Infections associated with Candida species cause the 4th most common type of bloodstream infection. Only three effective antifungal drugs exist, causing a real threat to the continued viability of antifungal therapy. C. glabrata is the second most commonly isolated species associated with candidemias and has both low intrinsic susceptibility to azole drugs and can readily acquire robust tolerance to this most commonly used class of antifungal compounds. Azole-resistant C. glabrata isolates are almost exclusively caused by point mutations in the gene encoding a transcription factor called PDR1. These point mutations lead to the production of a gain-of-function (GOF) form of the Pdr1 transcriptional regulator that in turn cause high level constitutive expression of target genes. A key Pdr1 target gene is the CDR1 locus that encodes an ATP-binding cassette transporter thought to act as a broad specificity drug efflux pump, preventing the accumulation of azole drugs in cells carrying these GOF PDR1 alleles. The goal of this application is to use a chemical genetic approach to identify small molecules that can inhibit activation of CDR1 by GOF forms of Pdr1. We will use two different chemical libraries to screen a C. glabrata strain containing a CDR1-luciferase (CDR1-LUC) gene fusion to identify compounds that are able to inhibit this central regulatory step in azole resistance acquisition in this pathogenic yeast. We have already validated our ability to readily detect expression changes in CDR1-LUC using a 384 well format assay that will be employed in the chemical library screen. Aim 1 will employ this CDR1- luciferase reporter strain to screen two different chemical libraries to identify small molecules that are able to block the normal constitutively high expression seen in the presence of a GOF allele of PDR1. Inhibitors of the luciferase enzymatic reaction and aggregation-dependent inhibitors will be eliminated. Compounds that satisfy these initial screening criteria will be tested for the ability to modulate the native CDR1 locus by RT-qPCR assays. Aim 2 will identify the action of compounds that modulate CDR1 expression by testing their ability to impact azole resistance of a collection of C. glabrata clinical isolates as well as a set of different PDR1 GOF alleles that we have previously characterized. The interaction of candidate compounds with a set of transcriptional Mediator complex mutations will also be analyzed to gain insight into how these molecules may impact Pdr1 transcriptional regulation. RNA-seq experiments will be carried out on several of the most promising compounds to examine the range of transcriptional changes these molecules cause and the similarity between their activities. Completion of this proposal will provide new chemical reagents that will be useful as potential adjuvants with fluconazole and powerful new probes for Pdr1-mediated activation of CDR1 expression.