Functional characterization of the antiepileptic drug candidate, padsevonil, on GABAA receptors.

OBJECTIVE: The antiepileptic drug candidate, padsevonil, is the first in a novel class of drugs designed to interact with both presynaptic and postsynaptic therapeutic targets: synaptic vesicle 2 proteins and γ-aminobutyric acid type A receptors (GABAA Rs), respectively. Functional aspects of padsevonil at the postsynaptic target, GABAA Rs, were characterized in experiments reported here. METHODS: The effect of padsevonil on GABA-mediated Cl- currents was determined by patch clamp on recombinant human GABAA Rs (α1ß2γ2) stably expressed in a CHO-K1 cell line and on native GABAA Rs in cultured rat primary cortical neurons. Padsevonil selectivity for GABAA R subtypes was evaluated using a two-electrode voltage clamp on recombinant human GABAA Rs (α1-5/ß2/γ2) in Xenopus oocytes. RESULTS: In recombinant GABAA Rs, padsevonil did not evoke Cl- currents in the absence of the agonist GABA. However, when co-administered with GABA at effective concentration (EC)20 , padsevonil potentiated GABA responses by 167% (EC50 138 nmol/L) and demonstrated a relative efficacy of 41% compared with zolpidem, a reference benzodiazepine site agonist. Similarly, padsevonil demonstrated GABA-potentiating activity at native GABAA Rs (EC50 208 nmol/L) in cultured rat cortical neurons. Padsevonil also potentiated GABA (EC20 ) responses in GABAA Rs expressed in oocytes, with higher potency at α1- and α5-containing receptors (EC50 295 and 281 nmol/L) than at α2- and α3-containing receptors (EC50 1737 and 2089 nmol/L). Compared with chlordiazepoxide-a nonselective, full GABAA R agonist-the relative efficacy of padsevonil was 60% for α1ß2γ2, 26% for α2ß2γ2, 56% for α3ß2γ2, and 41% for α5ß2γ2; no activity was observed at benzodiazepine-insensitive α4ß2γ2 receptors. SIGNIFICANCE: Results of functional investigations on recombinant and native neuronal GABAA Rs show that padsevonil acts as a positive allosteric modulator of these receptors, with a partial agonist profile at the benzodiazepine site. These properties may confer better tolerability and lower potential for tolerance development compared with classic benzodiazepines currently used in the clinic.

Discovery of a small molecule modulator of the Kv1.1/Kvß1 channel complex that reduces neuronal excitability and in vitro epileptiform activity.

AIMS: Kv1.1 (KCNA1) channels contribute to the control of neuronal excitability and have been associated with epilepsy. Kv1.1 channels can associate with the cytoplasmic Kvß1 subunit resulting in rapid inactivating A-type currents. We hypothesized that removal of channel inactivation, by modulating Kv1.1/Kvß1 interaction with a small molecule, would lead to decreased neuronal excitability and anticonvulsant activity. METHODS: We applied high-throughput screening to identify ligands able to modulate the Kv1.1-T1 domain/Kvß1 protein complex. We then selected a compound that was characterized on recombinant Kv1.1/Kvß1 channels by electrophysiology and further evaluated on sustained neuronal firing and on in vitro epileptiform activity using a high K+ -low Ca2+ model in hippocampal slices. RESULTS: We identified a novel compound able to modulate the interaction of the Kv1.1/Kvß1 complex and that produced a functional inhibition of Kv1.1/Kvß1 channel inactivation. We demonstrated that this compound reduced the sustained repetitive firing in hippocampal neurons and was able to abolish the development of in vitro epileptiform activity. CONCLUSIONS: This study describes a rational drug discovery approach for the identification of novel ligands that inhibit Kv1.1 channel inactivation and provides pharmacological evidence that such a mechanism translates into physiological effects by reducing in vitro epileptiform activity.

GRIN2B gain of function mutations are sensitive to radiprodil, a negative allosteric modulator of GluN2B-containing NMDA receptors.

De novo gain of function mutations in GRIN2B encoding the GluN2B subunit of the N-methyl-d-aspartate (NMDA) receptor have been linked with epileptic encephalopathies, including infantile spasms. We investigated the effects of radiprodil, a selective GluN2B negative allosteric modulator and other non-selective NMDA receptor inhibitors on glutamate currents mediated by NMDA receptors containing mutated GluN2B subunits. The experiments were performed in Xenopus oocytes co-injected with the following human mRNAs: GRIN1/GRIN2B, GRIN1/GRIN2B-R540H, GRIN1/GRIN2B-N615I and GRIN1/GRIN2B-V618G. Glutamate displayed slightly increased potency in the R540H variant, but not in N615I and V618G variants. However, the inhibition by Mg2+ was completely abolished in N615I and V618G variants. In fact, Mg2+ enhanced glutamate responses in those variants. The potency of radiprodil to block glutamate-evoked currents was not affected in any of the variants, while the effects by non-selective NMDA inhibitors were greatly reduced in some of the variants. Additionally, in the Mg2+ insensitive variants, radiprodil blocked glutamate-activated currents with the same potency as in the absence of Mg2+. The gain of function observed in the reported GRIN2B variants could be a key pathophysiological factor leading to neuronal hyper-excitability in epileptic encephalopathies. The GluN2B-selective inhibitor radiprodil fully retained its pharmacological profile under these conditions, while other non-selective NMDA receptor antagonists lost their potency. Consequently, our data suggest that radiprodil may be a valuable therapeutic option for treatment of pediatric epileptic encephalopathies associated with GRIN2B mutations.