Browsing by Subject "FKBP12"

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    Calcineurin Inhibitor CN585 Exhibits Off-Target Effects in the Human Fungal Pathogen Aspergillus fumigatus.
    (Journal of fungi (Basel, Switzerland), 2022-12) Juvvadi, Praveen R; Bobay, Benjamin G; Cole, D Christopher; Awwa, Monaf; Steinbach, William J
    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.
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    Leveraging Fungal Calcineurin-Inhibitor Structures, Biophysics and Dynamics to Design Selective and Non-Immunosuppressive FK506 Analogs
    (MBIO, 2020) Gobeil, Sophie M-C; Bobay, Benjamin; Juvvadi, Praveen; Cole, Christopher; Heitman, Joseph; Steinbach, William; Venters, Ronald; Spicer, Leonard
    Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically-accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal-specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungal-selective, non-immunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the M. circinelloides FKBP12 bound to FK506, and of the A. fumigatus, M. circinelloides and human FKBP12 proteins bound to the FK506 analog, APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes that will guide the rational design of inhibitors with enhanced fungal-selectivity.

    Significance statement

    Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of FK506 and a less immunosuppressive analog, APX879, that could be altered to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.