Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions

• Published in: Molecular immunology 2013-07, Vol.54 (3-4), p.355-367
• Main Authors: Chen, Gang, Panicker, Sandip, Lau, Kai-Yeung, Apparsundaram, Subramaniam, Patel, Vaishali A., Chen, Shiow-Ling, Soto, Rothschild, Jung, Jimmy K.C., Ravindran, Palanikumar, Okuhara, Dayne, Bohnert, Gary, Che, Qinglin, Rao, Patricia E., Allard, John D., Badi, Laura, Bitter, Hans-Marcus, Nunn, Philip A., Narula, Satwant K., DeMartino, Julie A.
• Format: Article
• Language: English
• Subjects: Anilides - pharmacology; Animals; Calcineurin - metabolism; Calcineurin Inhibitors; Calcium - metabolism; Calcium Channel Blockers - pharmacology; Calcium Channels - genetics; Calcium Channels - metabolism; CD4-Positive T-Lymphocytes - drug effects; CD4-Positive T-Lymphocytes - immunology; CD4-Positive T-Lymphocytes - metabolism; Cell Line; Cell Proliferation - drug effects; CHO Cells; CRAC; Cricetinae; Cyclosporine - pharmacology; Cytokines - genetics; Cytokines - metabolism; Gene expression; Gene Expression - drug effects; Gene Expression - genetics; Humans; Lymphocyte Activation - drug effects; Lymphocyte Culture Test, Mixed - methods; Membrane Proteins - genetics; Membrane Proteins - metabolism; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; ORAI1 Protein; Rats; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - metabolism; Stromal Interaction Molecule 1; T cell; Tacrolimus - pharmacology; Thiadiazoles - pharmacology
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2012.12.011

► Characterization of pharmacology and effects of a novel CRAC inhibitor, RO2959. ► Comparison of effects of RO2959 on T cells with that of YM58483 and CsA. ► Orai1-selective inhibition by RO2959 sufficient to decrease Ca2+ influx. ► RO2059 potently inhibited human T cell activation and effector functions. Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.