title: Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin.
abstract: Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Nav1.1-Nav1.9), Nav1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Nav1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX) on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR) membrane potential (FMP) blue dye. In HEK-293 cells heterologously expressing hNav1.7 α-subunit, ATX produced a robust membrane depolarization with an EC50 value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNav1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC50 values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNav1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNav1.7 antagonists.
authors.affiliation: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China. zhaofang0927@yahoo.com.|Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China. zhaofang0927@yahoo.com.
authors.forename: Fang
authors.initials: F
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China. xichun_li@163.com.|Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China. xichun_li@163.com.
authors.forename: Xichun
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China. liangjin1975@cpu.edu.cn.|School of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, China. liangjin1975@cpu.edu.cn.
authors.forename: Liang
authors.initials: L
authors.lastname: Jin
authors.affiliation: Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China. zhangfan20111112@126.com.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan. inoue@mol.f.u-tokyo.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inoue
authors.affiliation: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China. boyangyucpu@163.com.|Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China. boyangyucpu@163.com.
authors.forename: Boyang
authors.initials: B
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China. zycao1999@hotmail.com.|Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China. zycao1999@hotmail.com.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
mesh_terms: D000700:Analgesics / Q000008:administration & dosage / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D055808:Drug Discovery / Q000379:methods; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D055666:Lipopeptides / Q000494:pharmacology; D062556:NAV1.7 Voltage-Gated Sodium Channel / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D010456:Peptides, Cyclic / Q000494:pharmacology; D026941:Sodium Channel Blockers / Q000494:pharmacology; D061585:Voltage-Gated Sodium Channel Agonists / Q000494:pharmacology*; D061567:Voltage-Gated Sodium Channel Blockers / Q000008:administration & dosage / Q000494:pharmacology*; D061566:Voltage-Gated Sodium Channels / Q000187:drug effects
chemical_list: D000700:Analgesics; D055666:Lipopeptides; D062556:NAV1.7 Voltage-Gated Sodium Channel; D010456:Peptides, Cyclic; D026941:Sodium Channel Blockers; D061585:Voltage-Gated Sodium Channel Agonists; D061567:Voltage-Gated Sodium Channel Blockers; D061566:Voltage-Gated Sodium Channels; C121886:antillatoxin
keywords: FMPblue; antillatoxin; hNav1.7; membrane potential; rapid throughput