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Pathogenic variants in KCNT1 represent an important cause of treatment-resistant epilepsy, for which an effective therapy has been elusive. Reports about the effectiveness of quinidine, a candidate precision therapy, have been mixed. We sought to evaluate the treatment responsiveness of patients with KCNT1-related epilepsy. We performed an observational study of 43 patients using a collaborative KCNT1 patient registry. We assessed treatment efficacy based upon clinical seizure reduction, side effects of quinidine therapy, and variant-specific responsiveness to treatment. Quinidine treatment resulted in a > 50% seizure reduction in 20% of patients, with rare patients achieving transient seizure freedom. Multiple other therapies demonstrated some success in reducing seizure frequency, including the ketogenic diet and vigabatrin, the latter particularly in patients with epileptic spasms. Patients with the best quinidine response had variants that clustered distal to the NADP domain within the RCK2 domain of the protein. Half of patients did not receive a quinidine trial. In those who did, nearly half did not achieve therapeutic blood levels. More favorable response to quinidine in patients with KCNT1 variants distal to the NADP domain within the RCK2 domain may suggest a variant-specific response.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare early-onset developmental epileptic encephalopathy resistant to anti-epileptic drugs. The most common cause for EIMFS is a gain-of-function mutation in the KCNT1 potassium channel gene, and treatment with the KCNT1 blocker quinidine has been suggested as a rational approach for seizure control in EIMFS patients. However, variable results on the clinical efficacy of quinidine have been reported. In the present study, we provide a detailed description of the clinical, genetic, in vitro, and in vivo electrophysiological profile and pharmacological responses to quinidine of 2 EIMFS unrelated patients with a heterozygous de novo KCNT1 mutation: c.2849G>A (p.R950Q) in patient 1 and c.2677G>A (p.E893K) in patient 2. When expressed heterologously in CHO cells, KCNT1 channels carrying each variant showed gain-of-function effects, and were more effectively blocked by quinidine when compared to wild-type KCNT1 channels. On the basis of these in vitro results, add-on quinidine treatment was started at 3 and 16 months of age in patients 1 and 2, respectively. The results obtained reveal that quinidine significantly reduced seizure burden (by about 90%) and improved quality of life in both patients, but failed to normalize developmental milestones, which persisted as severely delayed. Based on the present experience, early quinidine intervention associated with heart monitoring and control of blood levels is among the critical factors for therapy effectiveness in EIMFS patients with KCNT1 gain-of-function mutations. Multicenter studies are needed to establish a consensus protocol for patient recruitment, quinidine treatment modalities, and outcome evaluation, to optimize clinical efficacy and reduce risks as well as variability associated to quinidine use in such severe developmental encephalopathy.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure–activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare, serious, and pharmacoresistant epileptic disorder often linked to gain-of-function mutations in the KCNT1 gene. KCNT1 encodes the sodium-activated potassium channel known as SLACK, making small molecule inhibitors of SLACK channels a compelling approach to the treatment of EIMFS and other epilepsies associated with KCNT1 mutations. In this manuscript, we describe a hit optimization effort executed within a series of 2-aryloxy-N-(pyrimidin-5-yl)acetamides that were identified via a high-throughput screen. We systematically prepared analogs in four distinct regions of the scaffold and evaluated their functional activity in a whole-cell, automated patch clamp (APC) assay to establish structure-activity relationships for wild-type (WT) SLACK inhibition. Two selected analogs were also profiled for selectivity versus other members of the Slo family of potassium channels, of which SLACK is a member, and versus a panel of structurally diverse ion channels. The same two analogs were evaluated for activity versus the WT mouse channel as well as two clinically relevant mutant human channels.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare devastating infantile epileptic encephalopathy that is characterized by a unique electroencephalopgraphy (EEG) signature of multifocal simultaneous seizures. Although no definite etiology is understood for EIMFS, mutations in certain ion channels, are implicated. Similarly, phenylalanyl-tRNA synthetase 2 (FARS2) deficiency is a rare, autosomal recessive disorder of dysfunctional mitochondrial translation causing refractory seizures, lactic acidosis, and developmental regression with a variety of EEG findings. However, an EIMFS-like pattern on EEG in FARS2 deficiency has only recently been reported once. Herein, we describe a seven-week-old male with seizures where whole exome sequencing (WES) revealed pathogenic FARS2 variants and an EIMFS pattern on EEG. This case provides an insight on a novel genetic mechanism for EIMFS. We encourage early consideration of WES when EIMFS is detected to evaluate for FARS2 deficiency, especially in the setting of profound lactic acidosis.

The potassium sodium-activated channel subtype T member 1 (KCNT1) gene encodes the Slack channel KNa1.1, which is expressed in neurons throughout the brain. Gain-of-function variants in KCNT1 are associated with a spectrum of epilepsy syndromes, and mice carrying those variants exhibit a robust phenotype similar to that observed in patients. Kcnt1 knockout (KO) mice, however, have a normal lifespan without any epileptic phenotype. To understand the molecular differences between these two models, we conducted a comprehensive proteomic analysis of the cerebral cortices of Kcnt1 KO and Kcnt1R455H/+ mice, an animal model bearing a cytoplasmic C-terminal mutation homologous to a human R474H variant that results in EIMFS. The greatest change observed in Kcnt1 KO mice compared to the wild-type mice was the increased expression of multiple proteins of the inner mitochondrial membrane. Electron microscopy studies of cortical mitochondria from Kcnt1 KO mice further confirmed a significant increase in the density of mitochondrial cristae compared to that in wild-type mice. Kcnt1 reduction by a murine-specific Kcnt1 antisense oligonucleotide (ASO) in Kcnt1R455H/+ mice partially corrected the proteomic dysregulations in the disease model. The results support the hypothesis that ASO-mediated KCNT1 reduction could be therapeutically useful in the treatment of KCNT1 epilepsies.