International audienc

The Ca 2؉ /calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6-diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K i value of 3.8 M. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.The activation and the precise interplay between signaling pathways are crucial for the successful initiation and progression of the immune response as a reaction of an antigen contact. In T cells the stimulation of the T cell receptor by a specific antigen leads to a calcium release from the intracellular stores and to a calcium release-activated Ca 2ϩ channel-mediated calcium influx into the cytoplasm which activates calmodulin and thereby the Ser/Thr-protein phosphatase calcineurin (1). Consequently, calcineurin represents a bottleneck in T cell receptor signaling and allows the modulation of T cell activation by low molecular compounds, such as cyclosporin A (CsA) 2 or tacrolimus (FK506) (2). The cyclic undecapeptide CsA and the macrolid FK506 bind to and inhibit the phosphatase activity of calcineurin only after interaction with their respective peptidyl prolyl cis/trans isomerases (PPIases), cyclophilins (Cyp), and FK506-binding proteins (FKBP) through a gain-of-function mechanism (3, 4). Based on their particular characteristic to bind the immunosuppressive drugs CsA and FK506, members of the Cyp and FKBP family of PPIases were also termed immunophilins (5). Among the many known PPIases, the most abundant isoforms, Cyp18 and FKBP12, were identified as major intracellular acceptor proteins for CsA and FK506, respectively. The PPIase activity of both enzymes is strongly inhibited by the immunosuppressive drugs (6), leading to many of serious side effects (e.g. nephrotoxicity, neurotoxicity, hypertension, fibrosis) that were observed in the prevention and therapy of graftversus-host reactions or autoimmune diseases (7-10). Therefore, CsA derivatives, such as [DAT-Sar] 3 CsA, were synthesized to obtain cyclophilin-independent calcineurin inhibitors (11). However, [DAT-Sar] ...

Dephosphorylation of NFAT by the Ca 2+ -calmodulin-dependent Ser/Thr protein phosphatase calcineurin is a bottleneck of T cell receptor-dependent activation of T cells. In dimeric complexes with immunophilins, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) block this process by inhibition of the enzymatic activity of calcineurin. We have identified the pyrazolopyrimidine compound NCI3 as a novel inhibitor of calcineurin-NFAT signaling. Similar to CsA and FK506, NCI3 inhibits dephosphorylation and nuclear translocation of NFAT, IL-2 production and proliferation of stimulated human primary T cells with IC 50 values from 2 to 4.5 lM. However, contrary to CsA and FK506, NCI3 neither blocks calcineurin`s phosphatase activity nor requires immunophilins for inhibiting NFAT activation. Our data suggest that NCI3 binds to calcineurin and causes an allosteric change interfering with NFAT dephosphorylation in vivo but not in vitro. NCI3 acts not only on the endogenous calcineurin but also on a C-terminally truncated, constitutively active version of calcineurin. The novel inhibitor described herein will be useful in better defining the cellular regulation of calcineurin activation and may serve as a lead for the development of a new type of immunosuppressants acting not by direct inhibition of the calcineurin phosphatase activity.