Abstracts

Rationale: Healthcare professionals are increasingly interested in repurposing approved drugs for the treatment of rare genetic conditions. The traditional approach to repurposing has included a literature review of approved compounds and testing only a few candidates. This approach leaves open the possibility that there may be other untested and potentially more effective epilepsy treatment alternatives amongst already approved drugs. Here we introduce an individualized approach for the identification of candidate drugs for repurposing in a rare genetic disease, focusing on SCN8A epileptic encephalopathy. Methods: We generated a HEK293 cell line containing wild-type (WT) SCN8A with the SCN8A R1872Q mutation carried in a patient presenting with epileptic encephalopathy. The R1872Q mutation was created by site-directed mutagenesis of WT SCN8A plasmid (5616G>A; CGG>CAG) and transfected into HEK293 cells. The DNA sequences and plasmid transcript levels were confirmed by Sanger sequencing and qRT-PCR. A clonal line was established and electrophysiological characterization was performed using the Molecular Devices PatchXpress platform for whole cell recordings. High throughput screening with > 1,000 approved drugs was carried out using stimulus (veratidine)-activated fluorescence-based Na+ flux FLIPR technology to identify active compounds. Results: Characterization of electrophysiological properties of SCN8A R1872Q cell lines revealed altered biophysical properties, the most pronounced of which was a slower rate of inactivation in the R1872Q variant as compared to wild-type (p < 0.05). This change in inactivation kinetics contributes to the hyperexcitable state in cells that constitutes a gain-of-function phenotype. Next, high throughput screening was carried out using a compound library of approved drugs using FLIPR-based screening. Of the compounds tested, 90 compounds produced greater than 63% inhibition (> 2 standard deviations from mean inhibition) of the stimulus-activated state and a further 80 compounds were able to produce an inhibition effect between 40 - 63%. Conclusions: Using this individualized screening approach, we identified a number of already approved compounds that inhibit the gain-of-function phenotype associated with the pathogenic SCN8A R1872Q mutation. Many of these compounds show acceptable side effect profiles, cross the brain blood barrier, and appear to be attractive candidates for evaluation in patients with SCN8A R1872Q epilepsy. These results not only identify new candidates for healthcare professionals to consider for treatment in patients with SCN8A R1872Q epilepsy, but also illustrate the importance of a systematic approach to the identification of candidate repurposed drugs as potential options for the treatment of rare genetic diseases. Funding: This work was funded by Pairnomix, LLC.