High throughput screening for polyadenylation inhibitors using the MLSCN library

描述（由申请人提供）：随着先进医疗技术的发展，念珠菌病已经成为一种重要的医院感染，在免疫功能低下的患者中引起相当大的发病率和死亡率。念珠菌血流感染的频率正在增加，并与高死亡率有关。口腔念珠菌病是艾滋病患者中极为常见的机会性感染。尽管这些感染很普遍，但治疗选择有限。除了新开发的棘白菌素外，目前使用的抗真菌药物受到毒性和天然或获得性耐药性的限制。因此，开发新的抗真菌药物具有重要意义。我们的长期目标是开发治疗真菌感染的新药。实现这一目标的困难在于，真菌使用的基因表达和细胞生长机制与哺乳动物细胞使用的机制即使不完全相同，也很相似。所有真核生物共有的一个基本过程是通过切割较长的前体分子和随后添加一束腺苷残基来修饰mrna的3'端。这种多聚(A)尾的获得对于成熟mRNA的积累、从细胞核输出、在蛋白质翻译中的利用以及在细胞不再需要mRNA时将其移除都是重要的。在过去的几年里，我们和其他人的研究已经确定了大多数，如果不是全部的话，这个加工复合体的亚基，并揭示了酵母酵母菌之间的显着保存。酿酒动物和后生动物。然而，我们也发现了显著的物种特异性差异，这表明可以找到唯一干扰真菌mRNA 3'端形成的抑制剂。在这项研究中，我们将筛选MLSCN小分子库中的mRNA聚腺苷化抑制剂。这种筛选利用了一种检测方法，其中酿酒酵母的3'端加工缺陷导致细胞生长所需的报告基因的产生。我们已经将该分析调整为384孔格式，可以通过自动板阅读器进行分析，并且它在中试屏幕中具有强大的性能。为了评估我们的hit化合物的活性谱，我们将测试它们对哺乳动物细胞和真菌病原体的生长抑制作用。我们还将使用体内和体外多聚腺苷化试验作为二次筛选，以确认命中确实针对mRNA 3'端形成。最后，我们将与MLPCN中心合作，设计和合成具有更高效力和特异性的衍生物。我们期望这项研究将产生一类新的抗真菌药物，从而解决对其他病原真菌抑制剂的迫切需求。一个额外的好处将是发现化学探针，以帮助我们了解真核mRNA聚腺苷化的分子机制。公共卫生相关性：念珠菌最近作为一种重要的机会性病原体出现，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其有限，使这一问题更加复杂的是，一些最好的抗真菌药物正在出现耐药性。通过利用真菌和人类细胞合成信使RNA的某些差异，我们建议对一类新型抗真菌药物进行高通量筛选，从而解决对其他病原真菌抑制剂的迫切需求。另一个好处是化学探针的发现将有助于我们了解真核mRNA聚腺苷化的分子机制。

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.