The development of cancer and fibrotic diseases has been shown to be highly dependent on disregulation of cap-dependent translation. Binding protein eIF4E to N7-methylated guanosine capped mRNA has been found to be the rate-limiting step governing translation initiation; and therefore represents an attractive target for drug discovery. Our group has found that 7-benzyl guanosine monophosphate (7Bn-GMP) is a potent antagonist of eIF4E cap binding (Kd = 0.8 uM). Recent X-ray crystallographic studies have revealed that the cap-dependent pocket undergoes a unique structural change in order to accommodate the benzyl group. Unfortunately, 7Bn-GMP is not cell permeable. Recently, we have prepared a tryptamine phosphoramidate prodrug of 7Bn-GMP, 4ei1, and shown that it is a substrate for human histidine triad nucleotide binding protein (hHINT1) and is inhibit eIF4E initiated epithelial-mesenchymal transition (EMT) by Zebra fish embryo cells. To assess the intracellular uptake of 4ei1 and conversion to 7Bn-GMP by cancer cells, we developed a sensitive assay using LC-ESI-MS/MS for the intracellular quantitation of 4ei1 and 7Bn-GMP. When incubated with the breast cancer cell line MDA-231; or lung cancer cell lines H460, H383 and H2009, 4ei1 was found to be rapidly internalized and converted to 7Bn-GMP. Since oncogenic mRNAs are predicted to have the highest eIF4E requirement for translation, we carried out chemosensitization studies with 4ei1. The prodrug was found to chemosensitize both breast and lung cancer cells to non-toxic levels of gemcitabine. Further mechanistic studies revealed that the expressed levels of eIF4E were substantially reduced in cells treated with 4ei1 in a dose dependent manner. The levels of eI4E could be restored by treatment with the proteasome inhibitor MG-132. Taken together, our results demonstrate that 4ei1 is likely to inhibit translation initiation by eIF4E cap binding by both antagonizing eIF4E cap binding and initiating eIF4E proteasomal degradation.
University of Minnesota M.S. thesis. August 2012. Major: Medicinal Chemistry. Advisors:Dr. Patrick Hanna, Dr. Carston Wagner. 1 computer file (PDF); vi, 95 pages.
Evaluation of Eukaryotic Initiation Factor 4E (eIF4E) antagonist 4Ei-1 in mammalian breast cancer and lung cancer cells : chemosensitizaiton with low cytotoxicity..
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