A fusion Cell-Permeable C16orf74 Peptide Selectively Disrupts Calcineurin-NFAT Interaction and Inhibits T-cell Activation Without Cytotoxicity

Calcineurin (Cn) is a protein phosphatase that initiates T-cell activation by dephosphorylating the transcription factor NFAT, driving its nuclear translocation and the transcription of immune-related genes. While clinical immunosuppressants like Cyclosporine A (CsA) potently inhibit Cn, they completely block its catalytic site, leading to non-specific inhibition and severe off-target toxicity. Selectively targeting the specific protein-protein interaction (PPI) between Cn and NFAT presents a safer therapeutic strategy. We previously identified the C16orf74 (C16) peptide as a high-affinity Cn-NFAT PPI inhibitor; however, its utility in cellular systems is restricted by poor membrane permeability. In this study, we evaluated cell-penetrating peptide (CPP) conjugates of C16 with an N-terminus transactivator of transcription (TAT) and polyarginine (R11) to enable efficient intracellular delivery. Structural modeling, fluorescence polarization displacement, and pull-down assays confirmed that the CPP–C16 conjugates retain the ability to compete with an NFAT-derived peptide and bind Cn. Fluorescence microscopy demonstrated efficient intracellular entry of TAT-C16 and R11-C16 in mammalian cells, and effective inhibition of NFAT nuclear translocation and attenuation of downstream NFAT-dependent transcriptional activity of the IL-2 gene in human T cells at concentrations of 10 µM or lower. Crucially, unlike CsA, the CPP-C16 peptides exhibited minimal cytotoxicity even at high concentrations of up to 50 µM, establishing a potential safe therapeutic window. These findings establish CPP-C16 conjugates as effective, cell-permeable, and non-toxic inhibitors of the Cn-NFAT signaling axis, providing the basis for the development of PPI-directed immunosuppressants.