Discovery and development of inhibitors of the Calcineurin-NFAT interaction

A central step in T-cell activation involves the interaction of the transcription factor ‘Nuclear Factor of Activated T-cell’ (NFAT) with the phosphatase calcineurin (Cn) a process which is also known as T-cell activation switch. Cn is a serine threonine protein phosphatase that depends on Ca²⁺ and calmodulin for its activity on a wide-range of substrate proteins. Its interaction with NFAT leads to the translocation of NFAT to the nucleus and to subsequent transcription of T-cell activating genes. Cyclosporine A (CsA) and FK506, two potent inhibitors of Cn where discovered so far, and are used for transplantation therapy by blocking Cn catalytic site and thus lowering the immune response. Although highly successful, CsA and FK506 have severe side effects as they are interfering with Cn enzymatic activity in a non-specific manner. The aim of this study is to discover and develop selective inhibitors of the Cn-NFAT interaction. Based on a structural model of the Cn-NFAT complex we performed virtual screening campaign and selected about 300 compounds for further in-vitro testing and validation by NMR and ITC. Indeed, several compounds presented direct binding to Cn at low micro-molar range. Promising binders were tested in-vitro by monitoring their ability to inhibit Jurkat cells stimulation with ionomycin and PMA treatment. Furthermore, expression levels of mRNA encoding cytokines like IL-2 and TNFα was inhibited after stimulation the cells with ionomycin and PMA in the presence of these compounds, suggesting that these compounds may be further developed as selective and safe immunosuppressive agents.