High-throughput screening against protein:protein interaction interfaces reveals anti-cancer therapeutics as potent modulators of the voltage-gated Na+ channel complex

Fernanda Laezza,Reid T Powell,David Brunell,Paul A Wadsworth,Nghi Nguyen,Aditya K Singh,Clifford Stephan,Oluwarotimi Folorunso,John Allen,Daniela D’Amico

doi:10.1038/s41598-019-53110-8

Abstract

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.

Highlights

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex
To Fibroblast growth factor 14 (FGF14) (CLuc-FGF14) and a chimera expressing CD4 fused to the Nav1.6 C-tail www.nature.com/scientificreports (CD4-Nav1.6-NLuc), and FGF14:Nav1.6 C-tail complex formation can be detected in the presence of the luciferase substrate, D-luciferin (Fig. 1a–c)
In order to utilize this system for high-throughput screening (HTS), we developed a double stable cell line that increased signal-to-noise ratio, decreased well-to-well variability, and circumvented the need for high volume transient transfections, which are labor-intensive and uneconomical

Summary

Introduction

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Interactions between FGF14 and Nav1.6 are regulated by kinase signaling pathways including glycogen synthase kinase 3 (GSK3) and casein kinase 2 (CK2), which directly phosphorylate serine/threonine (S/T) sites on FGF14 and/or Nav1.6 Targeting these kinases with inhibitors or short-hairpin RNA alters protein complex stability, Nav1.6 currents and excitability[13,16,19,27,32], while peptidomimetics targeting the FGF14V160 and FGF14Y158 residues, which are located at the FGF14:Nav1.6 PPI interface, reduce complex formation, exhibit state-dependent modulation of Nav1.6 currents and suppress excitability of medium spiny neurons in the nucleus accumbens (NAc)[20,28]. Our study provides a new practical tool to accelerate drug discovery for ion channels, and identifies the tyrosine kinase inhibitor lestaurtinib, an FDA approved anti-cancer drug, as a potential compound for repurposing toward CNS-related channelopathies

Methods

Results

Conclusion