Drug repurposing based on integrated in silico and in vitro approach to find a therapy for KCNA2-related disorders

Purpose: Variants in KCNA2 (Kv1.2) causing loss-, gain-of-function (LoF, GoF) and mixed LoF/GoF defects cause rare neurological disorders including developmental and epileptic encephalopathy (DEE) and ataxia, ranging from mild to severe (Masnada et al., Brain 2017,140(9):2337-2354; Imbrici et al., IJMS 2021;22(18):9913). Antiseizure medications have variable effects and drug-resistance occurs in most patients affected by DEE. 4-aminopyridine, a non-selective Kv1 blocker, is the only precision medicine approach trialled in some patients carrying GoF variants (Hedrich et al., Sci Transl Med. 2021;13(609):eaaz4957). Thus, new molecules acting on Kv1.2 need to be developed. This study aims to identify approved drugs to be repurposed in KCNA2-related disorders. Method: Using a homology model of Kv1.2 channel, multiple-grid structure-based virtual screening has been performed by using a collection of about 2000 approved drugs. The automated patch clamp platform, Patchliner (Nanion) has been used to validate in silico data on Kv1.2 channels and E236K mixed LoF/GoF mutant expressed in HEK293 cells. Results: A pool of repurposed drugs has been identified as promising modulators of Kv1.2 including paroxetine and fluvoxamine, selective serotonin reuptake inhibitors (SSRIs), and cannabidiol, recently approved as add on for rare epilepsies. Fluvoxamine blocked Kv1.2 with IC50 ⁓8.1±1.9 μM and E236K channels with IC50 ⁓12.2±4.6 μM. Similar block has been observed for paroxetine (IC50 ⁓8.9±4.4 μM for Kv1.2 and ⁓7.5±3.7 for E236K). Cannabidiol blocked both channels with lower affinity (IC50 ⁓40.3±15.4 μM for Kv1.2 and ⁓52.1±26.0 μM for E236K). Conclusion: Our results suggest that two SSRIs, paroxetine and fluvoxamine, are potential repurposing candidates for children harbouring KCNA2 GoF or mixed variants. These data support previous findings showing the effect of fluoxetine on other potassium channels variants (Ambrosino et al., Epilepsia 2023;64(7):e148-e155). Our data also suggest that cannabidiol, a drug with multitarget activity, may modulate Kv1.2 activity, albeit with a low affinity.