Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction

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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 inSaccharomyces cerevisiaeand in the clinically important human pathogenCandida glabrata5,6. The prevalence ofC. glabratais rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent7,8. Drug-resistant clinical isolates ofC. glabratamost commonly contain point mutations in Pdr1 that render it constitutively active9-14, suggesting that this transcriptional activation pathway represents a linchpin inC. glabratamultidrug resistance. Here we perform sequential biochemical andin vivohigh-throughput screens to identify small-molecule inhibitors of the interaction of theC. glabrataPdr1 activation domain with theC. glabrataGal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistantC. glabratato azole antifungalsin vitroand in animal models for disseminated and urinary tractC. glabratainfection. Determining the NMR structure of theC. glabrataGal11A 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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