Targeting the calcineurin-NFAT system - Developing novel dual-peptide as an immunosuppressant using directed-evolution optimization

The phosphatase calcineurin targets the nuclear factor NFAT as part of the cellular immune activation. Calcium influx activates calcineurin to dephosphorylate multiple serine residues within the 400 residue NFAT homology region; this triggers NFAT nuclear translocation. The binding of calcineurin-NFAT relies on the interaction between two sites in calcineurin, harboring the conserved motifs PxIxIT and LxVP. NFAT`s PxIxIT motif binds calcineurin`s catalytic domain at a location that is a distance from the catalytic site of calcineurin. The LxVP motif binds at the interface of calcineurin`s regulatory and catalytic domains; however, it can also bind to the same site on the catalytic domain as the PxIxIT, but with a lower affinity.

Owing to the important role of calcineurin it is the target for immune suppression drugs obscuring its catalytic site. However, the vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the discovery of protein-protein interaction inhibitors that will interfere with the calcineurin-NFAT complex formation while keeping calcineurin`s catalytic site free.

Recently, we developed new interaction assay that is based on the binding of calcineurin to the full length NFAT homology region, including the phosphorylation sites flanked by PxIxIT and LxVP binding motifs. Based on this setup, we are currently developing an optimized dual-peptide constructs by linking peptides derived from the PxIxIT and LxVP motifs. Furthermore, directed-evolution is being used for the optimization of this chimeric peptide and for the creation of a novel protein-protein interaction inhibitor that can act as a new immunosuppressant.