Complex eIF4E Research Articles

A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders.

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.

MRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Analogues of the mRNA 5′-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation.

The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a key control step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surface of eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageously compete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4E surface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understanding of translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumis melo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structural data together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonical motifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very important practical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, a universal eukaryotic bipartite mode of binding to eIF4E is proposed.

Abstract IA23: eIF4E as anti-cancer target

Abstract The interaction of the cap-binding protein eIF4E with eIF4G is central for recruiting the small ribosomal subunit to the 5' end of mRNA. This interaction is regulated by the competitive interaction with the 4E-binding proteins (4E-BPs). Genome analyses have shown that mRNAs coding for proto-oncogenes, transcription factors, translation factors, growth factors and their receptors have statistically long 5' UTRs with large negative free energies estimated with mfold. Translation of these genes thus, depends heavily on an efficient translation-initiation machinery. This is in contrast to 5' TOP mRNAs and highly expressed genes with expression controlled at the transcription level that have small negative free estimated with mfold. Consistently, overexpression of 4E-BP1 has been shown to suppress tumor growth in mouse xenografts. In a high-throughput screen of compound libraries we discovered a small molecule that mimics the action of 4E-BP1 by displacing eIF4G from eIF4E. Surprisingly, the compound does not displace 4E-BP1 but rather stabilizes its binding both in vitro and in vivo. To understand this effect we have solved crystal structures of complexes between eIF4E and several inhibitor analogs. In addition, we determined the structure of eIF4E in complex with a long fragment of 4E-BP1. These data revealed the dual mechanism of the compound displacing the tumor promoter eIF4G while stabilizing the tumor suppressor 4E-BP1. Furthermore, we investigated the activities of 4EGI-1 aggressive breast cancer cells that have properties of cancer stem cells and find that the compound specifically suppresses the aggressive properties of these cancer cells. In addition, we discovered that eIF4E is transcriptionally upregulated in hypoxia as it promoter contains hypoxia response elements utilized by Hif1a. Finally, we tested 4EGI-1 against a panel of ~1000 cancer cell lines and tested the global impact on Jurkat cells in a proteomics approach. Citation Format: Gerhard Wagner, Evangelos Papadopoulos, Tingfang Yi, Melissa Leger-Abraham, Naotaka Sekiyama. eIF4E as anti-cancer target. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA23.

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, (54)YXXXXLΦ(60) (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m(7)GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150-84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

Evaluating the Conformation and Binding Interface of Cap-Binding Proteins and Complexes via Ultraviolet Photodissociation Mass Spectrometry

We report the structural analysis of cap-binding proteins using a chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating solvent accessibility of proteins. Our methodology utilized a chromogenic probe (NN) to probe the exposed amine residues of wheat eukaryotic translation initiation factor 4E (eIF4E), eIF4E in complex with a fragment of eIF4G ("mini-eIF4F"), eIF4E in complex with full length eIF4G, and the plant specific cap-binding protein, eIFiso4E. Structural changes of eIF4E in the absence and presence of excess dithiothreitol and in complex with a fragment of eIF4G or full-length eIF4G are mapped. The results indicate that there are particular lysine residues whose environment changes in the presence of dithiothreitol or eIF4G, suggesting that changes in the structure of eIF4E are occurring. On the basis of the crystal structure of wheat eIF4E and a constructed homology model of the structure for eIFiso4E, the reactivities of lysines in each protein are rationalized. Our results suggest that chemical probe/UVPD mass spectrometry can successfully predict dynamic structural changes in solution that are consistent with known crystal structures. Our findings reveal that the binding of m(7)GTP to eIF4E and eIFiso4E appears to be dependent on the redox state of a pair of cysteines near the m(7)GTP binding site. In addition, tertiary structural changes of eIF4E initiated by the formation of a complex containing a fragment of eIF4G and eIF4E were observed.

Structural Insights into the Allosteric Effects of 4EBP1 on the Eukaryotic Translation Initiation Factor eIF4E

The eukaryotic translation initiation factor eIF4E plays key roles in cap-dependent translation and mRNA export. These functions rely on binding the 7-methyl-guanosine moiety (5′cap) on the 5′-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5′cap. Our understanding of the allosteric effects and structural underpinnings of 4EBP1 or eIF4G binding can be advanced by obtaining structural data on cap-free eIF4E bound to one of these proteins. Here, we report the crystal structure of apo-eIF4E and cap-free eIF4E in complex with a 4EBP1 peptide. We also monitored 4EBP1 binding to cap-free eIF4E in solution using NMR. Together, these studies suggest that 4EBP1 transforms eIF4E into a cap-receptive state. NMR methods were also used to compare the allosteric routes activated by 4EBP1, eIF4G, and the arenavirus Z protein, a negative regulator of cap binding. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G and differences from the negative regulator Z. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5′cap.

EIF4E: New Family Members, New Binding Partners, New Roles

Eukaryotic initiation factor 4E (eIF4E) has long been known as the cap-binding protein that participates in recruitment of mRNA to the ribosome. A number of recent advances have not only increased our understanding of how eIF4E acts in translation but also uncovered non-translational roles. New structures have been determined for eIF4E in complex with various ligands and for other cap-binding proteins. We have also learned that most eukaryotic organisms express multiple eIF4E family members, some involved in general translation but others having specialized functions, including repression of translation. A number of new eIF4E-binding proteins have been reported, some of which tether it to specific mRNAs.