Novel Cap Analogs and Interactions with Target Proteins

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DESCRIPTION (provided by applicant) This application for a Fogarty International Research Collaboration Award (FIRCA) is an extension of Grant R01GM20818, entitled "Regulation of Eukaryotic Protein Synthesis Initiation." The research will be done primarily in Poland at Warsaw University in collaboration with Edward Darzynkiewicz. The long-term goals of both the Parent Grant and the FIRCA project are to understand the steps that occur during the recruitment of eukaryotic mRNA to the ribosome and how they are regulated. This process determines both the overall rate of protein synthesis and also the spectrum of mRNAs translated. The four Specific Aims of the FIRCA application involve the 7-methylguanosine-containing cap of mRNA and the target protein which it interacts during cap-dependent initiation of protein synthesis, elF4E. The nematode C. elegans provides unparalleled advantages for studying such a complex biochemical process as initiation of protein synthesis. Surprisingly, it expresses five eIF4E isoforms, termed IFE proteins, that have differing cap-binding properties. In Specific Aim 1, we will synthesize and test in biological systems new classes of di- and oligonucleotide cap analogues, specifically a new series of anti-reverse cap analogues, dinucleotide cap analogues intended to be resistant to degradation by pyrophosphatases, and capped trinucleotides. In Specific Aim 2, we will synthesize and test new inhibitors of translation based on cap analogues that are able to cross the plasma membrane, in an attempt to target cap-dependent translation as an anticancer strategy. In Specific Aim 3, we will analyze thermodynamiC and solvent aspects of cap binding to elF4E isoforms of C. elegans. In Specific Aim 4, we will investigate the role of elF4E phosphorylation using chemically synthesized human Ser-209 phospho-elF4E. Because overexpression of elF4E causes malignant transformation of cells, and because naturally occurring tumors contain greatly elevated levels of elF4E, these studies have relevance to cancer, especially Specific Aim 2. They also have relevance for diseases caused by picornaviruses such as poliovirus (polio), rhinovirus (the common cold) and Coxsackievirus (heart failure), since picomaviruses shut down cap-dependent translation by proteolytically cleaving elF4G, the protein that tethers eIF4E to the 40S ribosomal subunit during initiation of translation.