Screening an In-House Isoquinoline Alkaloids Library for New Blockers of Voltage-Gated Na + Channels Using Voltage Sensor Fluorescent Probes: Hits and Biases.

Voltage-gated Na + (Na V ) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel Na V channel ligands. With the objective of discovering new blockers of Na V channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of Na V channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC 50 . These five alkaloids were then assayed using the Na + fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNa V 1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na + currents elicited by the hNa V 1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na + currents elicited by the hNav1.2 and hNav1.6 channels, with IC 50 values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block Na V channels, with promising therapeutical applications.