(1127-B) Fluorescence-based drug repurposing screen of the potassium channel, KV3.1 with V434L mutation.

Abstract: Fluorescence-based drug repurposing screen of the potassium channel, KV3.1 with V434L mutation.

The voltage-gated potassium channel, Kv3.1, encoded by the KCNC1 gene is expressed in central nervous system neurons such as cerebellar neurons and GABAergic interneurons. De novo mutations of KCNC1 can manifest in a variety of neurological disorders, including myoclonic epilepsy, ataxia, developmental epileptic encephalopathy or hypotonia. A recent study identified a particular mutation of the Kv3.1 channel, V434L, which exhibited a shift in the voltage dependence of activation to more hyperpolarised potentials1. Mutations of the Kv3.1 channel are extremely rare, and there is a lack of effective treatments for disorders associated with Kv3.1 channel mutation. As a result, the KCNC1 Foundation looked to find inhibitors of Kv3.1-V434L variant by screening compounds from the Broad Institute Repurposing Hub library.
Stable cell lines for both the wild-type and mutant Kv3.1 channels were created and confirmation of their biophysical properties was performed using the manual patch-clamp technique. Differences in the effects of two pharmacological tool molecules, 4-AP and AUT1, on the wild-type and mutant Kv3.1 currents were observed with the decision taken to screen the library against the Kv3.1-V434L variant.
A thallium-based fluorescence assay was developed and optimised on the FLIPRPenta to identify inhibitors of the Kv3.1-V434L variant. The Broad Repurposing Hub library of 6,718 compounds were screened in duplicate 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 Kv3.1-V434L variant in the thallium flux assay. IC50 values ranged from ~100 nM to 10 µM, with approximately 20% of the compounds tested having an IC50 < 1 µM.

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