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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Gene Expression Omnibus
Protein Data Bank
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.
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.
Extended data figures
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.