Structure-Guided Synthesis of FK506 and FK520 Analogs with Increased Selectivity Exhibit <i>In Vivo</i> Therapeutic Efficacy against Cryptococcus.
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2022-06
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Calcineurin is an essential virulence factor that is conserved across human fungal pathogens, including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals, and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which was less immunosuppressive yet maintained antifungal activity. We now report high-resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin-bound inhibitor complexes has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.
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Hoy, Michael J, Eunchong Park, Hyunji Lee, Won Young Lim, D Christopher Cole, Nicholas D DeBouver, Benjamin G Bobay, Phillip G Pierce, et al. (2022). Structure-Guided Synthesis of FK506 and FK520 Analogs with Increased Selectivity Exhibit In Vivo Therapeutic Efficacy against Cryptococcus. mBio, 13(3). p. e0104922. 10.1128/mbio.01049-22 Retrieved from https://hdl.handle.net/10161/28884.
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Scholars@Duke

Benjamin Bobay
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.

Maria Ciofani
Transcriptional Regulation of Proinflammatory Lymphocytes
IL-17-expressing CD4 T helper (Th17) cells are important members of the intestinal immune cell community that contribute to protection against bacterial and fungal infections, and maintenance of intestinal homeostasis. Although central to immunity, dysregulted Th17 cell function has been implicated in tissue inflammation and autoimmune disease (e.g. Inflammatory bowel disease, arthritis, and multiple sclerosis). In order to understand this balance between healthy and pathogenic responses, we are interested in defining the transcriptional regulatory mechanisms that govern (1) Th17 cell specification from naive T cell precursors and, (2) Th17 cell effector plasticity during inflammation. Combining genome-wide interrogation of regulatory information (transcription factor occupancy, chromatin accessibility, and transcriptional output) with gene-deficiency models in mice, we can dissect the contribution of key transcriptional regulators in proinflammatory T cell function.
We currently have open positions for students, postdoctoral fellows and a research technician.

Jiyong Hong
Research in the Hong group focuses on using chemical tools, in particular small molecules, to understand the signaling pathways in biology. We synthesize biologically interesting natural products and screen small molecule libraries to identify modulators of biological processes. Then, we explore their modes of action in order to investigate intracellular signaling pathways and identify novel targets for drug design. In addition, we design and develop unique and efficient synthetic strategies that will allow rapid access to molecular complexity and structural diversity. Through multidisciplinary approaches, including organic synthesis, molecular biology, and cell biology, the cellular components and molecular events that embody cancer, immune response, and GPCR signaling have systematically been explored. Compounds employed in these studies could also advance the development of novel therapeutics for the treatment of human diseases.
- Synthesis of Natural Products and Study of Mode of Action: We synthesize biologically interesting natural products and explore the modes of action in order to investigate intracellular signaling pathways and identify novel targets for drug design. Completed target molecules include largazole (a marine natural product with HDAC inhibitory activity), brasilibactin A (a cytotoxic siderophore), manassantins A and B (natural products with anti-HIF-1 activity), and subglutinols A and B (natural products with immunosuppressive activity).
- Development of Novel Synthetic Methodology: We design and develop unique and efficient synthetic strategies which will allow rapid access to molecular complexity and structural diversity. A specific area of interest includes the development of novel methods for the stereoselective synthesis of substituted tetrahydrofurans and tetrahydropyrans.
- Screen of Small Molecule Libraries for Identification of Small Molecule Modulators of Biological Processes: With the advent of combinatorial chemistry and other synthetic technologies, it is feasible to prepare large collections of synthetic organic molecules. These libraries are useful in providing molecules that can be used to probe relevant biological pathways. We are interested in identification of modulators of biological processes, including drug abuse and neurodegenerative diseases.
Through multidisciplinary approaches, the cellular components and molecular events that embody cancer, immune response, and neurodegenerative diseases are systematically explored. Compounds employed in these studies could also advance the development of novel therapeutics for the treatment of human diseases.
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