Chemical genetic analysis of Candida glabrata CDR1 expression

光滑念珠菌CDR1表达的化学遗传分析

• 批准号: 10588383
• 负责人: W Scott Moye-Rowley
• 金额: $ 21.92万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-04 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10588383
• 关键词: ATP-Binding Cassette Transporters; Adjuvant; Adjuvant Therapy; Alleles; Amino Acid Substitution; Amphotericin B; Antifungal Agents; Antifungal Therapy; Attention; Automobile Driving; Azole resistance; Azoles; Biochemical Reaction; Biological Assay; CDR1 gene; Candida; Candida albicans; Candida glabrata; Cells; Chemicals; Clinic; Clinical; Collection; Complex; Development; Disease; Drug Efflux; Exhibits; FDA approved; Firefly Luciferases; Fluconazole; Gene Expression; Gene Fusion; Genes; Genetic Transcription; Goals; Hyperactivity; Incidence; Infection; Lead; Libraries; Luc Gene; Luciferases; Mammalian Cell; Measures; Mediating; Mediator; Molecular Target; Multi-Drug Resistance; Mutation; Pathogenicity; Pharmaceutical Preparations; Phenotype; Play; Point Mutation; Predisposition; Process; Production; Proteins; Reagent; Reporter; Resistance; Resistant candida; Resources; Role; Sepsis; Specificity; Testing; Toxic effect; Transcription Coactivator; Transcriptional Activation; Transcriptional Regulation; Work; Yeasts; candidemia; chemical genetics; constitutive expression; efflux pump; experimental study; gain of function; genetic analysis; genetic approach; high throughput screening; inhibitor; insight; prevent; resistant strain; scaffold; screening; small molecule; small molecule inhibitor; small molecule libraries; transcription factor; transcriptome sequencing; trend

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.

与念珠菌属相关的感染导致第四种最常见的血流感染。 只有三种有效的抗真菌药物存在，导致一个真实的威胁，持续的生存能力，抗真菌药物 疗法C. glabrata是与念珠菌相关的第二个最常见的分离物种， 对唑类药物的固有敏感性较低， 常用的抗真菌化合物。抗唑C. glabrata分离物几乎 这完全是由编码一种叫做PDR 1的转录因子的基因的点突变引起的。这些 点突变导致产生Pdr 1转录的功能获得性（GOF）形式， 调节子，其又引起靶基因的高水平组成型表达。一个关键的Pdr 1靶基因是 编码ATP结合盒转运蛋白的CDR 1基因座被认为是广泛特异性的 药物外排泵，防止唑类药物在携带这些GOF PDR 1等位基因的细胞中积累。 这项应用的目标是使用化学遗传学方法来识别小分子， 通过GOF形式的Pdr 1抑制CDR 1的活化。我们将使用两个不同的化学库来筛选 C.包含CDR 1-荧光素酶（CDR 1-LUC）基因融合体的光滑菌株，以鉴定 能够抑制该致病酵母中唑类抗性获得的这一中心调节步骤。我们 我已经验证了我们使用384孔板容易检测CDR 1-LUC表达变化的能力 格式分析，将用于化学文库筛选。Aim 1将使用此CDR 1- 荧光素酶报告菌株筛选两个不同的化学文库，以鉴定 能够阻断在PDR 1的GOF等位基因存在下观察到的正常组成型高表达。 荧光素酶酶促反应的抑制剂和聚集依赖性抑制剂将被消除。 将测试满足这些初始筛选标准的化合物调节天然免疫应答的能力。 通过RT-qPCR测定的CDR 1基因座。目的2将鉴定调节CDR 1的化合物的作用 通过测试它们影响C.光滑临床分离株 以及一组不同的PDR 1 GOF等位基因，我们以前已经描述过。的相互作用 还将分析具有一组转录介体复合物突变的候选化合物， 深入了解这些分子如何影响Pdr 1转录调控。RNA-seq实验 将进行几个最有前途的化合物，以检查转录的范围 这些分子引起的变化及其活动之间的相似性。完成本提案 将提供可用作氟康唑的潜在佐剂的新化学试剂， Pdr 1介导的CDR 1表达激活的强大新探针。