High throughput screening for anti-fungal drugs that inhibit mRNA polyadenylation

高通量筛选抑制 mRNA 多腺苷酸化的抗真菌药物

• 批准号: 7359292
• 负责人: CLAIRE L MOORE
• 金额: $ 20.57万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7359292
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adenosine; Advanced Development; Animal Model; Antifungal Agents; Biological Assay; Blood; Candida; Candida albicans; Candidiasis; Cell Nucleus; Cells; Chemicals; Class; Collection; Complex; Defect; Development; Disease; Drug resistance; Effectiveness; Eukaryota; Eukaryotic Cell; Excision; Frequencies; Future; Gene Expression; Genes; Goals; Growth; Human; Immunocompromised Host; Infection; Institutes; Laboratories; Lead; Mammalian Cell; Medical; Messenger RNA; Modification; Morbidity - disease rate; Mycoses; Nosocomial Infections; Opportunistic Infections; Oral candidiasis; Organism; Patients; Pharmaceutical Preparations; Pharmacotherapy; Pilot Projects; Poly(A) Tail; Polyadenylation; Polyadenylation Pathway; Polymerase Chain Reaction; Prevalence; Process; Production; Proteins; RNA-Directed DNA Polymerase; Reader; Reporter; Research; Resistance; Saccharomyces cerevisiae; Screening procedure; Staining method; Stains; Stream; Techniques; Testing; Toxic effect; Transcript; Translations; Yeasts; base; cell growth; chemical genetics; fungus; high throughput screening; improved; in vitro Assay; in vivo; inhibitor/antagonist; mortality; novel; pathogen; small molecule; small molecule libraries

DESCRIPTION (provided by applicant): Accompanying the development of advanced medical techniques, candidiasis has emerged as a significant nosocomial infection that causes considerable morbidity and mortality among immunocompromised patients. Candida blood stream infections are increasing in frequency and are associated with high mortality. Oral candidiasis is an extremely common opportunistic infection in AIDS patients. Despite the prevalence of these infections, treatment options are limited. With the exception of the newly developed echinocandins, the antifungal drugs currently in use are limited by toxicity and natural or acquired resistance. Therefore, development of new antifungal drugs is of great importance. Our long-term goal is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. An essential process shared by all eukaryotes is the modification of the 3' ends of mRNAs by cleavage of longer precursor molecules and the subsequent addition of a tract of adenosine residues. Acquisition of this poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its removal when the mRNA is no longer needed by the cell. In the last few years, our research and that of others has identified most, if not all, of the subunits of this processing complex and revealed a remarkable conservation between the yeast Saccharomyces cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3' end formation could be found. In this study, we will develop a high throughput assay to screen S. cerevisiae for small-molecule inhibitors of mRNA polyadenylation. This assay will be based on an existing reporter construct used in our laboratory to detect defects in mRNA 3' end processing. Once the screen is optimized for a 384-well format, we will use it to screen a collection of 140,000 chemicals available through the Broad Institute of Harvard and M.I.T. We will use several in vivo and in vitro assays as secondary screens to confirm that hit molecules are indeed targeting mRNA 3' end formation. We will then determine if the candidate molecules inhibit growth and mRNA 3' end formation in the pathogen C. albicans, and construct a reporter for polyadenylation inhibitors that can be employed in additional large-scale screens directly in Candida. We expect that this study will yield a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. Candida has recently emerged as a significant opportunistic pathogen that causes considerable morbidity and mortality in immunocompromised patients. Unfortunately, treatment options for fungal diseases are extremely limited and, compounding this problem, resistance to some of the best anti-fungal drugs is emerging. By taking advantage of certain differences in how fungi and human cells synthesize messenger RNA, we propose to conduct a high throughput screen for a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi.

描述（由申请人提供）：伴随着高级医疗技术的发展，念珠菌病已成为一种重要的医院感染，在免疫功能低下的患者中引起了相当大的发病率和死亡率。念珠菌血流感染的频率增加，并且与高死亡率有关。口服念珠菌病是艾滋病患者中极为普遍的机会感染。尽管这些感染率普遍存在，但治疗方案仍有限。除了新开发的echinocandins外，目前正在使用的抗真菌药物受到毒性，自然或获得的抗性的限制。因此，新抗真菌药物的开发非常重要。 我们的长期目标是开发用于真菌感染的新药物疗法。实现这一目标的困难是，真菌将机制用于基因表达和细胞生长，即使与哺乳动物细胞使用的机制几乎相同。所有真核生物共有的一个基本过程是通过较长的前体分子的切割和随后添加腺苷残基的裂解来修饰mRNA的3'端。该poly（a）尾巴的获取对于成熟mRNA的积累，其从细胞核出口，其在蛋白质翻译中的利用以及当细胞不再需要mRNA时的去除很重要。在过去的几年中，我们的研究和其他研究已经确定了该加工复合物的最多（即使不是全部）的亚基，并揭示了酵母糖酵母酿酒酵母和后生动物之间的显着保护。但是，我们还发现了明显的物种特异性差异，这表明可以发现抑制剂独特地干扰真菌mRNA 3'末端形成。 在这项研究中，我们将开发高吞吐量测定，以筛选酿酒酵母，以实现mRNA聚腺苷酸化的小分子抑制剂。该测定法将基于我们实验室中使用的现有记者结构，以检测mRNA 3'末端处理中的缺陷。一旦对384孔格式进行了优化的屏幕，我们将使用它来筛选通过哈佛大学和M.I.T. Broad Institute提供的140,000种化学品的集合。我们将使用几个体内和体外测定作为次级筛选，以确认命中分子确实靶向mRNA 3'末端形成。然后，我们将确定候选分子在病原体白色念珠菌中是否抑制生长和mRNA 3'末端形成，并构建可用于直接在念珠菌中其他大型筛查中的聚腺苷酸化抑制剂的报告基因。我们预计这项研究将产生一类新型的抗真菌药物，从而解决对病原真菌额外抑制剂的紧迫需求。 念珠菌最近已成为一种重要的机会性病原体，可在免疫功能低下的患者中引起相当大的发病率和死亡率。不幸的是，真菌疾病的治疗选择极为有限，并且使这个问题更加复杂，对某些最好的抗真菌药物的抗性正在出现。通过利用真菌和人类细胞合成信使RNA的某些差异，我们建议对新型抗真菌药物进行高吞吐量筛选，从而解决对病原真菌的额外抑制剂的紧迫需求。