4EGI-1

4EGI-1
Names
	IUPAC name α-[2-[4-(3,4-Dichlorophenyl)-2-thiazolyl]hydrazinylidene]-2-nitro-benzenepropanoic acid
Identifiers
CAS Number	315706-13-9 ;
3D model (JSmol)	Interactive image ;
PubChem CID	6911989 ;
UNII	H57REU3DHP ;
	InChI InChI=1S/C18H12Cl2N4O4S/c19-12-6-5-10(7-13(12)20)15-9-29-18(21-15)23-22-14(17(25)26)8-11-3-1-2-4-16(11)24(27)28/h1-7,9H,8H2,(H,21,23)(H,25,26)/b22-14- Key: KFRKRECSIYXARE-HMAPJEAMSA-N;
	SMILES OC(=O)C(\CC1=CC=CC=C1[N+]([O-])=O)=N/NC1=NC(=CS1)C1=CC(Cl)=C(Cl)C=C1;
Properties
Chemical formula	C18H12Cl2N4O4S
Molar mass	451.28 g·mol−1
	Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). Infobox references

Last updated September 30, 2024

4EGI-1 is a synthetic chemical compound which has been found to interfere with the growth of certain types of cancer cells in vitro . Its mechanism of action involves interruption of the binding of cellular initiation factor proteins involved in the translation of transcribed mRNA at the ribosome. The inhibition of these initiation factors prevents the initiation and translation of many proteins whose functions are essential to the rapid growth and proliferation of cancer cells.

Reaction mechanism

4EGI-1 mimics the action of a class of cellular regulatory molecules that naturally inhibit the binding of two initiation factors necessary for interaction of transcribed mRNA with the subunits of ribosomal complexes. These naturally occurring regulatory molecules, or binding proteins (BPs), bind to eukaryotic initiation factor eIF4E, preventing its association with eIF4G, another initiation factor. These two proteins, under unregulated conditions, form a complex, known as eIF4F, which associates with the 5’ cap of mRNA and the ribosomal subunits. eIF4E BPs (4E-BPs), as small polypeptides, consist of the same amino acid sequence as the portion of eIF4G that interacts with eIF4E. 4EGI-1 thus prevents the proper association of mRNA, carrying the coded message of transcribed genes, with the ribosome, the cellular component necessary for the translation of those genes into functional proteins. Naturally occurring 4E-BPs are regulated by a protein kinase, mTOR, which through phosphorylation deactivates the binding affinity of 4E-BPs for the eIF4E protein.^[1]

Binding site specifics and effects of use

4EGI-1, like 4E-BP polypeptides, displaces eIF4G by associating with a binding site on eIF4E. Not only does the synthetic molecule prevent the association between the two initiation factors, but by binding to a different portion of eIF4E via the same motif, it has been shown to actually increase the binding affinity of eIF4E for endogenous (originating within an organism) 4E-BP1.^[1]

The Harvard research group leading the study screened 16,000 compounds, looking for one that would displace a fluorescein-labeled peptide derived from the eIF4G sequence that binds to the eIF4E form at the same site. Eventually they turned up 4EGI-1, which displaced eIF4G by binding to a smaller subset of its binding site (on eIF4E). The newly found molecule had the added advantage of enhancing 4E-BP1 binding, a surprise given that this molecule is also believed to bind eIF4E via the same motif. It appears that by displacing the eIF4G sequence without blocking the entire binding interface of eIF4E, 4EGI-1 is able to clear the “docking site” of the endogenous regulator.

Cap-dependent vs. initiation factor-independent translation

One caveat to the function of 4EGI-1 and thus the entire class of 4E-BP regulatory proteins is that both the synthetic and naturally occurring molecules are effective at inhibiting only cap-dependent translation, not initiation factor-independent translation.

Messenger RNAs (mRNAs) are transcribed from DNA, and serve as templates for the synthesis of proteins by ribosomal translation. Weak mRNAs contain long and highly structured untranslatable regions at their 5’ end. This lengthy region makes it difficult for enzymes to determine where transcription should begin. As a result, initiation factor proteins are required for translation of the message into protein. These weak mRNAs, or mRNAs that carry the code for proteins involved in the development of cancer cells, require cap-dependent translation which necessitates the cellular involvement of the eIFs. Examples of weak mRNAs include those that code for proliferation-related, and anti-apoptotic proteins. Strong mRNAs, in contrast, are translated with much less cellular machinery such as eIFs and generally code for biologically necessary proteins, such as those needed for the essential metabolic processes of a cell. Therapies such as the use of 4EGI-1 against cancer cells can thus be created such that their biologic targets include only the initiation factors involved in the production of weak mRNAs.

Cap-dependent translation involves a series of steps that join the small and large ribosomal subunits at the start codon of mRNA. The initiation factor complex eIF4F is dependent upon the presence of a 5’ mRNA cap upstream from the start codon in order to initiate translation.^[2]

Initiation factor independent translation does not require the association of initiation factors with the 5’ cap of mRNA. As an alternative, the associated ribosomal units are moved to the start location by internal ribosome entry site trans acting factors (ITAFs). It has been found that several cellular proteins that respond to apoptotic signals are translated in this fashion.

