Direct ligand-based control of C. albicans zinc cluster transcription factors

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C. albicans is a prevalent human fungal pathogen and a leading cause of hospital-acquired bloodstream infections. There is an approximately 40% mortality rate, and over 10,000 deaths per year in the U.S. associated with systemic candidiasis. Collectively, fungal pathogens account for more deaths worldwide (~1.6 million people annually), than tuberculosis and malaria combined. These mortality rates indicate that existing classes of antifungal drugs are insufficient to meet the need. The development of new drugs has been slow in coming. Two of the three main classes of antifungals (azoles and polyenes) in current use have been in the clinic for close to 40 years, and the discovery of the third class (echinocandins) dates to this same era. Moreover, the limited success achieved by azole drugs is now threatened by azole resistant C. albicans strains, which the CDC considers a ‘serious’ threat to human health in the U.S. The goal of this proposal is to address these needs by characterizing, and contributing to the development of a high-throughput ligand binding assay for, a new class of antifungal targets that play important roles in in both virulence and azole resistance. These proteins are known collectively as the zinc cluster transcription factors (ZCFs). ZCFs represent a fungal specific family of more than 80 sequence-specific DNA-binding transcriptional regulators bearing the Zn(II)2Cys6 motif. Despite their importance, most of the ZCFs currently lack a clearly characterized function; and even for those with an associated function, little is understood about the mechanism of their regulation. The central hypothesis of this proposal is that small-molecule inducers directly bind to ZCFs and trigger a conformational change that enables direct interactions between the ZCF transcriptional activation domain and a specific sub-unit of Mediator, a large multi-protein transcriptional co-activator complex. Our previous work showed that inducers and gain of function mutations in certain ZCFs lead to the recruitment of Mediator and the transcriptional activation of multi-drug efflux pump genes that confer azole resistance. The objective of this exploratory proposal is to characterize the domains that facilitate the inducer dependent interactions between ZCFs and Mediator, and leverage this information to create a ligand binding assay that can be applied to multiple ZCFs in C. albicans in a high-throughput manner to discover physiological ligands, and small-molecule antagonists that counteract ZCF promotion of virulence and drug resistance. Utilizing a platform similar to one successfully used for high-throughput ligand-binding assays screening for drugs that target mammalian nuclear receptors, the functional equivalent of fungal ZCFs, will help maximize the success and impact of this project.