Jonathan D. Lippiat

The Orthobunyavirus genus within the Peribunyaviridae family of enveloped arthropod-borne negative-sense RNA viruses includes species associated with serious or fatal disease in both animals and humans such as Schmallenberg, La Crosse and Oropouche viruses. Orthobunyaviruses (OBVs) are internalised into cells by endocytosis and release their genomes following fusion with late endosomal (LE) membranes, triggered by low pH of the luminal milieu. There is mounting evidence to suggest OBV endosomal escape is also influenced by potassium ions (K ⁺ ), which increase in concentration as endosomes mature. Endosomal K ⁺ flux is controlled by cellular K ⁺ channels, and we previously showed that K ⁺ channel blockade using broad spectrum pharmacological inhibition abrogates OBV infection, with virions trapped within the endosomes. However, the K ⁺ channels regulating this process are unknown. Herein, to identify the K ⁺ channels involved, we studied Bunyamwera virus (BUNV), the prototypical OBV, and screened a siRNA library targeting 342 human ion channels. We identified 19 K ⁺ channels whose knockdown inhibited BUNV gene expression by over 50%, with at least seven channels affecting BUNV at the entry stage, suggesting they exert a combinatorial influence. Of these seven channels, we used both pharmacological inhibition and genetic channel manipulation to show that OBV escape from CD63- and Rab7-positive LEs is controlled by KCNJ2 (K _IR 2.1), KCNJ13 (K _IR 7.1) and KCNMA1 (BK _Ca ). These studies add to the understanding of host factors influencing OBV entry, regulation of endosomal ion flux, and could aid in the design or repurposing of therapeutics for the treatment of OBV-associated disease.

Severe drug-resistant childhood epilepsy is caused by KCNT1 gain-of-function genetic variants, resulting in increased K _Na 1.1 channel activity. KCNT1 -associated epilepsy is thought to affect around 1 in 300,000 births worldwide. Current treatment for KCNT1 epilepsy only provides mild symptomatic relief and uses a cocktail of experimental medications which must be personalised for the individual and are often poorly tolerated. Critically, with many patients, no therapeutic benefit is achieved. We sought to address this by using large-scale virtual screening to accelerate the development of a molecule which binds directly to KCNT1 to supress overactivity. We purchased a total of 71 compounds and using a combination of fluorescent thallium flux assays and patch clamp electrophysiology, identified a series of eight structurally diverse, novel inhibitors of the K _Na 1.1 channel with potency in the low micromolar range. These provide potential starting points for further development of drugs to treat KCNT1 -associated epilepsy. Highlights: We have discovered a range of structurally distinct inhibitors of the KCNT1 ion channel using large-scale virtual screening These compound exhibit selectivity for the KCNT1 channel over other related ion channels These compounds could provide starting points for new treatments for KCNT1 related Epilepsy. Graphical

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

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 over-active channels poses a potential strategy. Methods: Using KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. Results: Eight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K ⁺ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. Interpretation: This study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments 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.

Kaposi’s sarcoma–associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi’s sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K ⁺ channel K _v 1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of K _v 1.3 and led to enhanced K ⁺ channel activity and hyperpolarization of the B cell membrane. Enhanced K _v 1.3 activity promoted intracellular Ca ²⁺ influx, leading to the Ca ²⁺ -driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by K _v 1.3 blockers or silencing. These findings highlight K _v 1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are poorly controlled with current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K⁺ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which responded either positively or negatively to 5 frontline epilepsy drugs most commonly administered to patients with KCNT1-epilepsy, often with little or no improvement of seizures. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila has the potential to model human KCNT1- epilepsy and can be used as a tool to assess new treatments for KCNT1- epilepsy.

The Na ⁺ -activated K ⁺ channel K _Na 1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na ⁺ ions bind and increase channel activity is not well understood. Analysis of K _Na 1.1 channel structures indicate that there is a large twisting of the βN-αQ loop in the intracellular RCK2 domain between the inactive and Na ⁺ -activated conformations, with a lysine (K885, human subunit numbering) close enough to form a salt bridge with aspartate (D839) in the Na ⁺ -activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within 4 Å of several acidic or polar residues. In carrying out mutagenesis and electrophysiology with human K _Na 1.1, we found alanine substitution of each of these residues resulted in almost negligible currents in the presence of up to 40 mM intracellular Na ⁺ . The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na ⁺ . Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active K _Na 1.1, D884I had Na ⁺ - dependence similar to wild-type K _Na 1.1, whilst increasing the side chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na ⁺ . We conclude that Na ⁺ binding results in a conformational change that accommodates D884 in the acid-rich pocket, which triggers further conformational changes in the RCK domains and channel activation. Statement of Significance: Sodium-activated potassium channels regulate neuronal excitability, and their dysfunction causes severe childhood disorders. Here, we identify a structural determinant in the intracellular domains that is responsible for triggering channel activation in response to sodium ion binding. An increase in the size of a particular amino acid renders the channel sodium-insensitive, whilst a decrease in size enables the channel to activate in the absence of sodium. This enhances our understanding of how this subclass of potassium channels respond to changes in the intracellular ionic environment. Furthermore, this may also further our understanding of the basis of human neurological disorders and their treatment.