Functional Drug Screening System (FDSS): Calcium mobilization responses to KCl depolarization were evaluated by measuring the intensity of calcium-mediated fluorescent signal in the presence of BD Calcium Assay Dye (BD Biosciences, Franklin Lakes, N.J., U.S.A.), utilizing a Functional Drug Screening System (FDSS) by Hamamatsu Corporation (Bridgewater, N.J. U.S.A.).Twenty-four hr prior to assay, cells were seeded in clear-base poly-D-lysine-coated 384-well plates (BD Biosciences) at a density of 5,000 cells per well in culture medium and grown overnight in 5% CO2 at 37° C. On the day of assay, growth media were removed, and cells were loaded with BD calcium assay dye (BD Biosciences) for 35 min at 37° C. under 5% CO2 and then for 25 min at room temp. Utilizing the FDSS, cells were exposed to representative compounds of Formula (I) at varying concentrations, and intracellular calcium was measured for 5 min prior to the addition of 50 mM KCl for an additional 3 min of measurement.
Inhibition of KCl-induced cytosolic voltage gated calcium channel opening in human SH-SY5Y cells at 10 uM by Fluo-4 AM dye based fluorescence assay relative to control
Inhibition of voltage gated calcium channel in human SH-SY5Y cells assessed as blockade of K+-evoked calcium uptake at 1 uM pretreated for 10 mins followed by 70 mM K+ addition by Fluo-4AM dye based fluorescence assay relative to control
Inhibition of voltage gated calcium channel in human SH-SY5Y cells assessed as blockade of K+-evoked calcium uptake at 3 uM pretreated for 10 mins followed by 70 mM K+ addition by Fluo-4AM dye based fluorescence assay relative to control
Inhibition of K+-induced voltage gated calcium channel opening in human SH-SY5Y cells assessed as decrease in Ca2+ level at 10 uM after 10 mins by Fluo-4 dye-based fluorescence assay relative to control
Inhibition of K+-induced voltage gated calcium channel opening in human SH-SY5Y cells assessed as decrease in Ca2+ level after 10 mins by Fluo-4 dye-based fluorescence assay
Inhibition of VGCC in human SHSY-5Y cells assessed as reduction of K+-evoked cytosolic ca2+ at 1 uM incubated for 10 mins prior to KCl addition by Fluo-4-AM probe-based fluorescence analysis
Inhibition of VGCC in human SHSY-5Y cells assessed as reduction of K+-evoked cytosolic ca2+ at 3 uM incubated for 10 mins prior to KCl addition by Fluo-4-AM probe-based fluorescence analysis