Compounds inhibiting the enzymatic exercise of cal cineurin are supposed to block the dephosphorylation of all protein substrates. Thus, only compounds target ing distinct calcineurin substrate interactions but not the general phosphatase action of calcineurin could be in a position to dissect the action of calcineurin on distinct substrates. Lately, different efforts were manufactured to identify this kind of com pounds, interfering particularly with calcineurin NFATc interactions in T cells. Dipyridamole, a drug clinically employed for stroke therapy, is advised to affect the interaction of NFATc with cal cineurin, as it competes with fluorescence labelled RCAN1 CIC peptide for binding to cal cineurin. Dipyridamole will not interfere with the phos phatase activity of calcineurin on RII phosphopeptide in cell absolutely free assays.
It suppresses ionomycin induced NFATc2 nuclear translocation in Jurkat T and U 2 osteosarcoma cell lines, and blocks subsequently NFATc dependent reporter gene and cytokine expression. Dipyrida mole inhibits TNF production in activated PBMC. NCI3, a pyrazolopyrimidine find more information derivative, will not influ ence the enzymatic exercise of calcineurin in cell totally free sys tems. Nonetheless, NCI3 inhibits NFATc dephosphorylation and nuclear translocation, IL 2 secretion and cell prolifer ation on stimulation of Jurkat or major human T cells. NFATc dependent reporter gene expression is extra delicate to NCI3 than NFB, whereas AP 1 dependent transcription is not influenced. These effects are dimin ished by calcineurin overexpression. An effect of NCI3 around the calcineurin substrate interface is postulated since it par tially displaces the VIVIT peptide, an oligopeptide derived from the PxIxIT calcineurin binding motif of NFATc.
INCA compounds really are a group of chemically unrelated substances selected within a screening for inhibition of NFATc calcineurin interaction. INCA one, 2 and 6 bind covalently but reversibly to calcineurin with the residue Cys266. Subse quently, steric alterations mask the binding web-site for NFATc and VIVIT peptide. INCA two, but not INCA 6, selelck kinase inhibitor inhibits the enzymatic action of calcineurin. INCA six inhibits the dephosphorylation of NFATc and its nuclear import in ionomycin stimulated Cl. 7W2 murine T cell line and, consequently, the expression of IFN and TNF. On the other hand, standard cytotoxicity continues to be reported for all INCA com pounds, ruling out their use in key cells. Inhibitors not acting right within the calcineurin molecule Inhibitors of calcineurin NFATc signalling might not only act on calcineurin itself, but in addition up or downstream of the calcineurin NFATc interaction or dephosphorylation processes. Between the choices are results of com lbs on calcium mobilization, to the nuclear translo cation of NFATc or on NFATc DNA binding.