Abstracts

Rationale: Activation of Kv7 channels is a proven mechanism for anticonvulsant activity across many animal models (Large et al., Epilepsia 2012). Dominant-negative mutations within Kv7.2 cause neonatal epileptic encephalopathies (KCNQ2-NEE) (Micelli et al., Proc Natl Acad Sci U S A, 2013; Orhan et al., Ann Neurol, 2014). Preliminary evidence suggests treatment with the Kv7.2/7.3 channel activator ezogabine (EZO) may reduce seziures and improve neurodevelopment in infants with KCNQ2-NEE (Millichap et al., Neurol Genet, 2016). Given EZO’s limited tolerability, off-target effects, and withdrawal from the market, we designed a novel Kv7.2/7.3 channel activator differentiated from EZO by structural class, improved potency, increased tolerability, selectivity over GABAA, and stability to photo-oxidation. Methods: Engineered cell lines expressing Kv7 channels were used to evaluate new compound activities in fluorescence and electrophysiological assays. Characterization of the half-activating voltage (V1/2) was measured by automated patch clamp technique with the QPatch HTX instrumentation. Positive allosteric modulation against the human α1β3ɣ2 GABAA receptor was evaluated on the IonFlux HT platform. KB-3061 was screened at 10 µM for off-target activities. Chemical stability was evaluated in a standard forced degradation photo-oxidation assay. Anticonvulsant activity was evaluated PO in rats dosed 0.3 - 30 mg/kg in the maximal electroshock seizure model (MES). Prior to electroshock, tolerability was assessed by neurological scoring (NS). Results: KB-3061 was discovered in a fluorescence-based assay to be a potent activator of Kv7.2/7.3 ion channels with an EC50 of 440 nM. When tested by QPatch, KB-3061 produced a -16 ± 1.1 mV (n=3) V1/2 shift at 1.1 µM. In the human α1β3ɣ2 GABA receptor PAM assay, KB-3061 had little to no effect at 10 µM. In photostability testing, KB-3061 was stable to visible light exposure up to 75 hrs. In Kv7.1/minK and hERG QPatch testing, KB-3061 produced no substantial inhibition up to 30 µM (23% and 9% inhibitions, respectively). In broad panel screening including GPCR, ion channel, transporter, and kinase targets, 10 µM KB-3061 exhibited no activity above the 25% significance threshold. In a rat MES model of epilepsy, KB-3061 provided 100% protection against seizures starting at 3 mg/kg with no change in NS up to 30 mg/kg (n = 6 per group). In comparison, EZO has a thallium flux EC50 of 1.1 uM, is completely degraded after 75 hr of light exposure, is active on the human α1β3ɣ2 GABA receptor, and provided 100% protection against seizures at 75 mg/kg with changes in NS starting at 45 mg/kg. Conclusions: KB-3061 is a potent Kv7.2/7.3 channel activator from a novel structural class and exhibits excellent in vivo anticonvulsant activity and tolerability over a range of doses tested. Compared to EZO, KB-3061 is more potent, better tolerated, selective against the α1β3ɣ2 GABA receptor, and stable to photo-oxidation. KB-3061’s in vivo profile coupled with its lack of off-target effects supports further development towards a new treatment for KCNQ2-NEE patients. Funding: NINDS U44NS093160