Screening for potassium channel modulators by a high through-put 86-rubidium efflux assay in a 96-well microtiter plate

Discovery and biological evaluation of potent, selective, orally bioavailable, pyrazine-based blockers of the Nav1.8 sodium channel with efficacy in a model of neuropathic pain

“…High-throughput mouse Na v 1.8 and hERG isotopic flux assays HEK293 cells stably expressing mouse Na v 1.8 sodium channels or CHO cells stably expressing hERG potassium channels were loaded overnight with an appropriate radiotracer, followed by stimulation using variations on protocols previously described. 33 Reference blockers for Na v 1.8 and hERG assays were tetracaine (30 lM) and terfenadine (30 lM), respectively. Concentration dependent activity was established via eight-point concentration-response curves and assays were performed in duplicate.…”

“…Inhibition of the hERG channel was also evaluated as a potential liability. 33 All of the final compounds, 15-36, demonstrated hERG IC 50 >10 lM, which was not considered to be a major liability. Selected compounds were evaluated for aqueous solubility.…”

“…33 The activity of selected compounds was confirmed by demonstrating inhibition of sodium currents in human Na v 1.8 (hNa v 1.8) expressing HEK293 cells using a conventional voltageclamp electrophysiology procedure. 25 Electrophysiological evaluation of inhibition of native TTX-r sodium current in acutely dissociated DRG neurons was carried out for a subset of these compounds.…”

“…In vitro characterization of pyrazine Na v 1.8 channel blockers[26][27][28][29][30][31][32][33][34][35][36] …”

Diazoxide‐sensitivity of the adenosine 5′‐triphosphate‐dependent K+ channel in mouse pancreatic β‐cells

“…In previous studies with intact cells the potency of diazoxide in activating the K-ATP-channel was determined by monitoring the 86Rb efflux from insulin-secreting tumour cells (HIT- T-15; Niki et al, 1989) or human medulloblastoma cells (Daniel et al, 1991). The diazoxide concentrations giving 50% stimulation of 86Rb efflux were found to be 45 and 100 AM, respectively.…”