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Mar. Drugs 2016, 14(2), 36; doi:10.3390/md14020036

Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin

1,2,†
,
1,2,†
,
1,3
,
2,* , 4
,
1,2
and
1,2,*
1
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, China
2
Jiangsu Provincial Key laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China
3
School of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, China
4
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Keith B. Glaser
Received: 23 December 2015 / Revised: 21 January 2016 / Accepted: 26 January 2016 / Published: 16 February 2016
View Full-Text   |   Download PDF [2832 KB, uploaded 16 February 2016]   |  

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. View Full-Text
Keywords: antillatoxin; FMPblue; membrane potential; hNav1.7; rapid throughput antillatoxin; FMPblue; membrane potential; hNav1.7; rapid throughput
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Zhao, F.; Li, X.; Jin, L.; Zhang, F.; Inoue, M.; Yu, B.; Cao, Z. Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin. Mar. Drugs 2016, 14, 36.

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