title: Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.
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 complex. 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 fungi. 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 glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, 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.
authors.affiliation: Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.|Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Joy L
authors.initials: JL
authors.lastname: Nishikawa
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Andras
authors.initials: A
authors.lastname: Boeszoermenyi
authors.affiliation: Institute of Microbiology, University Hospital Lausanne and University Hospital Center, Lausanne CH-1011, Switzerland.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Vale-Silva
authors.affiliation: Institute of Microbiology, Università Cattolica del Sacro Cuore, Rome 00168, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Torelli
authors.affiliation: Institute of Public Health, Università Cattolica del Sacro Cuore, Rome 00168, Italy.
authors.forename: Brunella
authors.initials: B
authors.lastname: Posteraro
authors.affiliation: Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.|Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Yoo-Jin
authors.initials: YJ
authors.lastname: Sohn
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Ji
authors.affiliation: Faculty of Chemistry and Pharmacy, Sofia University, Sofia 1164, Bulgaria.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Gelev
authors.affiliation: Institute of Microbiology, University Hospital Lausanne and University Hospital Center, Lausanne CH-1011, Switzerland.
authors.forename: Dominique
authors.initials: D
authors.lastname: Sanglard
authors.affiliation: Institute of Microbiology, Università Cattolica del Sacro Cuore, Rome 00168, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Sanguinetti
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.|Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Ruslan I
authors.initials: RI
authors.lastname: Sadreyev
authors.affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.|Supercomputing Facility for Bioinformatics &Computational Biology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
authors.forename: Goutam
authors.initials: G
authors.lastname: Mukherjee
authors.affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.|Supercomputing Facility for Bioinformatics &Computational Biology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.|Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
authors.forename: Jayaram
authors.initials: J
authors.lastname: Bhyravabhotla
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.|Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Buhrlage
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.|Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.
authors.forename: Nathanael S
authors.initials: NS
authors.lastname: Gray
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
authors.affiliation: Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.|Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Anders M
authors.initials: AM
authors.lastname: Näär
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Haribabu
authors.initials: H
authors.lastname: Arthanari
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D041221:Candida glabrata / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D002177:Candidiasis / Q000188:drug therapy / Q000382:microbiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000187:drug effects*; D026141:Drug Resistance, Multiple, Fungal / Q000187:drug effects; D015725:Fluconazole / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006834:Hydrazines / Q000493:pharmacokinetics / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D056892:Mediator Complex / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000935:Antifungal Agents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058337:GAL11 protein, S cerevisiae; D006834:Hydrazines; D056892:Mediator Complex; C085480:PDR1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C000605811:iKIX1 compound; D015725:Fluconazole; D013890:Thiourea; D007654:Ketoconazole