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

C.白色念珠菌是一种流行的人类真菌病原体，是医院获得性血流的主要原因 感染.死亡率约为40%，美国每年有超过10，000人死亡。 与系统性念珠菌病有关。总的来说，真菌病原体导致全球更多的死亡 （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 现有的抗真菌药物种类不足以满足需求。新药开发 来得很慢目前，三大类抗真菌药中的两类（唑类和多烯类） 使用已经在临床上接近40年，第三类（棘白菌素）的发现 可以追溯到同一个时代此外，唑类药物所取得的有限成功现在受到以下因素的威胁： 唑抗性C.白色念珠菌菌株，疾病预防控制中心认为这是对人类健康的“严重”威胁， 美国本提案的目标是通过描述和促进 开发一种高通量配体结合试验，用于一类新的抗真菌靶点， 在毒力和唑类耐药性中起重要作用。这些蛋白质统称为 锌簇转录因子（ZCF）。ZCF代表了一个真菌特异性家族， 序列特异性DNA结合转录调节子，具有Zn（II）2Cys 6基序。尽管他们 重要的是，大多数ZCF目前缺乏明确的功能特征;即使对于那些具有 相关的功能，很少有人了解他们的调节机制。核心假设 这一建议的一个重要方面是，小分子诱导剂直接结合ZCF并引发构象变化 使ZCF转录激活结构域和特定亚单位之间能够直接相互作用 Mediator是一种大型多蛋白转录辅激活因子复合物。我们之前的工作表明， 某些ZCF中的诱导物和功能突变的获得导致中介物的募集， 转录激活的多药外排泵基因赋予唑类耐药。的目标 这一探索性的建议是表征促进诱导剂依赖性相互作用的结构域 ZCF和Mediator之间的关系，并利用这些信息来创建可以 应用于C中的多个ZCF。以高通量的方式发现生理配体， 以及抵消ZCF促进毒力和耐药性的小分子拮抗剂。利用 一个类似于成功用于高通量配体结合试验筛选药物的平台 靶向哺乳动物核受体，功能相当于真菌ZCF，将有助于最大化 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.