Calcineurin Inhibitor CN585 Exhibits Off-Target Effects in the Human Fungal Pathogen <i>Aspergillus fumigatus</i>

Abstract

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 Aspergillus fumigatus, the most common cause of invasive aspergillosis. Unexpectedly, in contrast to FK506, CN585 exhibited off-target effect on A. fumigatus wild-type and the azole- and echinocandin-resistant strains. Unlike with FK506 and CsA, the A. fumigatus CN, FKBP12, CypA mutants (ΔcnaA, Δfkbp12, ΔcypA) 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 A. fumigatus CN complexes revealed differential potential binding sites between human CN versus A. fumigatus CN. Our results indicate CN585 may be a non-specific inhibitor of CN with a yet undefined antifungal mechanism of activity.

Keywords

• <i>Aspergillus</i>
• calcineurin
• CN585
• cyclosporin A
• cyclophilin
• FK506