Novel pyrazole compounds for pharmacological discrimination between receptor‐operated and store‐operated Ca²⁺ entry pathways

Abstract

Background and purposePyrazole 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 approachPyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca2+ 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 Ca2+ entry mediated by Orai channels. The consequences of inhibitory effects on Ca2+ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation.Key ResultsPyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca2+ 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 Ca2+ entry as compared with TRPC3-mediated Ca2+ 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 ImplicationsThe pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca2+ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca2+ channels.

Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

“…Pyr3 also inhibits SOCE due to Orai-1, at concentrations close to that for TRPC3 (our data and ref. 19), raising concerns regarding its selectivity. Selective TRPC6 inhibitors were recently reported by Urban et al (25) using the Chembionet library; however, their IC 50 for channel blockade ranged from 3 to 15 μM, and no assessment of hypertrophic modulation was reported.…”

“…Inhibitors such as SKF-96365, 2-aminoethoxydiphenyl borate (14), and spider venom GsMTx-4 (15) are nonselective. Pyr3 (ethyl-1-(4-(2,3,3-trichloro-acryl-amide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) has been identified as a selective TRPC3 antagonist (16,17), although members of the 5-(trifluoro-methyl)-1H-pyrazole family to which it belongs block Ca 2+ release-activated calcium (CRAC) channels, such as Orai channels (18,19).…”

TRP Channels: Their Function and Potentiality as Drug Targets

“…A pyrazole-derivative compound is also reported to inhibit SOCs as well as TRPC3 [ 169 ], but our fi ndings strongly suggest that a pyrazole-derivative compound (especially Pyr3) will become a promising seed for the treatment of chronic heart failure. …”

Ca2+ Signaling and Heart Rhythm

“…Some ryrazole derivatives have recently been suggested to be relatively selective blockers for TRPC channels (Schleifer et al, 2012). In WT mice, we showed that Pyr10, blocked the membrane depolarization of SAN cause by Ang II and attenuated the increase in pacemaker firing rate induced by Ang II (Figure 5).…”

“…Pyr3, a pyrazole derivative, has been suggested to be a blocker of TRPC3 (Kiyonaka et al, 2009) but it inhibits Orai1 mediated SOCE with similar potency (Salmon and Ahluwalia, 2010). Recently, a new selective blocker of TRPC3-ROCE blocker, Pyr10 has been developed and it displayed substantial selectivity for TRPC3-ROCE mediated responses over Orai1 mediated SOCE (Schleifer et al, 2012). We therefore studied the effect of Pyr10 on pacemaking and intracellular Ca 2+ after application of Ang II and/or the DAG derivative, OAG.…”