Identification of new KCNT1-epilepsy drugs by in silico, cell, and Drosophila modeling

Objective: Hyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and, in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep-related hypermotor epilepsy. There are currently no effective treatments for KCNT1 epilepsies, but suppressing overactive channels poses a potential strategy. Methods: Using the KCNT1 channel structure we in silico screened a library of known drugs for those predicted to block the channel pore to inhibit channel activity. Cellular KCNT1 channel inhibition was analyzed using electrophysiology and Drosophila bang-sensitive assays were used to analyze seizure suppression. Brain penetration of one drug was analyzed using liquid chromatography–mass spectrometry in a mouse. Results: Eight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with 4 drugs showing significant reduction of K⁺ current amplitudes. The action of the 4 drugs was then analyzed in vivo and 2 were found to reduce the seizure phenotype in humanized Drosophila KCNT1 epilepsy models. One drug, antrafenine, was shown to cross the blood–brain barrier in mice. Interpretation: This study identified a known drug, antrafenine, that reduces KCNT1 channel activity, reduces seizure activity in Drosophila, and crosses the blood–brain barrier in the mouse, suggesting its potential applicability as a new treatment for KCNT1 epilepsy. The sequential in silico, in vitro, and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified. ANN NEUROL 2025;98:1261–1274.