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.

A comprehensive approach to identifying repurposed drugs to treat SCN8A epilepsy

“…We utilized a stimulus‐activated, fluorescence‐based Na + flux assay with an FLIPR high‐throughput cellular screening system . Fluorescence‐based screening assays are frequently used for assessing ion channel function . After preincubation with the Na + indicator dye, ANG‐2, clonal cell lines were tested for their abilities to generate a fluorescent readout in response to veratridine, a neurotoxin that causes persistent opening of sodium channels.…”

“…11 Fluorescence-based screening assays are frequently used for assessing ion channel function. 12,13 After preincubation with the Na + indicator dye, ANG-2, clonal cell lines were tested for their abilities to generate a fluorescent readout in response to veratridine, a neurotoxin that causes persistent opening of sodium channels. Veratridine CRCs were generated using the Na v 1.6 R1872Q clonal cell line, revealing a robust dynamic range (Figure 2A) consistent with historical data generated on wild-type Na v 1.6 channels and suitable for screening.…”

Sodium channels and pain: from toxins to therapies

“…Similarly, grayanotoxin produced by plants from the family Ericaceae (Maejima et al ., ) (Figure G), aconitine from the plant Aconitum (Borcsa et al ., ) and antillatoxin produced by marine cyanobacterium (Cao et al ., ) are potent Na V channel activators. Veratridine and antillatoxin in particular have been used in programmes for the discovery of Na V channel modulators due to its ability to specifically activate these channels in neuroblastomas and recombinant cells lines and facilitate assay development (Vetter et al ., ; Cardoso et al ., ; Zhao et al ., ). In addition, veratridine and grayanotoxin have been used in in vivo studies of pain mediated by Na V channels (Gingras et al ., ; Cardoso et al ., ).…”

Bringing new dimensions to drug discovery screening: impact of cellular stimulation technologies

“…If inactivation is removed using channel openers, sodium channels stay in an open state, and the sodium influx via open channels will result in membrane depolarization. The most widely used sodium channel opener is veratridine [16,17], although a few alternatives are also available [18–20]. This approach might be effective only if MOAs of novel drug candidates are orthogonal to the MOA of veratridine.…”