Treatment of Breast and Lung Cancer Cells with a N-7 Benzyl Guanosine Monophosphate Tryptamine Phosphoramidate Pronucleotide (4Ei-1) Results in Chemosensitization to Gemcitabine and Induced eIF4E Proteasomal Degradation

Abstract

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 μ/M). 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, 4Ei-1, and shown that it is a substrate for human histidine triad nucleotide binding protein (hHINT1) and inhibits eIF4E initiated epithelial–mesenchymal transition (EMT) by Zebra fish embryo cells. To assess the intracellular uptake of 4Ei-1 and conversion to 7Bn-GMP by cancer cells, we developed a sensitive assay using LC-ESI-MS/MS for the intracellular quantitation of 4Ei-1 and 7Bn-GMP. When incubated with the breast cancer cell line MDA-231 or lung cancer cell lines H460, H383 and H2009, 4Ei-1 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 4Ei-1. The prodrug was found to chemosensitize both breast and lung cancer cells to nontoxic levels of gemcitabine. Further mechanistic studies revealed that the expressed levels of eIF4E were substantially reduced in cells treated with 4Ei-1 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 4Ei-1 is likely to inhibit translation initiation by eIF4E cap binding by both antagonizing eIF4E cap binding and initiating eIF4E proteasomal degradation.

Targeting the eIF4F Translation Initiation Complex: A Critical Nexus for Cancer Development

“…Accordingly, one compound, 4Ei-1, inhibited cap-dependent translation in vitro and in vivo when injected into zebrafish embryos (168). 4Ei-1 chemosensitized breast and lung cancer cells to nontoxic levels of the cytotoxic drug gemcitabine (169). 4Ei-1 reduced proliferation and repressed colony formation in mesothelioma cells and sensitized these to pemetrexed, a folate antimetabolite (170).…”

Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins

“…An example of as mallmolecule translation inhibitor is N-7-benzyl guanosine monophosphate, which has been delivered to cultured lung and breastc ancerc ells as the tryptamine phosphoramidate pronucleotide, 4Ei-1, and shown to increasec hemosensitivity to gemcitabine. [17] Targeting eIF4E is also important for improving gene therapy by increasing the translation efficiency of therapeuticm RNA, whichc an be achieved by introducing chemically modifiedc ap analogues at the mRNA 5'-end. The desired features of the compounds are to bind tightly with eIF4E and increaset he translation efficiency.D inucleotidec ap analogues with thiophosphate modificationsp ossessing superiort ranslation properties have already been reported.…”

Effect of different N7 substitution of dinucleotide cap analogs on the hydrolytic susceptibility towards scavenger decapping enzymes (DcpS)

“…Both cap-binding proteins, DcpS and eIF4E exhibit flexibility in the 7-methylguanine binding site which enables accommodation of larger substituents. N7-benzylated cap analogs have been proposed as new families of eIF4E inhibitors with potential therapeutic applications in cancer [29]. Therapeutic activity of cap analogs depends on their stability in cellular conditions and DcpS enzymes can effectively compete for cap binding with eIF4E.…”