The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells

Abstract

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] ...

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“…9, D-F). To further confirm these observations, we utilized the potent calcineurin inhibitor CN585, which interacts directly with calcineurin (27). Similar to CSA, treatment with CN585 dose-dependently reduced IL-1␤ secretion with a minimal effect on TNF␣ or LDH release (Fig.…”

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“…26 Thus, we sought to determine whether calcineurin indeed plays a role in their mechanism of action. To this end, we used several established calcineurin modulators such cyclosporin A, developed as an immuno-suppressant drug due to the crucial role that calcineurin plays in T-cell activation ( Figure 5).…”