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

描述(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导致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一种非常常见的机会性感染。尽管这些感染盛行，但治疗选择有限。除了新开发的棘球菌素类药物外，目前使用的抗真菌药物都受到毒性和自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使用的机制相似，如果不是几乎相同的话。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并随后添加一段腺苷残基来修饰mRNAs的3‘端。获得这种Poly(A)尾巴对于成熟mRNA的积累、它从细胞核中输出、它在蛋白质翻译中的利用以及当细胞不再需要它的时候它的移除都是重要的。在过去的几年里，我们和其他人的研究已经确定了这个加工复合体的大部分(如果不是全部)亚单位，并揭示了酿酒酵母和后生动物之间的显著保守。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mRNA3‘末端形成的抑制剂。 在这项研究中，我们将建立一种高通量的方法来筛选酿酒酵母的小分子mRNA多腺苷基化抑制物。本试验将基于我们实验室使用的现有报告结构，以检测mRNA3‘末端加工中的缺陷。一旦屏幕针对384孔的格式进行了优化，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14万种化学物质。我们将使用几种体内和体外测试作为二级筛选，以确认HIT分子确实是针对mRNA 3‘末端形成的。然后，我们将确定候选分子是否抑制病原体白念珠菌的生长和mRNA3‘端的形成，并构建可直接在念珠菌中用于额外大规模筛选的多聚腺苷基化抑制剂的报告。我们预计，这项研究将产生一类新的抗真菌药物，从而解决对病原真菌更多抑制剂的迫切需求。 念珠菌最近已经成为一种重要的机会性病原体，在免疫功能低下的患者中会导致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，使这一问题雪上加霜的是，对一些最好的抗真菌药物正在出现抗药性。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，从而解决对更多病原真菌抑制剂的迫切需求。

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.