Abstracts

Rationale: KCNQ2 epileptic encephalopathy arises due to mutations in the Kv7.2 voltage-gated potassium (K+) channel. Mutations conferring either a gain or loss of function in KCNQ2 often lead to early infantile epileptic encephalopathy (EIEE) that is usually refractory to conventional anti-epileptic drugs (AEDs). This highlights an acute need to find new therapeutic options for refractory KCNQ2 epilepsy patients. Methods: A wild-type or R201C mutant KCNQ2 gene, and a copy of the wild-type KCNQ3 gene, were transfected into CHO cells to generate cell models capable of producing an M-like K+ current. The cell lines were evaluated electrophysiologically to characterize the effect of the mutation on K+ related currents. A rubidium (86Rb+) efflux assay was adapted for high-throughput screening (HTS); Rb+ serves as a charge and size proxy for K+ flow. Exposure to KCl to stimulate channel opening, produced a concentration-dependent release of 86Rb+ from pre-loaded cells. Co-incubation of either cell line with XE-991, a KCNQ channel blocker, completely inhibited 86Rb+ efflux. Wild-type and R201C cell lines were screened against the Prestwick library (a collection of 1,280 approved, off-patent drugs), as well as a panel of AEDs and known K+ channel modulators. Results: Patch clamp evaluation demonstrated a voltage-dependent increase in outward K+ current for both cell lines. However, the R201C mutation conferred a significant leftward shift in the voltage-current relationship with activation (opening) at lower voltages and a significantly slower deactivation rate compared to wild-type cells resulting in excess K+ conductance. HTS was carried out with both cell lines using the 86Rb+ efflux assay and KCl to stimulate channel opening. Of the 1,320 compounds screened at 10 μM, 26 significantly reduced efflux from the wild-type channel and 36 significantly reduced efflux from the R201C mutant cell line. The entire 1,320 compound library that was screened demonstrated a surprisingly dichotomous distribution with the most active drugs inhibiting either the wild-type or the R201C cell line; only two compounds significantly inhibited 86Rb+ efflux from both cell lines. Re-screening of the lead compounds in the R201C cell line confirmed their activities in a concentration-dependent manner. The drug with the greatest inhibitory activity against the mutant cell line was paroxetine (an SSRI antidepressant; IC50 = 6.90 µM; maximum inhibition = 90%); other drugs with potent and significant inhibition of 86Rb+ efflux included acitretin (a retinoid), norgestimate (a steroidal) and hexestrol (a synthetic estrogen). Patch clamp evaluation of the lead compounds in the R201C cell line confirmed a concentration-dependent reduction in K+ current. Conclusions: This study demonstrates the utility of precision genetic modeling by replicating an underlying genetic mutation of EIEE in a cell model suitable for HTS. We identified a total of 60 compounds with inhibitory activity against K+ efflux, many of which have not been previously described to possess any activity against Kv7.2 channels. These compounds may hold therapeutic value for KCNQ2 patients with EIEE. Funding: This study was supported by Pairnomix, LLC.