Calcineurin inhibitors complex inhibition

Cyclosporine and tacrolimus belong to a class of immunosuppressants called the calcineurin inhibitors. These agents are considered by many to be the cornerstone of medical immunosuppression. The calcineurin inhibitors work by complexingwith cytoplasmic proteins (cyclosporine with cyclophylin and tacrolimus with FK binding protein 12). These complexes then inhibit calcineurin phosphatase, which results in reduced IL-2 gene transcription. The final outcome is a decrease in IL-2 synthesis and a subsequent reduction in T cell activation.7 11 20 21... 

Example of Scheme C Inhibition of Calcineurin by FKBP-Inhibitor Complexes... 

Cyclosporine binds to an intracellular protein, cyclophilin. Cyclophilins and similar binding proteins are now referred to collectively as immunophilins and their enzymatic activities are relevant to the actions of immunosuppressants such as cyclosporine and tacrolimus. This complex inhibits the phosphatase activity of calcineurin, which in turn prevents dephosphorylation and translocation of NFAT. NFAT is required to induce a number of cytokine genes, including that for interleukin-2, which serves as a T-cell growth and differentiation factor (Krensky et al., 2005 Matsuda and Koyasu, 2000). Cyclosporine also increases expression of TGF-p, which is a potent inhibitor of IL-2 stimulated cell proliferation and generation of cytotoxic T lymphocytes (Khanna et al., 1994). 

Sirohmus is a macrocychc lactone produced by the bacteria Streptomyces hygroscopious. Like the calcineurin inhibitors cyclosporine and tacrolimus its mechanism of action involves formation of a complex with an immunophiUn, in this case, FKBP-12. Unlike cyclosporine and tacrolimus, sirohmus does not affect calcineurin activity but binds to and inhibits the mammalian kinase, target of rapamycin (mTOR.). mTOR is a key enzyme in cell-cycle progression. When inhibited this kinase blocks cell cycle progression at the G1 to S phase transition (Dumont and Su, 1996 Sehgal, 2003). 

Topical calcineurin inhibitors, including tacrolimus and pime-crolrmus, have added a new dimension to treatment of AD. Unlike corticosteroids, these agents offer a more long-term option, as they can be used on all parts of the body for prolonged periods without fear of corticosteroid-induced adverse effects. These agents form a complex that results in inhibition of calcineurin, which normally initiates T-cell activation. Through inhibition, the complex subdues the inflammatory component of AD (Table 97-3). ... 

Cyclosporine A (CSA), sirolimus, and tacrolimus are commonly used immunosuppressive drugs. Cyclosporine and tacrolimus are calcineurin inhibitors, whereas sirolimus is a mammalian target of rapamycin (mTOR) inhibitor. Calcineurin inhibitors bind to immunophilins, and drug-immunophilin complexes inhibit calcineurin activity, which in turn prevents nuclear translocation of the nuclear factor of activated T cells (NFAT) (1,2). This results in inhibition of activation and proliferation of CD4 and CDS lymphocytes by inhibiting IL-2 production. The mTOR protein is a... 

Sirolimus (rapamycin) (Vezina et al. 1975) is widely used to prevent rejection in organ transplant. It is especially usefiil in kidney transplants because, different from cyclosporine and tacrolimus, it is not a calcineurin inhibitor and therefore is less toxic to the kidney. Sirolimus inhibits T-cell and B-cell activation. It binds to the immunophilin FKproteinl2, and this binary complex inactivates a serine-threonine kinase (mTOR) termed the mammalian target of rapamycin (Huang et al. 2003). The final effect is the arrest at phase G1 of cell cycle progression. This effect occurs not only in T cell and B cells, but it has been observed in many tumor cell lines. Semisynthetic derivatives of rapamycin, suitable for i.v. administration, have been developed as antitumor agents. Temsirolimus was approved by FDA in 2007 for advanced kidney cancer treatment (Hudes 2009). Everolimus was also approved for kidney cancer treatment in 2009 and for organ rejection prophylaxis in 2010. At present, phase III clinical trials are under way in a variety of tumors (Dansey 2006). 

Addition of the L-732,531 FKBP binary complex to a calcineurin activity assay resulted in increasingly nonlinear progress curves with increasing binary complex concentration. The htting of the data to Equation (6.3) revealed an inhibitor concentration effect on v-, as well as on vs and obs, consistent with a two-step mechanism of inhibition as in scheme C of Figure 6.3. Salowe and Hermes analyzed the concentration-response effects of the binary complex on v, and determined an IC50 of 0.90 pM that, after correction for I.S I/A (assuming competitive inhibition), yielded a A) value for the inhibitor encounter complex of 625 nM. 

Although CsA and FK506 are extremely selective inhibitors of calcineurin, some of the studies above indicate that these drugs could possibly have calcineurin-independent elfects. Moreover, calcineurin shows phosphatase activity towards a wide variety of phosphoprotein substrates, and inhibition of calcineurin by immunosuppressant-immunophilin complexes blocks phosphatase activity toward a broad spectrum of phos-phoproteins. Inhibitors that blocked calcineurin mediated dephosphorylation of a specific substrate (such as NF-AT, or specific isoforms of NF-AT) without affecting the dephosphorylation of other substrates would be of great therapeutic as well as academic interest. The search for... 

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