The Discovery Of Nav1.2 Small Molecule Inhibotors And Its Antiepileptic Activity

Voltage-gated sodium channel is an important protein in the process of bioelectric signal transduction,and it has been reported as an essential target of neuroexcitability disorders such as epilepsy and arrhythmia.Nav1.2 is an isoform that specifically and highly expressed in glutamatergic neurons in the central nervous system.Previous studies have shown that Nav1.2 channel is associated with epilepsy,autism,and intellectual disability.Gain of Nav1.2 function could contribute to neuronal hyperexcitability.Mutations of the gene SCN2A encoding Nav1.2 channel have been associated to a wide spectrum of epileptic disorders,including benign familial neonatal-infantile seizures（BFNIS）,developmental and epileptic encephalopathy（DEE）.And Nav1.2 channel could be a potential anti-epileptic target.However,there are few small molecule inhibitors of Nav1.2 channel,these identified Nav1.2 channel antagonists are generally encountered with high IC₅₀ values in vitro or no antiepileptic activity in vivo.Therefore,there is an urgent need to discover new Nav1.2 small molecule inhibitors,for exploring the feasibility of targeting Nav1.2 channel in the treatment of epilepsy.In this work,we used the Ion Works Barracuda system to carry out high-throughput screening of Nav1.2 inhibitors.Then,we cooperated with medicinal chemists to conduct structure modification,and found several potent inhibitors of Nav1.2 channel,from which we selected some molecules to evaluate their anti-epileptic effects in the maximal electroshock seizure（MES）model,and found that several molecules showed anti-epileptic effects in vivo.In addition,we found that Lvguidingan,a new anti-epileptic drug in phaseⅡdevelopment in China,is an inhibitor of the Nav1.2 channel.Lvguidingan（10-30μM）dose-dependently suppresses the spontaneous firing with reduction of action potential amplitudes in cultured hippocampal neurons.Subtype selectivity study showed that Lvguidingan preferentially inhibits the central neuron system distributed Nav1.2channel.Similar to other anti-epileptic drugs targeted on Nav channels,Lvguidingan also exhibited state-and use-dependent inhibition on Nav1.2 channel.Moreover,Lvguidingan caused significant depolarizing shift of channel activation and hyperpolarizing shift of channel inactivation that would prompt the Nav1.2 channel into a nonfunctional state.Notably,application of Lvguidingan（30μM）could override the increased currents of epilepsy-associated Nav1.2 gain-of-function mutations.Taken together,our study indicated that Lvguidingan might act as a lead compound for the identification of selective inhibitors.Further modification of Lvguidingan-based derivatives might be an effective strategy for the development of new antiepileptic drugs targeting the Nav1.2 isoform.Lvguidingan-produced suppression on the increased current densities of the gain-of-function mutations indicated that it might be an alternative therapy for Nav1.2-caused epileptic encephalopathies.We found that vinpocetine,which is clinically used for the treatment of cerebrovascular diseases,is also an inhibitor of Nav1.2 channel.Vinpocetine（1-30μM）significantly inhibits the firing of action potentials in hippocampal neurons,causing obvious depolarization of neuronal membrane potentials.After further study on potassium currents in hippocampal neurons,we found that vinpocetine inhibits the delayed rectifier potassium current,but has no effect on the transient outward potassium current.The inhibitory effect of vinpocetine on Nav1.2 channel is also state-dependent and use-dependent.By cooperating with medicinal chemists to modify the structure of vinpocetine,we discovered two derivatives,VC11 and VC13,with higher efficacy.