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.

与念珠菌物种相关的感染引起了第四种最常见的血液感染类型。 只有三种有效的抗真菌药物，造成了抗真菌持续生存能力的真正威胁 治疗。 C. glabrata是与念珠菌和 对硫唑药物的内在敏感性均低，并且很容易获得对此最大的耐受性 通常使用的抗真菌化合物类别。抗唑的C. glabrata分离株几乎是 仅由编码转录因子PDR1的基因中的点突变引起。这些 点突变导致PDR1转录的功能获得（GOF）形式的产生 调节剂反过来引起靶基因的高水平本构表达。关键的PDR1靶基因是 编码ATP结合的盒式转运蛋白的CDR1基因座被认为是广泛的特异性 药物外排泵，防止了载有这些GOF PDR1等位基因的细胞中硫唑药物的积累。 该应用的目的是使用化学遗传方法来识别可以 通过PDR1的GOF形式抑制CDR1的激活。我们将使用两个不同的化学库来筛选 C. glabrata菌株含有CDR1-卢西酶（CDR1-LUC）基因融合，以鉴定化合物 能够抑制这种致病性酵母中唑的耐药性采集的中心调节步骤。我们 已经验证了我们使用384井轻松检测CDR1-LUC中表达变化的能力 将在化学库屏幕中使用的格式测定。 AIM 1将采用此CDR1- 荧光素酶报告基菌株以筛选两个不同的化学文库，以鉴定小分子 能够阻止在PDR1的GOF等位基因存在下看到的正常组成性高表达。 将消除荧光素酶酶促反应和聚集依赖性抑制剂的抑制剂。 满足这些初始筛选标准的化合物将测试调节天然的能力 RT-QPCR分析的CDR1基因座。 AIM 2将确定调节CDR1的化合物的作用 通过测试其影响一系列C. glabrata临床分离株的偶氮抗性的能力来表达表达 以及我们以前表征的一组不同的PDR1 GOF等位基因。相互作用 具有一组转录介质复合物突变的候选化合物也将被分析为 了解这些分子如何影响PDR1转录调控。 RNA-seq实验 将在几种最有希望的化合物上进行转录范围 改变这些分子引起的及其活动之间的相似性。完成此提案 将提供新的化学试剂，这些试剂将作为氟康唑和氟康唑的潜在佐剂有用 PDR1介导的CDR1表达激活的强大新探针。