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Abstract
Grant Number: 1R03DA026555-01 Project Title: High throughput screening for polyadenylation inhibitors using the MLSCN library
PI Information: Name Title KUMAMOTO, CAROL A. PROFESSOR MOORE, CLAIRE L. (Contact) claire.moore@tufts.edu PROFESSOR Abstract: 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.
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Institution: TUFTS UNIVERSITY BOSTON Office of the Vice Provost BOSTON, MA 02111 Fiscal Year: 2008 Department: MOLECULAR BIOLOGY AND MICROBIOLOGY Project Start: 01-SEP-2008 Project End: 31-AUG-2009 ICD: NATIONAL INSTITUTE ON DRUG ABUSE IRG: ZRG1