Abstract

There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, we establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders antifungal-resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating the protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for treating fungal infectious disease that evades resistance.

  • Compound

    (3S,6R,9S,12R,15S,18R)-3,9,15-tribenzyl-6,12,18-triisopropyl-4,10,16-trimethyl-1,7,13-trioxa-4,10,16-triazacyclooctadecane-2,5,8,11,14,17-hexaone

  • Compound

    4-(((3S,6R,9S,12R,15S,18R)-9,15-dibenzyl-6,12,18-triisopropyl-4,10,16-trimethyl-2,5,8,11,14,17-hexaoxo-1,7,13-trioxa-4,10,16-triazacyclooctadecan-3-yl)methyl)phenyl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate

  • Compound

    (2R)-2{[N-(tert-butoxycarbonyl)-N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid

  • Compound

    (2R)-2{[N-(tert-butoxycarbonyl)-N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid 2-(trimethylsilyl) ethyl ester

  • Compound

    (2R)-2{[N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid 2-(trimethylsilyl)ethyl ester

  • Compound

    (2R)-2{[N-(tert-butoxycarbonyl)-N-methyl-L-phenylalanyl]oxy}-3-methylbutanoic acid

  • Compound

    (2R)-2{[N-[(2R)-2{[N-(tert-butoxycarbonyl)-N-methyl-L-phenylalanyl]oxy}-3-methylbutanoyl]-N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid 2-(trimethylsilyl)ethyl ester

  • Compound

    (2R)-2{[N-[(2R)-2{[N-(tert-butoxycarbonyl)-N-methyl-L-phenylalanyl]oxy}-3-methylbutanoyl]-N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid 2-(trimethylsilyl)ethyl ester

  • Compound

    (2R)-2{[N-[(2R)-2{[N-[(2R)-2-[[N-(tert-butoxycarbonyl)-N-methyl-L-phenylalanyl]oxy]-3-methylbutanoyl]-N-methyl-L-phenylalanyl]oxy}-3-methylbutanoyl]- N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid 2-(trimethylsilyl)ethyl ester

  • Compound

    (2R)-2{[N-[(2R)-2{[N-[(2R)-2-[[N-(tert-butoxycarbonyl)-N-methyl-L-phenylalanyl]oxy]-3-methylbutanoyl]-N-methyl-L-phenylalanyl]oxy}-3-methylbutanoyl]-N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid

  • Compound

    (2R)-2{[N-[(2R)-2{[N-[(2R)-2-[[N-methyl-L-phenylalanyl]oxy]-3-methylbutanoyl]-N-methyl-L-phenylalanyl]oxy}-3-methylbutanoyl]-N-methyl-O-benzyl-L-tyrosyl]oxy}-3-methylbutanoic acid

  • Compound

    (3S,6R,9S,12R,15S,18R)-3,9-dibenzyl-15-(4-(benzyloxy)benzyl)-6,12,18-triisopropyl-4,10,16-trimethyl-1,7,13-trioxa-4,10,16-triazacyclooctadecane-2,5,8,11,14,17-hexaone

  • Compound

    (3S,6R,9S,12R,15S,18R)-3,9-dibenzyl-15-(4-hydroxybenzyl)-6,12,18-triisopropyl-4,10,16-trimethyl-1,7,13-trioxa-4,10,16-triazacyclooctadecane-2,5,8,11,14,17-hexaone

