen in this patient population, including an increase in anxiety-like behaviors, deficits in sensorimotor gating and the presence of spontaneous seizures. Furthermore, the identification and extensive characterization of VU0606170 provides compelling evidence that selective inhibition of Slack channel activity can be achieved with small molecules and that inhibition of Slack channel activity in neurons produces efficacy consistent with an anti-epileptic effect. All in all, these studies provide a promising foundation for the drug discovery efforts for these important channels and provide a framework for evaluating Slack inhibitors in vivo.