Two Novel CRAC Channel Inhibitors Targeting the Orai Pore

Abstract

Store-operated channels (SOCs) represent a widespread route for Ca2+ entry into the cell, the best characterized amongst them being the Ca2+ release-activated Ca2+ (CRAC) channel. As the molecular composition of CRAC channels has been a long standing mystery, the identification of selective inhibitors has been considerably hampered. Diverse agents such as La3+, Gd3+, 2-APB and imidazole derivatives inhibit CRAC currents, however, in a non-selective manner limiting their usefulness as CRAC blockers. The two key molecular components of the CRAC channels: STIM1 and Orai1 which were identified 5-6 years ago, represent promising targets for the elucidation of novel and selective CRAC blockers. Here, we present two novel inhibitors, GSK-7975A and GSK-5503A, which fully inhibited Orai1 and Orai3 currents with a substantially slower rate of onset than La3+. Inhibition of Orai currents occurred with an IC50 of ∼ 4μM and exhibited limited reversibility upon wash-out. Blockage of currents through the less Ca2+-selective pore mutant Orai1 E106D (or 2-APB stimulated Orai3) was significantly reduced suggesting that the selectivity filter is a direct or allosteric target of these GSK CRAC channel blocking compounds. Furthermore, FRET experiments indicated that neither STIM1-STIM1 oligomerization nor STIM1-Orai1 coupling was affected by these compounds. The endogenous CRAC current of RBL cells was also inhibited by these compounds whereas amongst other Ca2+-selective channels, L-type Ca2+ currents exhibited only slight inhibition and TRPV6 currents were fully blocked. In summary, the elucidation of these novel CRAC current blockers represents an important step forward in the identification of CRAC channel-selective drug molecules. (supported by the Austrian Science Fund (FWF): T466 and P22565)