HTS Screening for Small-Molecule Inhibitors of miRNA-Mediated mRNA Deadenylation

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DESCRIPTION (provided by applicant): The processes by which genes are transcribed to produce messenger RNA (mRNA) molecules, and then translated into proteins are central to the life and growth of cells. These key cellular functions are frequently altered in cancers and other diseases, and we are interested in increasing our knowledge of these processes as a means of ultimately discovering new therapeutic options in this area. We are focused on the poly (A)-binding protein (PABP) which binds to the mRNA tail and plays a central role in protein translation and mRNA degradation. PABP is essential for cellular protein production and is a major control site that functions via binding interactions with other regulatory and signaling proteins. We have produced high-resolution images of these PABP interactions and have identified two critical binding pockets that are also potential drug binding sites. Our research plan is to screen large chemical libraries in cooperation with the NIH to discover drug-like compounds that will bind to these pockets. Our screen is a competition assay between the regulatory domain of PABP and a small protein fragment, derived from a key interacting partner that is fluorescently labeled. Since initial screening hits are frequently false positives, we will filter out non-specific hits and insoluble compounds with control assays, and confirm direct PABP binding using sensitive detection methods that can measure protein-drug interactions. The binding sites of validated hit compounds will be mapped onto the surface of PABP using high resolution NMR imaging to see how the chemical compounds interact, and examine the means by which they interfere with PABP regulatory interactions. If we identify drug-like compound with good potency we will test them in cellular extracts and whole cells to see how they alter the processes of translation and mRNA degradation. We will also directly examine changes in PABP interactions in response to compound addition. Our hypothesis is that small chemical probes we will serve as useful tools in unraveling the complex processes of translation and mRNA degradation. An increased understanding of cellular mechanics will provide insight into which PABP interactions would be the best therapeutic targets in diseases associated with too much or too little protein production. PUBLIC HEALTH RELEVANCE: The processes by which DNA genes are transcribed to produce messenger RNA molecules, and then translated into proteins is central to the life and growth of cells. These functions are frequently altered in cancer and other diseases and we are interested in examining a potential therapeutic target called the poly (A)-binding protein, which plays a major role in translation and messenger RNA stability. We intend to screen for chemical compounds that will modulate this protein to produce a probe that will help investigate this key biological process, and hopefully in time lead to new drugs for patients.