Small-molecule NFAT signaling inhibitor with dual NFATc1/NFATc2 activity and limited calcineurin catalytic inhibition.

Calcineurin-nuclear factor of activated T cells (NFAT) signaling plays a central role in immune activation and represents a major therapeutic target for immunosuppression. Although calcineurin inhibitors such as tacrolimus are clinically effective, their long-term use is limited by severe systemic toxicities associated in part with broad inhibition of calcineurin catalytic activity. In this study, we investigated whether small-molecule inhibition of NFAT signaling could suppress NFAT activation while limiting direct inhibition of calcineurin catalytic activity. We designed and synthesized fourteen analogs (2-15) based on a small-molecule inhibitor previously identified and derived from the PxIxIT sequence. We assessed the inhibitory activities of structurally modified analogs against NFATc1 and NFATc2 and compared their isoform-specific inhibitory profiles. Our investigation revealed that analog 14 produced closely matched inhibition of NFATc1 and NFATc2, suppressed NFAT-dependent cytokine production, and showed cellular immunosuppressive activities. Importantly, 14 showed only limited inhibition of calcineurin catalytic activity at concentrations associated with its NFAT-inhibitory effects. At concentrations matched for IL-2 suppression, 14 modestly reduced Jurkat T-cell proliferation and activation. It also showed a favorable preliminary safety-related profile across the cellular and zebrafish models tested, including longer-term assessments of cell viability, oxidative stress, and renal injury. Collectively, these findings establish 14 as an early-stage lead compound for selective modulation of NFAT signaling while limiting calcineurin catalytic inhibition, providing a foundation for further pharmacological development.