Characterization of a novel CRAC inhibitor that potently blocks 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.
Inflammation Discovery, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA. gang.chen21@gmail.com
Gang
G
Chen
Sandip
S
Panicker
Kai-Yeung
KY
Lau
Subramaniam
S
Apparsundaram
Vaishali A
VA
Patel
Shiow-Ling
SL
Chen
Rothschild
R
Soto
Jimmy K C
JK
Jung
Palanikumar
P
Ravindran
Dayne
D
Okuhara
Gary
G
Bohnert
Qinglin
Q
Che
Patricia E
PE
Rao
John D
JD
Allard
Laura
L
Badi
Hans-Marcus
HM
Bitter
Philip A
PA
Nunn
Satwant K
SK
Narula
Julie A
JA
DeMartino
D000813:Anilides / Q000494:pharmacology; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D016572:Cyclosporine / Q000494:pharmacology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D007959:Lymphocyte Culture Test, Mixed / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D000071740:ORAI1 Protein; D051381:Rats; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1; D016559:Tacrolimus / Q000494:pharmacology; D013830:Thiadiazoles / Q000494:pharmacology
C476308:4-methyl-4'-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)-1,2,3-thiadiazole-5-carboxanilide; D000813:Anilides; D065095:Calcineurin Inhibitors; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D016207:Cytokines; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011948:Receptors, Antigen, T-Cell; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D013830:Thiadiazoles; D016572:Cyclosporine; D019703:Calcineurin; D002118:Calcium; D016559:Tacrolimus