Potent Inhibition of NFAT Activation and T Cell Cytokine Production by Novel Low Molecular Weight Pyrazole Compounds

Abstract

Engagement of the T cell antigen receptor (TcR)1 with the antigen-major histocompatibility complex on antigen-presenting cells triggers a complex TcR signaling cascade that leads to T cell activation and cytokine secretion (1). During this process, T cells express the autocrine growth factor interleukin 2 (IL-2), which promotes T cell proliferation by interacting with the IL-2 receptor, which is also up-regulated on activated T cells. The transcriptional regulation of the IL-2 gene has been extensively analyzed at the IL-2 promoter, a 275-bp region located upstream of the transcriptional start site of the gene (2, 3). Several transcription factors have been identified to bind elements within this regulatory region, including AP-1, NF-B, and the nuclear factor of activated T cells (NFAT) (2).The transcription factor NFAT plays an essential role in IL-2 expression. Binding sites for NFATs have also been found within the promoter regions of several other cytokine genes, including IL-3, IL-4, IL-5, IL-8, IL-13, tumor necrosis factor ␣, granulocyte-macrophage colony-stimulating factor, and ␥-IFN (4, 5). NFAT is a complex composed of a cytoplasmic subunit and an inducible nuclear component comprised of AP-1 (Fos/ Jun) family members. At least four structurally related NFAT cytoplasmic subunit members, NFATp/NFAT1, NFATc/ NFAT2, NFAT3, and NFATX/NFATc3/NFAT4, have been identified (5). NFAT proteins share a conserved domain located toward the C terminus (6) that binds DNA and also participates in cooperative protein-protein interactions with AP-1 transcription factors (7,8). Immediately N-terminal to the DNA-binding domain is a second conserved module of ϳ300 residues known as the NFAT homology (NFAT-h) region. The N terminus of NFAT, including the NFAT-h region, regulates nuclear/cytoplasm trafficking in response to changes in intracellular Ca 2ϩ concentrations. In resting T cells, the protein is retained in the cytoplasm and its NFAT-h domain is heavily phosphorylated. Engagement of the TcR or treatment of cells with the Ca 2ϩ ionophore activates the Ca 2ϩ /calmodulin-dependent Ser/Thr phosphatase, calcineurin. CaN dephosphorylates the NFAT-h domain, resulting in translocation of NFAT to the nucleus (9).

Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2

“…Unfortunately, all CRAC channel blockers described so far, including the most potent ones, SK&F 96365 and econazole (11,12) or 2-aminoethyldiphenyl borate (13)(14)(15)(16)(17)(18), have IC 50 values in the micromolar range and are nonspecific. Recently, Djuric and colleagues (19,20) have described pyrazole derivatives that interfere specifically with the expression of Ca 2ϩ -dependent cytokine production following TCR stimulation, but they could not detect inhibition of TCR-dependent Ca 2ϩ signals in T-cells (21). On the contrary, Ishikawa et al (22) could demonstrate that one of the pyrazole derivatives named BTP2 (or YM-58483) is a potent inhibitor of store-operated influx in Jurkat T-cells.…”

“…Because other channels and transporters in T-cells were unaffected, BTP2 seems to be the first specific and potent CRAC channel inhibitor. Pyrazole derivatives were only recently introduced as immunomodulators by Djuric and colleagues (19,20) and were found to be potent inhibitors of nuclear factor of activated T-cells activation and cytokine production. They were later described to inhibit Ca 2ϩ -dependent gene expression in mononuclear cells from peripheral blood (21).…”

The role of nuclear factor of activated T cells in pulmonary arterial hypertension

“…Specific NFAT inhibitors such as modulatory calcineurin-interacting proteins which restrains calcineurin activity has been used to treat cardiac hypertrophy and failure. 55 NFAT inhibitor (A-285222) inhibits NFAT without affecting calcineurin activity and has no off-target side effects in either primates 56,57 or rodents. 58 Therefore, in PAH, NFAT inhibitors might reverse pulmonary vascular remodeling through their effects on PA-SMCs, and inflammatory cells.…”

The Immunosuppressive Effect of Gamisanghyulyunbueum through Inhibition of Mitogen-Activated Protein Kinase and Nuclear Factor Activation in MOLT-4 Cells

“…31) Pyrazole compounds are a potent inhibitor of NFAT activation and T cell cytokine production. 32) We tried to chase the main acting compound from GSHYBE. Hence we fractionated GSHYBE and HPLC assay (data not shown).…”