Calcineurin Inhibitor CN585 Exhibits Off-Target Effects in the Human Fungal Pathogen .

Calcineurin (CN) is an attractive antifungal target as it is critical for growth, stress response, drug resistance, and virulence in fungal pathogens. The immunosuppressive drugs, tacrolimus (FK506) and cyclosporin A (CsA), are fungistatic and specifically inhibit CN through binding to their respective immunophilins, FK506-binding protein (FKBP12), and cyclophilin (CypA). We are focused on CN structure-based approaches for the development of non-immunosuppressive FK506 analogs as antifungal therapeutics. Here, we examined the effect of the novel CN inhibitor, CN585, on the growth of the human pathogen , the most common cause of invasive aspergillosis. Unexpectedly, in contrast to FK506, CN585 exhibited off-target effect on wild-type and the azole- and echinocandin-resistant strains. Unlike with FK506 and CsA, the CN, FKBP12, CypA mutants (Δ, Δ, Δ) and various FK506-resistant mutants were all sensitive to CN585. Furthermore, in contrast to FK506 the cytosolic to nuclear translocation of the CN-dependent transcription factor (CrzA-GFP) was not inhibited by CN585. Molecular docking of CN585 onto human and CN complexes revealed differential potential binding sites between human CN versus CN. Our results indicate CN585 may be a non-specific inhibitor of CN with a yet undefined antifungal mechanism of activity.