Abstract

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex1,2. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi3,4. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata5,6. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent7,8. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active9,10,11,12,13,14, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease.

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Accessions

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

Coordinates and NMR resonance assignments have been deposited in the Protein Data Bank (PDB code 4D7X) and Biological Magnetic Resonance Data Bank (BMRB code 25372). RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession GSE74361.

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Acknowledgements

We are grateful to P. Coote, E. Papadopoulos, R. Oh and R. E. Luna for helpful discussions and advice with data analysis and manuscript preparation. We acknowledge the ICCB-Longwood Screening Facility at Harvard Medical School for assistance with the high-throughput screens and access to the compound libraries, and the MGH Next Gen sequencing core for RNA-seq library construction. Mouse plasma and microsomal stability experiments were carried out at the Scripps Research Institute and iKIX1 pharmacokinetic parameters were assessed by Sai Life Sciences Limited. We acknowledge support from the National Institute of Health (grants GM047467 to G.W. and A.M.N. and EB002026 to G.W.). J.L.N. was supported by an NSERC fellowship.

Author information

Affiliations

  1. Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA

    • Joy L. Nishikawa
    • , Yoo-Jin Sohn
    •  & Anders M. Näär
  2. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Joy L. Nishikawa
    • , Yoo-Jin Sohn
    •  & Anders M. Näär
  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Andras Boeszoermenyi
    • , Sara J. Buhrlage
    • , Nathanael S. Gray
    • , Gerhard Wagner
    •  & Haribabu Arthanari
  4. Institute of Microbiology, University Hospital Lausanne and University Hospital Center, Lausanne CH-1011, Switzerland

    • Luis A. Vale-Silva
    •  & Dominique Sanglard
  5. Institute of Microbiology, Università Cattolica del Sacro Cuore, Rome 00168, Italy

    • Riccardo Torelli
    •  & Maurizio Sanguinetti
  6. Institute of Public Health, Università Cattolica del Sacro Cuore, Rome 00168, Italy

    • Brunella Posteraro
  7. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Fei Ji
    •  & Ruslan I. Sadreyev
  8. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Fei Ji
  9. Faculty of Chemistry and Pharmacy, Sofia University, Sofia 1164, Bulgaria

    • Vladimir Gelev
  10. Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

    • Ruslan I. Sadreyev
  11. Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

    • Goutam Mukherjee
    •  & Jayaram Bhyravabhotla
  12. Supercomputing Facility for Bioinformatics & Computational Biology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

    • Goutam Mukherjee
    •  & Jayaram Bhyravabhotla
  13. Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India

    • Jayaram Bhyravabhotla
  14. Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Sara J. Buhrlage
    •  & Nathanael S. Gray

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Contributions

J.L.N., A.B., G.W., A.M.N. and H.A. conceived and designed the studies. A.B. and H.A. performed experiments relating to protein structure, small molecule screening and small molecule-protein interaction and data analysis. J.B. and G.M. performed the docking and free energy calculations. V.G., S.J.B. and N.S.G. designed the synthesis for iKIX1 and its analogues. J.L.N. performed the in vivo small molecule screen, luciferase, ChIP, transcription, efflux, spot plating, combination index and mammalian cell culture (HepG2) experiments. Y.-J.S. performed transcription and efflux experiments. J.L.N. prepared samples for RNA-seq analysis; bioinformatic analysis was carried out by F.J. and R.I.S.; L.A.V.-S. and D.S. designed and performed moth survival and adhesion assays. R.T., B.P. and M.S. designed and executed mouse fungal burden and UTI model studies. J.L.N., A.B., G.W., A.M.N. and H.A. wrote the manuscript with input from the team.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gerhard Wagner or Anders M. Näär or Haribabu Arthanari.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods and Supplementary References.

  2. 2.

    Supplementary Table 1

    This file contains RNA-Seq data for C. glabrata as RPKM (reads per kilobase per million mapped reads); key CgPdr1 targets are in bold. Genes are ranked by log2 fold change of expression between KET and iKIX1/KET treated cells (column logFC).

  3. 3.

    Supplementary Table 2

    This file contains RNA-Seq data for S. cerevisiae as RPKM (reads per kilobase per million mapped reads); key ScPdr1 targets are in bold. Genes are ranked by log2 fold change of expression between KET and iKIX1/KET treated cells (column logFC).

  4. 4.

    Supplementary Table 3

    This file contains gene ontology (GO) analysis of differentially expressed genes in S. cerevisiae upon iKIX1 treatment. Oxidative stress response gene expression is prominently elevated.

Excel files

  1. 1.

    Supplementary Tables 4-6

    This file contains Supplementary Tables 4-6. Supplementary Table 4 contains a list of strains used in the study, Supplementary Table 5 contains a list of plasmids used in the study and Supplementary Table 6 contains a list of primers used in the study.

About this article

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DOI

https://doi.org/10.1038/nature16963

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