Abstracts

Rationale: Perampanel (PMP) is a third-generation anticonvulsant which is clinically used for adjunctive therapy for refractory partial-onset and secondarily generalized tonic-clonic seizures. PMP is thought to dampen hyperactivity in an epileptic brain through AMPA receptor (AMPAR) inhibition because it acts as a potent and selective noncompetitive negative allosteric modulator (NAM) of AMPARs. The recent structural resolution of the binding site for PMP in AMPARs revealed the residues critical for its NAM activity (Yelshanskaya et al., Neuron, 2020; 91, 1305-1315). We noted that key residues required for binding of PMP to AMPARs are largely conserved in the kainate receptor (KAR) subunits GluK4 and GluK5. We therefore hypothesized that PMP could also inhibit heteromeric KARs in a subunit-dependent manner and that this pharmacological action could in part contribute to its anticonvulsant activity, because GluK5 mRNA is expressed abundantly in many neuronal populations in the CNS.

Methods: We examined PMP NAM activity on recombinant and neuronal KARs. To test if PMP inhibits recombinant KARs, we recorded glutamate-evoked currents from HEK293 cells transfected with KAR subunits in the absence and presence of PMP at concentrations between 30 nM and 30 mM. PMP NAM activity on neuronal KARs was determined by recording KAR-mediated excitatory postsynaptic currents (KAR-EPSCs) at hippocampal mossy fiber-CA3 pyramidal cell synapses (MF synapses) in acute brain slices from 2-week-old C57Bl/6 mice. This synaptic pathway was chosen because postsynaptic MF KARs are known to contain GluK5 subunits (Contractor et al., J Neurosci., 2003; 23, 422-429) and because recurrent circuits in CA3 make this region prone to synchronized firing.

Results: We found that PMP is a subunit-dependent inhibitor of heteromeric KARs. PMP inhibited heteromeric GluK1/GluK5 and GluK2/GluK5 KARs with much higher affinity (IC₅₀: 2.8 mM and 0.85 mM, respectively) than homomeric GluK1 or GluK2 KARs (IC₅₀: was ~19 mM and ~26 mM, respectively), consistent with a strong subunit-dependence of NAM activity. In the hippocampal slices, PMP inhibited KAR-EPSCs at the MF synapses in a dose-dependent manner, suggesting that it is also effective on neuronal KARs containing GluK5 subunits. The amplitude of KAR-EPSCs after 10 min bath-application were 74% for vehicle, 58% for 1 mM PMP, 35% for 10 mM PMP, and 21% for 30 mM PMP compared to the control conditions, respectively. These data demonstrate that PMP inhibits recombinant and neuronal GluK5-containing KARs in a concentration range equivalent to that for AMPARs.

Conclusions: We demonstrate in these studies that PMP inhibits KARs, contrary to previous reports (Ceolin et al., Neurochemistry International., 2012; 61, 517-522, Fukushima et al., Epilepsy Res., 2020; 167, 106452), and that NAM activity requires a particular subunit, GluK5. Our results suggest that KAR inhibition could underlie some aspects of PMP anticonvulsant activity or adverse effects, which are significant with this drug. Drug discovery aimed at more selective NAMs that discriminate between AMPARs and KARs could yield drugs with an improved therapeutic profile.

Funding: Please list any funding that was received in support of this abstract.: Grant from NIH (R01NS105502) to G.T.S.