Abstract

Rationale: The Kv3.1 channel, a voltage-gated potassium channel encoded by the KCNC1 gene, is found in neurons of the central nervous system, including cerebellar and GABAergic interneurons. De novo mutations in KCNC1 are linked to various neurological conditions such as myoclonic epilepsy, developmental epileptic encephalopathy, or hypotonia. A recent study identified a mutation of the K_V3.1 channel, V434L, which exhibited a shift in the voltage dependence of activation to more hyperpolarising potentials¹. Due to the rarity of K_V3.1 channel mutations and the lack of effective treatments for associated disorders, the KCNC1 Foundation sought to identify inhibitors of the K_V3.1-V434L variant. Metrion Biosciences designed drug repurposing screens using compounds from The Broad Institute Repurposing Hub library.

Methods: Manual patch-clamp technique was used to evaluate channel pharmacology using cells transiently transfected with wild-type and V434L mutant channel. A monoclonal cell line for K_V3.1 V434L was developed using automated patch-clamp, and a Fluorescent Imaging Plate Reader (FLIPR) thallium flux assay. FLIPR high throughput screening assays against the mutant channel were performed to identify hit compounds using compound plates which included 6,718 compounds from The Broad Institute.

Results: Metrion confirmed that the KCNC1 V434L mutation results in gain-of-function, rendering the mutant channel active at more hyperpolarising voltages. Wild-type and V434L currents exhibited different pharmacological profiles using the K_V3.1 modulators 4-aminopyridine and AUT1. This difference led Metrion to generate a monoclonal V434L cell line, which was used to develop and optimise a thallium-flux assay on the FLIPR^Penta platform, suitable for identifying mutant channel modulators. The Broad library of compounds was screened at 10 µM. The screen was robust, with the assay metrics for signal-to-background and robust Z’ having mean values (± SD) of 3.48 ± 0.42 and 0.72 ± 0.07, respectively. A good correlation was observed between the replicate testing of the compounds with 202 compounds (3% hit rate) found to inhibit more than 60% of the signal in both replicate plates. Eighty compounds were subsequently progressed to full concentration-response testing against the K_V3.1-V434L variant in the thallium flux assay. IC₅₀ values ranged from ~100 nM to 10 µM, with ~20% of the compounds tested having an IC₅₀ <1 µM. By combining the information on compound potency and stage of drug development, the most promising compounds were selected for testing in an orthogonal patch-clamp assay to assess activity in the K_V3.1-V434L variant.

Conclusions: Sixteen compounds were identified with <1 µM IC₅₀ values, including 5 compounds launched for other indications. Identification of functional inhibitors of K_V3.1 V434L validates the strategy to screen the repurposing library as a fast and cost-effective approach to discover potential new therapies for patients with this mutation.

¹ Clatot J. Ginn N. Costain G. Goldberg E. doi: 10.1002/acn3.51707