Chemical genetics to identify NFAT inhibitors: Potential of targeting calcium mobilization in immunosuppression

Abstract

The development of more selective immunosuppressive agents to mitigate transplant rejection and autoimmune diseases requires effective strategies of blocking signaling pathways in T cells. Current immunosuppressive strategies use cyclosporin A (CsA) or FK506 to inhibit calcineurin, which dephosphorylates and promotes the nuclear import of nuclear factor of activated T cells (NFAT) transcription factors. These nuclear NFATs then transactivate cytokine genes that regulate proliferative responses of T cells. Both CsA and FK506 have debilitating side effects, including nephrotoxicity, hypertension, diabetes, and seizures, that argue for the development of alternative or complementary agents. To this end, we developed cell-based assays for monitoring NFAT dynamics in nonlymphoid cells to identify small molecules that inhibit NFAT nuclear import. Interestingly, we found that the majority of these small molecules suppress NFAT signaling by interfering with ''capacitative'' or ''store-operated'' calcium mobilization, thus raising the possibility that such mobilization processes are relevant targets in immunosuppression therapy. Further, these small molecules also show dose-dependent suppression of cytokine gene expression in T cells. Significantly, the IC50 of CsA in primary T cells was reduced by the addition of suboptimal concentrations of these compounds, suggesting the possibility that such small molecules, in combination with CsA, offer safer means of immunosuppression.capacitative calcium entry ͉ store-operated calcium channels ͉ cyclosporin A D rugs that destroy T cells or block their antigen-dependent activation have been the mainstays of treatment of organ transplant rejection (1). The latter group is represented chief ly by the natural products cyclosporin A (CsA) and FK506, both of which act by suppressing the protein phosphatase calcineurin (2-4). Calcineurin's activity depends on calcium signaling and plays an essential role in T cell signal transduction by controlling the nuclear import of the nuclear factor of activated T cells (NFAT) family of transcription factors. The NFATs are Rel-related transcription factors encoded by four genes (NFATc1-4) that are widely expressed in cells of the immune, cardiovascular, and nervous systems (4 -6). In resting cells, NFATs are localized to the cytoplasm due to a phosphorylation-dependent intramolecular masking of their nuclear location signals (NLSs) (7). During calcium signaling, calcineurin unmasks these NLSs, resulting in a rapid nuclear import of the dephosphorylated NFATs and transcriptional activation of cytokine genes (8). Besides unmasking the NLSs on the NFATs, calcineurin also masks their nuclear export signals, thereby preventing the futile cycling of these transcription factors across the nuclear envelope, thus ensuring their transcriptional functions in the nucleus (9, 10). NFAT proteins are essential for T cell activation and therefore represent potential targets for new immunosuppressive therapies. Interestingly, recent data from mouse knockout studie...

Coordination of NF-κB and NFAT Antagonism by the Forkhead Transcription Factor Foxd1

“…Regardless of the specific pathways, the present findings are consistent with the long-established clinical efficacy of the NF-AT inhibitors cyclosporin A and FK506 in human and murine systemic autoimmunity (24 -28) as well as the immunosuppressive utility of specific NF-AT inhibitors (29). They further are consistent with prior demonstrations of dysregulated NF-B activity in both human and murine lupus (30 -32), the inflammatory phenotypes of animals deficient in IB activity (33), and the ability of ectopic expression of inhibitory regulators of NF-B to suppress autoimmunity in vivo (31).…”

TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling

“…The association of SOCE with NFAT signaling has been well documented in nonexcitable cell types (20,21,38). However, relatively little is known of the potential roles of SOCE in excitable cells.…”

Small Molecules, Big Players: the National Cancer Institute's Initiative for Chemical Genetics

“…For example, a fluorescence polarization assay was used to identify a series of inhibitors of calcineurin-NFAT signaling [inhibitors of NFAT-calcineurin association (INCAs)] by blocking the targeting of calcineurin to its substrate NFAT (29). Cellbased assays used to monitor NFAT dynamics in nonlymphoid cells led to the discovery of small molecules that inhibit translocation of NFAT to the nucleus, including ChemB-143072 (30). The psammaplysenes-natural products derived from the marine sponge, Psammaplysilla-were discovered in a screen to compensate for lost tumor suppressor functionality in cells deficient in phosphatidylinositol phosphate 3 ¶-phosphatase (a phosphatase that works in opposition to phosphatidylinositol-3 ¶-kinase).…”