ABSTRACT
The Ca2+/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 mum. 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.
Subject(s)
Calcineurin Inhibitors , Enzyme Inhibitors/pharmacology , Immunosuppressive Agents/pharmacology , Pyrimidines/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Calcineurin/metabolism , Cell Proliferation/drug effects , Cytokines/biosynthesis , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/immunology , Enzyme Inhibitors/metabolism , Humans , Immunization , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/immunology , Immunosuppressive Agents/metabolism , Intracellular Space/drug effects , Intracellular Space/metabolism , Jurkat Cells , Leukocytes, Mononuclear/immunology , NFATC Transcription Factors/metabolism , Phosphorylation/drug effects , Pyrimidines/chemistry , Pyrimidines/immunology , Pyrimidines/metabolism , Substrate Specificity , T-Lymphocytes/cytologyABSTRACT
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 microM. 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.
