High throughput screening for polyadenylation inhibitors using the MLSCN library

使用 MLSCN 文库高通量筛选聚腺苷酸化抑制剂

• 批准号: 7563077
• 负责人: CAROL A. KUMAMOTO
• 金额: $ 2.5万
• 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: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7563077
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adenosine; Advanced Development; Antifungal Agents; Biological Assay; Blood; Candida; Candida albicans; Candidiasis; Cell Nucleus; Cells; Chemicals; Chemistry; Class; Collection; Complex; Coupled; Defect; Development; Disease; Drug resistance; Eukaryota; Eukaryotic Cell; Excision; Frequencies; Gene Expression; Goals; Growth; Human; Immunocompromised Host; In Vitro; Infection; Institutes; Knowledge; Laboratories; Lead; Libraries; Mammalian Cell; Medical; Messenger RNA; Modification; Molecular; Morbidity - disease rate; Mycoses; Nosocomial Infections; Opportunistic Infections; Oral candidiasis; Organism; Pathogenesis; Patients; Performance; Pharmaceutical Preparations; Pharmacotherapy; Poly(A) Tail; Polyadenylation; Polyadenylation Pathway; Polymerase Chain Reaction; Prevalence; Process; Production; Proteins; Public Health; RNA-Directed DNA Polymerase; Reader; Reporter; Research; Resistance; Saccharomyces; Saccharomyces cerevisiae; Sampling; Screening procedure; Specificity; Staining method; Stains; Stream; Synthesis Chemistry; Techniques; Testing; Toxic effect; Transcript; Translations; United States; Work; Yeasts; base; cell growth; chemical genetics; design; fungus; high throughput screening; in vitro Assay; in vivo; inhibitor/antagonist; mortality; novel; pathogen; repository; small molecule; small molecule libraries; tool

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 screen the MLSCN Small Molecule Repository for inhibitors of mRNA polyadenylation. This screen utilizes an assay in which defects in 3' end processing in S. cerevisiae lead to production of a reporter required for cell growth. We have adapted this assay to a 384-well format that can be analyzed by an automated plate reader, and it gives robust performance in pilot screens. To assess the spectrum of activity of our hit compounds, we will test them for growth inhibition of mammalian cells and fungal pathogens. We will also use in vivo and in vitro assays for polyadenylation as secondary screens to confirm that hits are indeed targeting mRNA 3' end formation. Finally, we will work with the MLPCN Center to design and synthesize derivatives with increased potency and specificity. 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. An added benefit will be the discovery of chemical probes to help us understand the molecular mechanism of eukaryotic mRNA polyadenylation. PUBLIC HEALTH RELEVANCE: 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. An additional benefit will be the discovery of chemical probes that will help us understand the molecular mechanism of eukaryotic mRNA polyadenylation.

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，并揭示了酵母糖疗法之间的显着保护。酿酒酵母和后生动物。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，我们将筛选MLSCN小分子存储库中的mRNA聚腺苷酸化抑制剂。该屏幕利用了一种测定法，其中在酿酒酵母中3'末端加工中的缺陷导致产生细胞生长所需的记者。我们已经将此测定法调整为384孔格式，该格式可以由自动板读取器分析，并在试验屏幕中提供了稳健的性能。为了评估热门化合物的活性谱，我们将测试它们以抑制哺乳动物细胞和真菌病原体的生长抑制。我们还将在体内和体外测定中用于多腺苷酸化作为次级筛选，以确认命中确实是靶向mRNA 3'末端形成。最后，我们将与MLPCN中心合作，以增加效力和特异性设计和合成衍生物。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的其他抑制剂的紧迫需求。额外的好处是发现化学探针，以帮助我们了解真核mRNA聚腺苷酸化的分子机制。公共卫生相关性：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，真菌疾病的治疗方案极为有限，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。另一个好处是发现化学探针，这将有助于我们了解真核mRNA聚腺苷酸化的分子机制。