Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs.

Abstract

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 fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus 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. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE 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 the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

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Published Version (Please cite this version)

10.1128/mbio.03000-21

Publication Info

Gobeil, Sophie M-C, Benjamin G Bobay, Praveen R Juvvadi, D Christopher Cole, Joseph Heitman, William J Steinbach, Ronald A Venters, Leonard D Spicer, et al. (2021). Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs. mBio, 12(6). p. e0300021. 10.1128/mbio.03000-21 Retrieved from https://hdl.handle.net/10161/28645.

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Scholars@Duke

Bobay

Benjamin Bobay

Assistant Professor in Radiology

I am the Assistant Director of the Duke University NMR Center and an Assistant Professor in the Duke Radiology Department. I was originally trained as a structural biochemist with an emphasis on utilizing NMR and continue to use this technique daily helping collaborators characterize protein structures and small molecules through a diverse set of NMR experiments. Through the structural characterization of various proteins, from both planta and eukaryotes, I have developed a robust protocol of utilizing computational biology for describing binding events, mutations, post-translations modifications (PTMs), and/or general behavior within in silico solution scenarios. I have utilized these techniques in collaborations ranging from plant pathologists at the Swammerdam Institute for Life Sciences department at the University of Amsterdam to biomedical engineers at North Carolina State University to professors in the Pediatrics department at Duke University. These studies have centered around the structural and functional consequences of PTMs (such as phosphorylation), mutation events, truncation of multi-domain proteins, dimer pulling experiments, to screening of large databases of ligands for potential binding events. Through this combination of NMR and computational biology I have amassed 50 peer-reviewed published articles and countless roles on scientific projects, as well as the development of several tutorials concerning the creation of ligand databases and high-throughput screening of large databases utilizing several different molecular dynamic and computational docking programs.

Venters

Ronald Venters

Assistant Professor in Radiology

I am Director of the Duke University NMR Center and a faculty member in the Duke Radiology Department (https://radiology.duke.edu/faculty/divisions/research-admin/). I have over 35 years of experience in the structure determination and dynamics of large biological macromolecules and in NMR methods development.  I am actively involved with training investigators in these areas and in assisting them with their projects.

Spicer

Leonard D. Spicer

University Distinguished Service Professor Emeritus

The focus of this laboratory is the study of structure/function relationships in biological macromolecules and their binding interactions. The principal method we use for system characterization is magnetic resonance spectroscopy. One specific area of interest is the structural characterization of functional domains in proteins which regulate the transcription of DNA coding for biosynthetic enzymes. The system under current investigation is the methionine repressor protein metJ, its corepressor S-adenosylmethionine, and the cognate sequence DNA. This protein, which functions as a dimer, exhibits a recently described DNA binding motif involving insertion of two beta strands into the major groove with additional stabilization of the complex arising from helix contacts at the dimer-dimer interface. We are using a full complement of heteronuclear 3D and 4D NMR methods to aid in the assignment of the main chain of the metJ repressor. We have recently reported a thermodynamic analysis of the binding interactions of metJ with its cognate DNA and corepressor SAM. We are now developing methods to measure fast proton exchange rates to complement our planned solution structural characterization. We have just initiated another project in collaboration with scientists at the Pacific Northwest National Laboratory to study macromelecular structures of DNA repair proteins in the nucleotide excision repair pathway. The first components of this critical supramacromolecular assembly we are investigating involve the DNA binding domain of the XPA protein for which we are determining the global fold in solution by NMR. Our program also includes a systematic approach to characterizing the conformational preferences of a number of sequentially related peptides developed by Dr. Barton Haynes' laboratory as candidate vaccines for HIV. The peptides consist of a fusion of two noncontiguous segments of the HIV protein gp120. Our goal is to establish whether structural conformers in solution contribute to peptide immunogenicity. We have finished a careful conformational analysis of the initial four peptides and are now correlating the conformer similarities and differences with immunogenic properties. We have also rationally designed several new peptides based on structural criteria and corresponding structural homology to the heavy fragment of IgA proteins. Initial NMR analysis and immunogenic response to three of the designed mutants indicate the rational design of preferred conformers was successful, but raised some novel questions regarding function of immunogenic peptides. We have also just begun a study of solution conformations of the hypoglycosylated tumor specific epitope repeat unit of human mucin and a promising mutant identified by Dombrowski and Wright. This epitope is common to breast and other adenocarcinomas and regulation of tumor specific lymphoid cells responding to this immunogen may be an important step in tumor control. Another protein under investigation is a functional core packing mutant of thioredoxin. We have fully characterized backbone chain dynamics to assess the impact of this mutation on molecular motions and are currently determining its high resolution tertiary structure. Currently, we are also using this mutant to demonstrate a new approach to global fold determination using a minimum set of long range NMR constraints. Finally, as an essential part of these studies, we are developing and have reported new 3- and 4-dimensional NMR experiments and heteronuclear filters for application to large protein systems and binding complexes.

Finally, the core activities of the NMR Center staff have continued to progress rapidly and enhancements to the state-of-the-art instrumentation have again been incorporated. A new deuteration strategy for assignment and study of large proteins by NMR has been developed and used to characterize one of the largest protein monomer reported to date, human carbonic anhydrase. We have also shown that we can observe the longest range distance constraints to date from NOESY correlations which are important in determining tertiary structure of proteins and we are examining the efficacy of structure determinations based on using these critical but limited constraints.


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