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Acknowledgements

We thank the Donnelly Sequencing Centre for whole genome sequencing; the 2013 MGY360H1 class for performing and analyzing whole-genome sequencing data for a subset of the resistant mutants in the ABC16 background; D. Kim for assistance with genome sequencing analysis of the resistant mutants in the yor1Δ background; and all members of the Cowen lab for discussions. T.S.-G. is supported by the Ontario Graduate Scholarship and University of Toronto Open Fellowship. L.E.C. is supported by the Canadian Institutes of Health Research (CIHR) Operating Grants (MOP-86452 and MOP-119520), the Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery Grants (06261 and 462167), an NSERC E.W.R. Steacie Memorial Fellowship (477598), and a Canada Research Chair in Microbial Genomics and Infectious Disease. J.H. is supported by the DUKE PO1 (AI104533-01) and RO1 (AI112595-02) for antifungal drug discovery. Y.-S.B is supported by the General International Collaborative R&D program funded by Ministry of Trade, Industry and Energy (MOTIE) in Republic of Korea (N0001720). A.A.L.G. is supported by US Institutes of Health Grants (R01 CA090265 and P41 GM094060). A.-C.G. is supported by a CIHR Foundation Grant, Genome Canada Genomics Innovation Network (GIN) Node and Technical Development Grants, and a Canada Research Chair in Functional Proteomics. J.-P.L was supported by a postdoctoral fellowship from the CIHR and by a TD Bank Health Research Fellowship at the Lunenfeld–Tanenbaum Research Institute. P.C. is supported by the Italian Government (FA). F.T. is supported by a postdoctoral fellowship from MIUR.

Author information

Affiliations

  1. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

    • Tanvi Shekhar-Guturja
    • , Frederick P Roth
    • , Anne-Claude Gingras
    •  & Leah E Cowen
  2. Natural Products Center, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of Arizona, Tucson, Arizona, USA.

    • G M Kamal B Gunaherath
    • , E M Kithsiri Wijeratne
    •  & A A Leslie Gunatilaka
  3. Lunenfeld–Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

    • Jean-Philippe Lambert
    • , Frederick P Roth
    •  & Anne-Claude Gingras
  4. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.

    • Anna F Averette
    • , Soo Chan Lee
    • , Taeyup Kim
    •  & Joseph Heitman
  5. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

    • Yong-Sun Bahn
  6. Department of Biotechnology and Biosciences, University of Milano–Bicocca and SYSBIO, Centre of Systems Biology, Milan, Italy.

    • Farida Tripodi
    •  & Paola Coccetti
  7. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.

    • Ron Ammar
  8. Institute of Biochemistry, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.

    • Katja Döhl
    •  & Lutz Schmitt
  9. Department of Molecular Biology, University of Geneva, Geneva, Switzerland.

    • Karolina Niewola-Staszkowska
    •  & Robbie J Loewith
  10. Institute of Microbiology, University Hospital Lausanne and University Hospital Center, Lausanne, Switzerland.

    • Dominique Sanglard
  11. Department of Medicine, University of Wisconsin, Madison, Wisconsin, USA.

    • David Andes
  12. Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, USA.

    • David Andes
  13. Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.

    • Corey Nislow

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Contributions

L.E.C. and T.S.-G. conceived of and designed the study. T.S.-G. designed and performed selection experiments, drug-susceptibility assays, qRT-PCR, β-galactosidase activity assays, and western blots. R.A. and C.N. analyzed whole-genome sequencing data for a subset of the beauvericin-resistant strains in the ABC16 background. F.P.R. analyzed whole-genome sequencing data for a subset of the beauvericin-resistant strains in the ABC16 background. D.S. designed and performed the azole accumulation experiment. L.S. and K.D. designed and performed the in vitro Pdr5 ATPase and rhodamine 6G transport assays. G.M.K.B.G., E.M.K.W., and A.A.L.G. synthesized the biotinylated beauvericin analog. A.-C.G. and J.-P.L. designed the AP–MS experiment; J.-P.L. performed and analyzed AP–MS data under supervision of A.-C.G. F.T. and P.C. designed and performed CK2 kinase activity assays. R.J.L. and K.N.-S. designed and performed experiments to monitor TORC1 and TORC2 activity via Sch9 and Ypk1 phosphorylation, respectively. Y.-S.B., A.F.A., and J.H. designed the C. albicans mouse study; Y.-S.B., A.F.A., S.C.L., T.K., and J.H. performed and analyzed the mouse study. D.A. designed and performed the C. albicans biofilm study.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Leah E Cowen.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–12.

  2. 2.

    Supplementary Note 1

    Isolation of beauvericin (1) and synthesis of biotinyloxybeauvericin

  3. 3.

    Supplementary Note 2

    Plasmid and strain construction

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DOI

https://doi.org/10.1038/nchembio.2165

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