Products Cited in Publications Worldwide Targeting the 7-Methyl Guanosine Triphosphate Cap Binding Site on eIF4E With Ribavirin and Analogs

Targeting the 7-Methyl Guanosine Triphosphate Cap Binding Site on eIF4E With Ribavirin and Analogs

Mohini, Amrita Talukder

Abstract

Eukaryotic Translation Initiation Factor (eIF4E) is a 5’-mRNA cap-binding protein. Upon the binding of the 5’-mRNA cap to eIF4E, translation initiation occurs as the 40S ribosomal complex begins translating the mRNA codon sequence. The eIF4E protein is elevated in 30% of cancers and has oncogenic potential through multiple mechanisms. Several epigenetic changes and overexpression of oncogenic proteins lead to the overexpression of eIF4E. Effects of overexpression of eIF4E at the protein level can be labeled as a hallmark of cancer and tumorigenesis. For example, when eIF4E is overexpressed, it facilitates the nuclear export of many oncogenic mRNAs coding for proteins such as c-myc, Mcl-1, VEGF, and cyclin D1. The 5’-mRNA cap structure consists of a 7-methyl guanosine ring which interacts with the tryptophan residues in the binding pocket of the eIF4E protein. Ribavirin, a guanosine analog, is an antiviral drug that is in ongoing clinical trials for anti-cancer effects. The triphosphate metabolite of ribavirin is thought to have anti-proliferative and anti-migratory effects in cancer cells in patients suffering from breast, head, and neck cancers, carcinomas, and hepatic cancer. It is proposed in various studies that one possible mechanism of action of ribavirin is through direct binding and inhibition of the eIF4E protein. Thus, ribavirin can block the binding of the 5’-cap of mRNA, inhibiting translation initiation. We observe that ribavirin or ribavirin 5'-triphosphate (RTP) does not show significant inhibition in our in vitro studies. We use fluorescence polarization (FP), tryptophan fluorescence quenching, and cell lysate pulldown assays to examine the extent of binding of eIF4E with ribavirin and RTP. In the fluorescence polarization assay, we utilize the ability of an inhibitor to bind to eIF4E, competing with a fluorescently labeled cap mimic. The fluorophore (tracer) is the EDA-m7GDP-ATTO-488 molecule, designed to have binding affinity for the cap binding site on eIF4E, where the 7-methyl guanosine cap of an endogenous mRNA would bind. Upon competitive inhibition, an inhibitor is likely to displace this tracer fluorophore molecule and bind at the said site, increasing the tumbling effect and reducing fluorescence polarization. On the other hand, the tryptophan quenching assay works on the principle that any inhibitor binding at the cap site would reduce the fluorescence signal emitted by the tryptophan 56 and 102 present in the binding pocket of eIF4E. Unlike the FP assay, the quenching assay does not require a fluorophore. Moreover, in our pull-down assay, we assess whether resin beads immobilized to m7GTP and bound to eIF4E could be used to test interaction of an inhibitor with the m7GTP:eIF4E interface. Western blot is used to visualize the resultant eluates after an inhibitory test molecule such as RTP is incubated with the m7GTP: eIF4E system for sufficient time. For comparison, we use a mRNA cap mimic, 7-methyl guanosine triphosphate (m7GTP), which shows complete inhibition through binding to the cap site on eIF4E. Assay data will be presented for m7GTP, ribavirin, RTP, and analogs, where we conclude that neither ribavirin nor RTP showed significant inhibition in the three assays. While there have been some controversies regarding the binding of ribavirin and RTP with eIF4E at the cap site, our study aims to clarify and contribute to the ongoing debate surrounding ribavirin’s mechanism of action.

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Ribavirin

A11178536791-04-598%

Ribavirin

10.0/1mg

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