Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression

Baumgrass R, Zhang Y, Erdmann F, Thiel A, Weiwad M, Radbruch A and Fischer G
J Biol Chem (2004) 279(4): 2470-2479
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Nucleofectorâ„¢ I/II/2b
The immunosuppressive drug cyclosporin A (CsA) is believed to inhibit antigen-specific activation of lymphocytes by blocking T cell receptor signaling cascades through targeting the Ca2+/calmodulin dependent Ser/Thr phosphatase calcineurin. CsA binds to and inhibits calcineurin only after interaction with cyclophilin 18 through a gain of function mechanism. The authors describe CsA derivatives, in which a single hydrogen atom of sarcosine at position 3 (Sar3) has been replaced by either alkyl or alkylthio groups, bypassing the requirement for Cyp18 binding in calcineurin inhibition. NFAT is a substrate of calcineurin and directly dephosphorylated at multiple sites. To further reveal the role of NFAT in this mechanism T cells were preincubated with a CsA derivative ([Dat Sar]3 CsA) or normal CsA at different concentrations and nucleofected with a NFAT-luciferase reporter plasmid. Cells were then stimulated and luciferase activity was determined. In both cases, luciferase activity was dose dependent to CsA treatment indicating that like CsA, [Dat Sar]3 CsA targets the NFAT pathway.
Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.