Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents

Praveen R Juvvadi ,Anna Floyd Averette ,Michael Trzoss ,David Fox ,Joshua D Wheaton ,Ronald A Venters ,Maria Ciofani ,S Gobeil ,Mitchell W Mutz ,Michael J Hoy ,Maria A Schumacher ,Benjamin G Bobay ,Zanetta Chang ,Leonard D Spicer ,Blake C Barrington ,Ying-Lien Chen ,Xiaoming Li ,Joseph Heitman ,Deborah Cole ,Jackie Lin ,Soo Chan Lee ,William J Steinbach

doi:10.1038/s41467-019-12199-1

Abstract

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

Highlights

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive
Crystal structures of CN–FK506-FKBP12 ternary complexes from four fungi were determined by molecular replacement to resolutions between 1.85 and 3.30 Å (Table 1)
A flexible hinge in CnA separates the catalytic domain from the BBH, which forms the composite surface for FKBP12-FK506-complex binding

Summary

Introduction

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506FKBP12-complex identify a Phe[88] residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. X-ray structures, NMR-based inhibitor binding studies, site-directed mutagenesis of the A. fumigatus CN–FK506FKBP12 complex, and molecular dynamic (MD) simulations reveal a key residue, Phe[88], which is not conserved in the mammalian FKBP12 (His88), as essential for binding and inhibiting fungal CN. This enabled the rational design and development of a less immunosuppressive FK506 analog, APX879, with broad-spectrum in vitro antifungal activity and efficacy in a cryptococcal infection model. This study broadens translational insight into the development of novel non-immunosuppressive CN inhibitors for effective antifungal targeting

Methods

Results

Conclusion