ABSTRACT: FK506 (tacrolimus) is an FDA-approved immunosuppressant indicated for the prevention of allograft rejections in patients undergoing organ transplants. In mammals, FK506 inhibits the calcineurin-nuclear factor of activated T cells (NFAT) pathway to prevent T-cell proliferation by forming a ternary complex with its binding protein, FKBP12, and calcineurin. FK506 also exerts antifungal activity by inhibiting calcineurin, which is essential for the virulence of human-pathogenic fungi. Nevertheless, FK506 cannot be used directly as an antifungal drug due to its immunosuppressive action. In this study, we analyzed the cytotoxicity, immunosuppressive activity, and antifungal activity of four FK506 analogs, 31-O-demethyl-FK506, 9-deoxo-FK506, 9-deoxo-31-O-demethyl-FK506, and 9-deoxo-prolyl-FK506, in comparison with that of FK506. The four FK506 analogs generally possessed lower cytotoxicity and immunosuppressive activity than FK506. The FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against Cryptococcus neoformans and Candida albicans, which are two major invasive pathogenic yeasts, due to the inhibition of the calcineurin pathway. Furthermore, the FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against the invasive filamentous fungus Aspergillus fumigatus Notably, 9-deoxo-31-O-demethyl-FK506 and 31-O-demethyl-FK506 exhibited robust synergistic antifungal activity with fluconazole, similar to FK506. Considering the antifungal efficacy, cytotoxicity, immunosuppressive activity, and synergistic effect with commercial antifungal drugs, we selected 9-deoxo-31-O-demethyl-FK506 for further evaluation of its in vivo antifungal efficacy in a murine model of systemic cryptococcosis. Although 9-deoxo-31-O-demethyl-FK506 alone was not sufficient to treat the cryptococcal infection, when it was used in combination with fluconazole, it significantly extended the survival of C. neoformans-infected mice, confirming the synergistic in vivo antifungal efficacy between these two agents.
