Cyclosporin complex with cyclophilin

Fig. 11 Separation of human cyclophilin 18 (1) and the corresponding complex with the immunosuppressant cyclosporin A (2) by CE. A fixed concentration of cyclophilin (6.1 /xM) and increasing concentrations of cyclosporin (0 /xM, A, 2 /xM, B, 4 /xM, C, 7 /xM CsA, D) dissolved in DMSO (3) were incubated prior to the CE separation. (Reprinted with permission from Ref. 42. Copyright 1999 of Elsevier Science.)...

NMR investigations have also been made on the complex of cyclosporin A with another cyclophilin, cyclophilin B, which is highly homologous to cyclophilin A. The only difference between the complexes exists in the vicinity of residue 11, suggesting that selectivity could be achieved in a derivative of cyclophilin A by modification at just this position (Erickson and Fesik, 1992). 

Fig. 4.1 Mechanism of action of cyclosporine. Cyclosporine readily diffuses into the cytoplasm of the target cells where it binds to cyclophilins. The cyclosporine-cyclophilin complex stably associates with calcineurin and inhibits calcineurin activity. Calcineurin is a Ca2+-dependent enzyme— serine/threonine phosphatase— which after activation by Ca2+, dephosphorylates a cytosolic component of NFAT (NFATc, cytosolic factor of activated T cells). After dephosphorylation, NFATc migrates from the cytoplasm to the nucleus where it associates with NFATn and induces transcription of several cytokine genes including IL-2. Cyclosporine inhibits calcineurin activity after associating with cyclophilins, resulting in the inhibition of IL-2 production and other cytokines (see Color Insert)...

Lamos and colleagues reported the modification of cyclophilin A (CypA) by binding immunosuppressive cyclosporin A with a benzophenone-Dj j and a biotin moiety (compound 89, Fig. 11a) [91]. As a proof of principle, they used a 1 1 mixture of this TIP and its nondeuterated isoster for the selective PAL and pulldown of CypA among three other proteins. Subsequent tryptic digestion of the elutes and LC-MS/MS analysis allowed the identification of 11 CypA characteristic peptides, two of which were modified with the probe, as evidenced from the double, 11 Da separated, peaks in the mass spectra. The large 11-Da mass difference allowed easy visual recognition of labeled peptides in the mass spectra, which makes this a powerful method for determination of the modification site after PAL however, application in more complex systems still remains to be done. 

Cyclosporine provides maintenance immunosuppression by inhibition of the activation of T lymphocytes via a multifaceted mechanism. The drug, a fat soluble 11 amino acid cyclic polypeptide, crosses the lymphocyte membrane freely where it forms a pharmacologically active complex with the intracellular immunophilin receptor cyclophilin. This complex, but not cyclosporine by itself, inhibits the Ca /calmodulin-activated form of serine/threonine phosphatase calcineurin, thereby inhibiting the activation of NFAT cells. The latter action is considered to be the key step... 

Figure 9. (A) (left) Residues comprising the rotamase domain of cyclo-philin A. Residues that appear to be involved in the mechanism of enzymatic activity are colored green Trp-121 (yellow) is important for cyclosporin A binding, (right The solvent accessible surface of the cyclo-philin A domain, from the complex with dipeptide Ala-Pro. (B) Lipophilic (left and electrostatic (right) potential surfaces of the cyclophilin Adomain.

FRAP inhibits its function in T lymphocytes, which results in impaired interleukin-2 receptor signaling [31-34], and this is thought to be the basis for the immunosuppressive actions of rapamycin [30]. Another example of an immunosuppressant that acts in this manner is cyclosporin A, which interacts with cyclophilin to form a complex that then binds calcineurin [30]. 

Mechanism of action These peptide antibiotics interfere with T cell function by binding to immunophyllins, small cytoplasmic proteins that play critical roles in T cell responses to TCR activation and to cytokines. Cyclosporine binds to cyclophilin and tacrolimus binds to FK-binding protein (FKBP), both complexes inhibiting calcineurin, a cytoplasmic phosphatase. Calcineurin regulates the ability of the nuclear factor of activated T cells (NF-AT) to translocate to the nucleus and increase the production of cytokines. Cyclophilin and tacrolimus both inhibit the production of cytokines that normally occurs in response to TCR activation. Sirolimus also binds to FKBP, inhibiting the re-... 

The immimosuppressive effect of cyclosporin and FK506 could not initially be explained by these observations. Only with the discovery that cyclosporin and FK506 achieve their immunosuppressive effect via inhibition of calcineurin did it become clear that the immimosuppression is mediated by a complex reaction chain involving calcineurin. It was shown that the complexes of cyclosporin/cyclophilin and FK506/ FK506 binding protein bind to calcineurin and inhibit the phosphatase activity of the latter. 

Figure 16 (a) The structure of calcmeurin with suhunit A in cyan. The catalytic site containing a Zn-Fe hinding site is in dark red. Subunit B in orange contains four calciimi (green) ions hound to the EF-hand motifs. Both N- and C-terminal domains of calcineurin B interact to the same extended a-helix of calcineurin A. (h) The inhibitor complex cyclosporin A (red) and cyclophilin (violet) interacts with both calcineurin subunits and blocks the catal)dic site of calcineurin ... 

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