Initial Characterization of the Indole-3-Carboxamide Bic-154 as a Fast Onset and Reversible ORAI Channel Blocker

Calcium ion elevations are required for human T-lymphocyte proliferation in response to antigen recognition by a T-cell receptor. Calcium influx through the plasma membrane is necessary for efficient T-cell proliferation and effector function. The calcium channels responsible for Ca2+ influx in lymphocytes have been identified and Orai interacting with ER Ca2+ sensor STIM were shown to be crucial for persistent calcium mobilization. Loss-of-function mutations in Orai1 or STIM1 result in severe combined immunodeficiency (SCID) with muscle hypotonia. Suppression of calcium influx through Orai/STIM channels gives rise to various lymphoproliferative defects. Thus, deletion of Orai or STIM in mice results in lymphoproliferative disorders and hair loss. Gain-of-function mutations in Orai or STIM resulting in constitutively activated channels cause tubular aggregate myopathy (TAM). Despite the importance of Orai/STIM pathway in multiple signaling pathways and diseases, small molecule high affinity specific inhibitors of these channels are lacking. In this work we examined a class of indole-3-carboxamides for their potential to inhibit store-operated Ca2+ entry. They were tested in vitro for their ability to inhibit Orai1 mediated Ca2+ entry and currents. At 10 μM and higher the less lipophilic BIC-154 was the most potent blocker in the group. We used a constitutively active Orai1 variant implicated in TAM, which does not require STIM1 for function. BIC-154 rapidly and reversibly inhibited Orai1 currents. BTP2 (YM-58483) and 2-APB also inhibited Orai1 mutant channels in the same cells. TRPM7, a Ca2+/Mg2+ permeable channel highly expressed in lymphocytes was insensitive to these compounds tested at 100 μM. Future investigations will test their effectiveness in T-cell blastogenesis and proliferation assays. We anticipate that Orai1-specific compounds will be useful for proof of concept studies of the contribution of this channel to human disease