Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5.

Abstract

The NaV1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding NaV1.7, resulting in either loss-of-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of NaV1.7. A full-deck high-throughput screen was developed for both NaV1.7 and cardiac NaV1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in NaV1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with NaV1.7 pharmacological selectivity over NaV1.5 as confirmed in electrophysiology.

• Authors: Michael Finley, Jason Cassaday, Tony Kreamer, Xinnian Li, Kelli Solly, Greg O'Donnell, Michelle Clements, Antonella Converso, Sean Cook, Chris Daley, Richard Kraus, Ming-Tain Lai, Mark Layton, Wei Lemaire, Donnette Staas, Jixin Wang
• Journal: Journal of biomolecular screening (J Biomol Screen) Vol. 21 Issue 5 Pg. 480-9 (Jun 2016) ISSN: 1552-454X [Electronic] United States
• PMID: 26861708 (Publication Type: Journal Article)
• Copyright: © 2016 Society for Laboratory Automation and Screening.

Chemical References

• NAV1.5 Voltage-Gated Sodium Channel
• NAV1.7 Voltage-Gated Sodium Channel
• SCN5A protein, human
• SCN9A protein, human

Topics

• High-Throughput Screening Assays (methods)
• Humans
• Kinetics
• Membrane Potentials (drug effects)
• Molecular Targeted Therapy
• NAV1.5 Voltage-Gated Sodium Channel (drug effects, metabolism)
• NAV1.7 Voltage-Gated Sodium Channel (drug effects, metabolism)
• Neuralgia (drug therapy)
• Neurons (drug effects, pathology)
• Pain (drug therapy)
• Rectum (abnormalities)