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.