In silico-assisted design of selective KCNT1 blocker: toward new epilepsy therapies

Potassium (K+ ) channels are the the most diverse and wide-ranging class of voltage- and/or ligand-gated ion channels, which facilitate potassium flow through the cell membrane, playing a crucial role in several cellular functions, including modulation of Ca2+ signaling and cell proliferation, migration and neuronal excitability control. 1 KCNT1 (K+ channel subfamily T member 1) is a sodium-activated potassium channel primarly expressed in the nervous system where it regulates neuronal excitability by modulating depolarization following repetitive firing of action potentials.2 Gain of function mutations in the KCNT1 gene are are associated with severe neurological disorders, including drug-resistant forms of childhood epilepsy, such as Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) and Autosomal Dominant Sleep-Related Hypermotor Epilepsy (ADSHE).3 Quinidine is a well-known KCNT1 blocker, but its clinical application is limited due its severe drawbacks, as the prolongation of the QT interval on the ECG. 4 With the aim of identify novel KCNT1 blockers, in the present work we developed a homology model of human KCNT1 quinidine binding site, in order to screen an in house molecular library. Among these compounds, 20 structures were selected and tested in a fluorescence-based assay performed on CHO cells stably expressing KCNT1 channels. Five compounds (namely CPK4, 13, 16, 18 and 20) demonstrated KCNT1 blocking activity. Subesequently, patch-clamp experiments confirmed that these compounds exhibited higher potency and selectivity over KCNT1 compared to quinidine, with reduced activity on hERG and Kv7.2 channels. Additionally, in vitro metabolism assay indicated high stability for CPK20. Lastly, this compound also counteracted the gain-of-function effects of two recurrent epilepsy-associated KCNT1 variants (G288S and A934T), highlighting its potential for further therapeutic development.