Rapid characterization PRAX-330 of effects on hNav1.2 GOF mutants

Mutations in genes encoding voltage-gated sodium (NaV) channels are associated with a variety of epilepsy syndromes with diverse clinical severity.1-12 Diverse patterns of NaV channel dysfunction may underlie differences in disease severity, clinical course, and pharmacological responses among the associated epilepsies. Mutations in SCN2A and SCN8A are associated with early-onset epileptic encephalopathy (EOEE). Early investigations suggest that gain-of-function (GOF) is common among EOEE-associated mutations in both genes, but loss-of-function (LOF) defects have also been observed. Knowing which variants can be pharmacologically corrected by specific drugs or investigational compounds would enable clinical studies to determine the efficacy of in vitro-predicted gene- and variant-specific treatments. There are no valid computational methods to predict the pharmacological effects of antiepileptic drugs on epilepsy-associated variants, hence this information has to be obtained experimentally. Importantly, the FDA recently approved an expanded indication for the cystic fibrosis drug ivacaftor based entirely on in vitro assay data demonstrating which types of CFTR mutations were responsive to the medication.13 This represents a paradigm shift in the drug approval process for rare genetic diseases. The availability of a robust, rigorous, and standardized platform for testing drugs on specific ion channel variants will be valuable for achieving the goal of precision medicine in genetic epilepsy and may contribute to future drug approvals. We propose to experimentally determine the effects of PRAX-330 on a panel of recurrent SCN2A mutations associated with EOEE. We will employ automated patch clamp recording on the Nanion Syncropatch 768PE platform. Variants will be engineered in recombinant human NaV1.2 plasmids and expressed heterologously in HEK-293T cells. For some variants that are associated with very early onset epilepsy, we will engineer both canonical and neonatal expressed splice isoforms. Electrophysiological experiments will first determine the biophysical properties of each variant in the absence of compound to establish the primary functional defects. Some previously characterized variants with established GOF defects will be included as controls. Next, we will assay variant channels in the absence and presence of PRAX-330 at a screening concentration (250 nM) to determine general effects of the compound on each variant. We will specifically examine effects of PRAX-330 on the time course of fast inactivation, level of peak and persistent current (tonic block), voltage-dependence of fast inactivation, conductance-voltage relationships, time course of recovery from fast inactivation, and frequency-dependent block of peak current. Assays will be performed at a holding potential -120 mV and -90 mV. Based on results from these preliminary measurements, we will examine 7-point concentration-response relationships for the effect of PRAX-330 on the major functional defects exhibited by individual variants. The IC50 values will be compared between variants and wildtype channels. We predict that some variants will exhibit enhanced PRAX-330 sensitivity, and that the compound will selectively correct certain types of GOF biophysical properties exhibited by variant channels.