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

• Published in: Scientific reports Vol. 9; no. 1; pp. 16890 - 15
• Main Authors: Wadsworth, Paul A, Folorunso, Oluwarotimi, Nguyen, Nghi, Singh, Aditya K, D'Amico, Daniela, Powell, Reid T, Brunell, David, Allen, John, Stephan, Clifford, Laezza, Fernanda
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 15.11.2019; Nature Publishing Group UK
• Subjects: Antineoplastic Agents - isolation & purification; Antineoplastic Agents - pharmacology; Channelopathy; Dose-response effects; Drug Evaluation, Preclinical - methods; Enzyme inhibitors; Fibroblast growth factors; Fibroblast Growth Factors - agonists; Fibroblast Growth Factors - antagonists & inhibitors; Fibroblast Growth Factors - chemistry; HEK293 Cells; High-throughput screening; High-Throughput Screening Assays - methods; Humans; Interfaces; Macromolecules; Mental disorders; Multiprotein Complexes - agonists; Multiprotein Complexes - antagonists & inhibitors; Multiprotein Complexes - chemistry; NAV1.6 Voltage-Gated Sodium Channel - drug effects; NAV1.6 Voltage-Gated Sodium Channel - metabolism; Neuromodulation; Protein Binding; Protein Interaction Maps - physiology; Protein-tyrosine kinase; Proteins; Regulatory proteins; Risk factors; Sodium channels (voltage-gated); Toxicity; Voltage-Gated Sodium Channel Agonists - isolation & purification; Voltage-Gated Sodium Channel Agonists - pharmacology; Voltage-Gated Sodium Channel Blockers - isolation & purification; Voltage-Gated Sodium Channel Blockers - pharmacology; Voltage-Gated Sodium Channels - drug effects; Voltage-Gated Sodium Channels - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-53110-8

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.