Background/Objectives: Recent studies, typically using patient cerebrospinal fluid (CSF), have suggested that different autoantibodies (Aabs) acting on their respective receptors, may underlie neuropsychiatric disorders. The GluN1 (NR1) subunit of the N-methyl-D-aspartate receptor (NMDAR) has been identified as a target of anti-NMDAR Aabs in a number of central nervous system (CNS) diseases, including encephalitis and autoimmune epilepsy. However, the role or the nature of Aabs responsible for effects on neuronal excitability and synaptic plasticity is yet to be established fully. Methods: Peptide immunisation was used to generate Aabs against selected specific GluN1 extracellular sequences based on patient-derived anti-NMDAR Aabs that have been shown to bind to specific regions within the GluN1 subunit. ‘Protein A’ purification was used to obtain the total IgG, and further peptide purification was used to obtain a greater percentage of NMDAR-target specific IgG Aabs. The binding and specificity of these anti-NMDAR Aabs were determined using a range of methodologies including enzyme-linked immunosorbent assays, immunocytochemistry and immunoblotting. Functional effects were determined using different in vitro electrophysiology techniques: two-electrode voltage-clamps in Xenopus oocytes and measures of long-term potentiation (LTP) in ex vivo hippocampal brain slices using multi-electrode arrays (MEAs). Results: We show that anti-NMDAR Aabs generated from peptide immunisation had specificity for GluN1 immunisation peptides as well as target-specific binding to the native protein. Anti-NMDAR Aabs had no clear effect on isolated NMDARs in an oocyte expression system. However, peptide-purified anti-NMDAR Aabs prevented the induction of LTP at Schaffer collateral-CA1 synapses in ex vivo brain slices, consistent with causing synaptic NMDAR hypofunction at a network level. Conclusions: This work provides a solid basis to address outstanding questions regarding anti-NMDAR Aab mechanisms of action and, potentially, the development of therapies against CNS diseases.

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 gamma-aminobutyric acid type A receptors (GABA(A)Rs), respectively. Functional aspects of padsevonil at the postsynaptic target, GABA(A)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 GABA(A)Rs (alpha 1 beta 2 gamma 2) stably expressed in a CHO-K1 cell line and on native GABA(A)Rs in cultured rat primary cortical neurons. Padsevonil selectivity for GABA(A)R subtypes was evaluated using a two-electrode voltage clamp on recombinant human GABA(A)Rs (alpha 1-5/beta 2/gamma 2) in Xenopus oocytes. Results In recombinant GABA(A)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 GABA(A)Rs (EC50 208 nmol/L) in cultured rat cortical neurons. Padsevonil also potentiated GABA (EC20) responses in GABA(A)Rs expressed in oocytes, with higher potency at alpha 1- and alpha 5-containing receptors (EC50 295 and 281 nmol/L) than at alpha 2- and alpha 3-containing receptors (EC50 1737 and 2089 nmol/L). Compared with chlordiazepoxide-a nonselective, full GABA(A)R agonist-the relative efficacy of padsevonil was 60% for alpha 1 beta 2 gamma 2, 26% for alpha 2 beta 2 gamma 2, 56% for alpha 3 beta 2 gamma 2, and 41% for alpha 5 beta 2 gamma 2; no activity was observed at benzodiazepine-insensitive alpha 4 beta 2 gamma 2 receptors. Significance Results of functional investigations on recombinant and native neuronal GABA(A)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.

Summary 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 Kvβ1 subunit resulting in rapid inactivating A‐type currents. We hypothesized that removal of channel inactivation, by modulating Kv1.1/Kvβ1 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/Kvβ1 protein complex. We then selected a compound that was characterized on recombinant Kv1.1/Kvβ1 channels by electrophysiology and further evaluated on sustained neuronal firing and on in vitro epileptiform activity using a high K + ‐low Ca 2+ model in hippocampal slices. Results We identified a novel compound able to modulate the interaction of the Kv1.1/Kvβ1 complex and that produced a functional inhibition of Kv1.1/Kvβ1 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.

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.

Background and PurposeAvailable medications for chronic pain provide only partial relief and often cause unacceptable side effects. There is therefore a need for novel molecular targets to develop new therapeutics with improved efficacy and tolerability. Despite encouraging efficacy data in rodents with inhibitors of the neuronal glycine transporter-2 (GlyT2), there are also some reports of toxicity and their development was discontinued.Experimental ApproachIn order to clarify the possibility of targeting GlyT2 for the treatment of pain, we have used an integrated approach comprising in vitro pharmacology, selectivity, bioavailability, in vivo efficacy and safety assessment to analyse the properties and efficacy of ALX-1393 and Org-25543, the two published GlyT2 inhibitors from which in vivo data are available.Key ResultsWe report that these compounds have a different set of undesirable properties that limit their usefulness as pharmacological tools. Importantly, we discover that inhibitors of GlyT2 can exert an apparent reversible or irreversible inhibition of the transporter and describe a new class of reversible GlyT2 inhibitors that preserves efficacy while avoiding acute toxicity.Conclusions and ImplicationsOur pharmacological comparison of two closely related GlyT2 inhibitors with different modes of inhibition provides important insights into their safety and efficacy profiles, uncovering that in the presence of a GlyT2 mechanism-based toxicity, reversible inhibitors might allow a tolerable balance between efficacy and toxicity. These findings shed light into the drawbacks associated with the early GlyT2 inhibitors and describe a new mechanism that might serve as the starting point for new drug development.