Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca(2+) entry pathways

Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation. Experimental approach: Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca(2+) entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca(2+) entry mediated by Orai channels. The consequences of inhibitory effects on Ca(2+) signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation. Key

Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation. Experimental approach: Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca(2+) entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca(2+) entry mediated by Orai channels. The consequences of inhibitory effects on Ca(2+) signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation. Key

Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca(2+) entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca(2+) entry as compared with TRPC3-mediated Ca(2+) entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation. Conclusions and implications: The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca(2+) entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca(2+) channels.