Potent inhibition of NFAT activation and T cell cytokine production by novel low molecular weight pyrazole compounds.
NFAT (nuclear factor of activated T cell) proteins are expressed in most immune system cells and regulate the transcription of cytokine genes critical for the immune response. The activity of NFAT proteins is tightly regulated by the Ca(2+)/calmodulin-dependent protein phosphatase 2B/calcineurin (CaN). Dephosphorylation of NFAT by CaN is required for NFAT nuclear localization. Current immunosuppressive drugs such as cyclosporin A and FK506 block CaN activity thus inhibiting nuclear translocation of NFAT and consequent cytokine gene transcription. The inhibition of CaN in cells outside of the immune system may contribute to the toxicities associated with cyclosporin A therapy. In a search for safer immunosuppressive drugs, we identified a series of 3,5-bistrifluoromethyl pyrazole (BTP) derivatives that block Th1 and Th2 cytokine gene transcription. The BTP compounds block the activation-dependent nuclear localization of NFAT as determined by electrophoretic mobility shift assays. Confocal microscopy of cells expressing fluorescent-tagged NFAT confirmed that the BTP compounds block calcium-induced movement of NFAT from the cytosol to the nucleus. Inhibition of NFAT was selective because the BTP compounds did not affect the activation of NF-kappaB and AP-1 transcription factors. Treatment of intact T cells with the BTP compounds prior to calcium ionophore-induced activation of CaN caused NFAT to remain in a highly phosphorylated state. However, the BTP compounds did not directly inhibit the dephosphorylation of NFAT by CaN in vitro, nor did the drugs block the dephosphorylation of other CaN substrates including the type II regulatory subunit of protein kinase A and the transcription factor Elk-1. The data suggest that the BTP compounds cause NFAT to be maintained in the cytosol in a phosphorylated state and block the nuclear import of NFAT and, hence, NFAT-dependent cytokine gene transcription by a mechanism other than direct inhibition of CaN phosphatase activity. The novel inhibitors described herein will be useful in better defining the cellular regulation of NFAT activation and may lead to identification of new therapeutic targets for the treatment of autoimmune disease and transplant rejection.
Trevillyan
J M
JM
Global Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA. james.m.trevillyan@abbott.com
Chiou
X G
XG
Chen
Y W
YW
Ballaron
S J
SJ
Sheets
M P
MP
Smith
M L
ML
Wiedeman
P E
PE
Warrior
U
U
Wilkins
J
J
Gubbins
E J
EJ
Gagne
G D
GD
Fagerland
J
J
Carter
G W
GW
Luly
J R
JR
Mollison
K W
KW
Djuric
S W
SW
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D019169:Jurkat Cells; D007959:Lymphocyte Culture Test, Mixed; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D000814:Aniline Compounds; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011720:Pyrazoles; D014157:Transcription Factors; D010749:Phosphoprotein Phosphatases; D002118:Calcium