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

描述（由申请人提供）：随着先进医疗技术的发展，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造成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，并且与高死亡率相关。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，但治疗选择仍然有限。除新开发的棘白菌素外，目前使用的抗真菌药物均受到毒性和天然或获得性耐药性的限制。因此，开发新型抗真菌药物具有重要意义。 我们的长期目标是开发针对真菌感染的新药物疗法。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所使用的机制相似，甚至几乎完全相同。所有真核生物共有的一个重要过程是通过切割较长的前体分子并随后添加一系列腺苷残基来修饰 mRNA 的 3' 末端。这种 Poly(A) 尾巴的获得对于成熟 mRNA 的积累、从细胞核中输出、在蛋白质翻译中的利用以及当细胞不再需要 mRNA 时将其去除非常重要。在过去的几年中，我们和其他人的研究已经鉴定了该加工复合物的大部分（如果不是全部）亚基，并揭示了酿酒酵母和后生动物之间显着的保守性。然而，我们还发现了显着的物种特异性差异，表明可以找到独特地干扰真菌 mRNA 3' 末端形成的抑制剂。 在本研究中，我们将开发一种高通量测定法来筛选酿酒酵母中 mRNA 多腺苷酸化的小分子抑制剂。该测定将基于我们实验室使用的现有报告构建体，以检测 mRNA 3' 末端加工中的缺陷。一旦屏幕针对 384 孔格式进行优化，我们将使用它来筛选哈佛大学和麻省理工学院布罗德研究所提供的 140,000 种化学物质的集合。我们将使用几种体内和体外测定作为二次筛选，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是否抑制病原体白色念珠菌的生长和 mRNA 3' 末端形成，并构建聚腺苷酸化抑制剂的报告基因，该报告基因可直接用于念珠菌的额外大规模筛选。我们预计这项研究将产生一类新型抗真菌药物，从而解决对病原真菌额外抑制剂的迫切需求。 念珠菌最近已成为一种重要的机会病原体，导致免疫功能低下患者的发病率和死亡率相当高。不幸的是，真菌疾病的治疗选择极其有限，而且使这个问题变得更加复杂的是，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，从而解决对病原真菌额外抑制剂的迫切需求。

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.