Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.

Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.