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

白色念珠菌是一种流行的人类真菌病原体，也是医院获得性血流的主要原因。 感染。在美国，死亡率约为40%，每年有超过1万人死亡。 与系统性念珠菌病有关。总的来说，真菌病原体在世界范围内造成了更多的死亡 (每年约160万人)，比结核病和疟疾的总和还要多。这些死亡率表明 现有的几类抗真菌药物不足以满足需求。新药的开发 来得很慢。目前主要的三类抗真菌药物中的两类(氮唑类和多烯类) 在临床上已经使用了近40年，并发现了第三类(棘豆多糖类) 可以追溯到同一个时代。此外，唑类药物取得的有限成功现在受到以下威胁 耐药白念珠菌菌株，疾控中心认为这种菌株对人类健康构成严重威胁 美国这项提案的目标是通过描述和促进 一类新型抗真菌靶标高通量配基结合分析方法的建立 在毒力和唑类耐药性中都发挥着重要作用。这些蛋白质统称为 锌簇转录因子(ZCFs)。ZCFs代表了超过80个真菌专一性家族 带有锌(II)2Cys6基序的序列特异性DNA结合转录调控因子。尽管他们 重要的是，目前大多数ZCFs缺乏明确的功能特征；即使对于那些有 相关功能，对它们的调控机制知之甚少。中心假说 这一提议的核心是小分子诱导剂直接与ZCFs结合并引发构象变化 这使得ZCF转录激活结构域与特定亚单位之间能够直接相互作用 Mediator是一种大型的多蛋白转录共激活复合体。我们之前的工作表明， 某些ZCFs功能突变的诱导者和获得导致中介的招募和 与唑类耐药性有关的多药外排泵基因的转录激活。的目标是 这一探索性的建议是描述促进诱导子依赖相互作用的区域 在ZCFs和Mediator之间，并利用该信息创建配体结合分析，该分析可以 以高通量的方式应用于白色念珠菌中的多个ZCFs以发现生理配体， 和小分子拮抗剂，抵消ZCF对毒力和耐药性的促进作用。利用 一种类似于成功用于高通量配基结合分析筛选药物的平台 靶向哺乳动物核受体，相当于真菌ZCFs的功能，将有助于最大限度地 这个项目的成功和影响。

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.