title: 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: Merck Research Labs, Screening and Protein Sciences, North Wales, PA, USA michael.finley@merck.com.
authors: Michael
authors: M
authors: Finley
authors: Merck Research Labs, Screening and Protein Sciences, North Wales, PA, USA.
authors: Jason
authors: J
authors: Cassaday
authors: Merck Research Labs, Screening and Protein Sciences, North Wales, PA, USA.
authors: Tony
authors: T
authors: Kreamer
authors: Merck Research Labs, Screening and Protein Sciences, North Wales, PA, USA.
authors: Xinnian
authors: X
authors: Li
authors: Merck Research Labs, Screening and Protein Sciences, North Wales, PA, USA.
authors: Kelli
authors: K
authors: Solly
authors: Merck Research Labs, Screening and Protein Sciences, North Wales, PA, USA.
authors: Greg
authors: G
authors: O'Donnell
authors: Merck Research Labs, Pharmacology, West Point, PA, USA.
authors: Michelle
authors: M
authors: Clements
authors: Merck Research Labs, Chemistry, West Point, PA, USA.
authors: Antonella
authors: A
authors: Converso
authors: Merck Research Labs, Pharmacology, West Point, PA, USA CNS Discovery Research, Teva Pharmaceuticals, West Chester, PA, USA.
authors: Sean
authors: S
authors: Cook
authors: Merck Research Labs, Pharmacology, West Point, PA, USA.
authors: Chris
authors: C
authors: Daley
authors: Merck Research Labs, Pharmacology, West Point, PA, USA.
authors: Richard
authors: R
authors: Kraus
authors: Merck Research Labs, Pharmacology, West Point, PA, USA.
authors: Ming-Tain
authors: MT
authors: Lai
authors: Merck Research Labs, Chemistry, West Point, PA, USA.
authors: Mark
authors: M
authors: Layton
authors: Merck Research Labs, Pharmacology, West Point, PA, USA.
authors: Wei
authors: W
authors: Lemaire
authors: Merck Research Labs, Chemical Modeling and Informatics, West Point, PA, USA.
authors: Donnette
authors: D
authors: Staas
authors: Merck Research Labs, Pharmacology, West Point, PA, USA.
authors: Jixin
authors: J
authors: Wang
mesh_terms: D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D058990:Molecular Targeted Therapy; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000187:drug effects / Q000378:metabolism*; D062556:NAV1.7 Voltage-Gated Sodium Channel / Q000187:drug effects / Q000378:metabolism*; D009437:Neuralgia / Q000188:drug therapy*; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D010146:Pain / Q000188:drug therapy; D012007:Rectum / Q000002:abnormalities
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel; D062556:NAV1.7 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C486866:SCN9A protein, human
keywords: high-throughput screen; membrane potential; sodium channel