Techniques of discovery

When attempting to identify biological molecules that would disrupt the formation of the F complex, researchers developed a high-throughput fluorescence polarization (FP)-binding assay. In this assay, a small peptide of a known sequence was synthesized and tagged with a fluorescent molecule. This traceable peptide of sequence KYTYDELFQLK binds to the binding site of endogenous 4E-BPs on eIF4E. 16,000 compounds of known chemical composition were then tested in this assay. Compounds that displace the labeled peptide from eIF4E would yield a decrease in fluorescence polarization. The sequence of 4EGI-1 was such that it displaced the labeled peptide, thus demonstrating its affinity for the complex binding site on eIF4E.^[3]

Related Research Articles

In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.

An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis. Initiation of eukaryotic translation nearly always occurs at and is dependent on the 5' cap of mRNA molecules, where the translation initiation complex forms and ribosomes engage the mRNA. IRES elements, however allow ribosomes to engage the mRNA and begin translation independently of the 5' cap.

Ribosome shunting is a mechanism of translation initiation in which ribosomes bypass, or "shunt over", parts of the 5' untranslated region to reach the start codon. However, a benefit of ribosomal shunting is that it can translate backwards allowing more information to be stored than usual in an mRNA molecule. Some viral RNAs have been shown to use ribosome shunting as a more efficient form of translation during certain stages of viral life cycle or when translation initiation factors are scarce. Some viruses known to use this mechanism include adenovirus, Sendai virus, human papillomavirus, duck hepatitis B pararetrovirus, rice tungro bacilliform viruses, and cauliflower mosaic virus. In these viruses the ribosome is directly translocated from the upstream initiation complex to the start codon (AUG) without the need to unwind RNA secondary structures.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recapping.

In molecular biology, initiation factors are proteins that bind to the small subunit of the ribosome during the initiation of translation, a part of protein biosynthesis.

Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the initiation phase of eukaryotic translation. These proteins help stabilize the formation of ribosomal preinitiation complexes around the start codon and are an important input for post-transcription gene regulation. Several initiation factors form a complex with the small 40S ribosomal subunit and Met-tRNA_i^Met called the 43S preinitiation complex. Additional factors of the eIF4F complex recruit the 43S PIC to the five-prime cap structure of the mRNA, from which the 43S particle scans 5'-->3' along the mRNA to reach an AUG start codon. Recognition of the start codon by the Met-tRNA_i^Met promotes gated phosphate and eIF1 release to form the 48S preinitiation complex, followed by large 60S ribosomal subunit recruitment to form the 80S ribosome. There exist many more eukaryotic initiation factors than prokaryotic initiation factors, reflecting the greater biological complexity of eukaryotic translation. There are at least twelve eukaryotic initiation factors, composed of many more polypeptides, and these are described below.

<span class="mw-page-title-main">Hepatitis A virus internal ribosome entry site (IRES)</span>

This family represents the internal ribosome entry site (IRES) of the hepatitis A virus. HAV IRES is a 450 nucleotide long sequence located in the 735 nt long 5’ UTR of Hepatitis A viral RNA genome. IRES elements allow cap and end-independent translation of mRNA in the host cell. The IRES achieves this by mediating the internal initiation of translation by recruiting a ribosomal 40S pre-initiation complex directly to the initiation codon and eliminates the requirement for eukaryotic initiation factor, eIF4F.

<span class="mw-page-title-main">Hepatitis C virus internal ribosome entry site</span>

The Hepatitis C virus internal ribosome entry site, or HCV IRES, is an RNA structure within the 5'UTR of the HCV genome that mediates cap-independent translation initiation.

Poly(A)-binding protein is an RNA-binding protein which triggers the binding of eukaryotic initiation factor 4 complex (eIF4G) directly to the poly(A) tail of mRNA which is 200-250 nucleotides long. The poly(A) tail is located on the 3' end of mRNA and was discovered by Mary Edmonds, who also characterized the poly-A polymerase enzyme that generates the poly(a) tail. The binding protein is also involved in mRNA precursors by helping polyadenylate polymerase add the poly(A) nucleotide tail to the pre-mRNA before translation. The nuclear isoform selectively binds to around 50 nucleotides and stimulates the activity of polyadenylate polymerase by increasing its affinity towards RNA. Poly(A)-binding protein is also present during stages of mRNA metabolism including nonsense-mediated decay and nucleocytoplasmic trafficking. The poly(A)-binding protein may also protect the tail from degradation and regulate mRNA production. Without these two proteins in-tandem, then the poly(A) tail would not be added and the RNA would degrade quickly.

The 5' cap of eukaryotic messenger RNA is bound at all times by various cap-binding complexes (CBCs).

Eukaryotic translation initiation factor 4E, also known as eIF4E, is a protein that in humans is encoded by the EIF4E gene.

Eukaryotic translation initiation factor 4 gamma 1 is a protein that in humans is encoded by the EIF4G1 gene.

Eukaryotic translation initiation factor 4 gamma 3 is a protein that in humans is encoded by the EIF4G3 gene. The gene encodes a protein that functions in translation by aiding the assembly of the ribosome onto the messenger RNA template. Confusingly, this protein is usually referred to as eIF4GII, as although EIF4G3 is the third gene that is similar to eukaryotic translation initiation factor 4 gamma, the second isoform EIF4G2 is not an active translation initiation factor.

Eukaryotic initiation factor 4A-I is a 46 kDa cytosolic protein that, in humans, is encoded by the EIF4A1 gene, which is located on chromosome 17. It is the most prevalent member of the eIF4A family of ATP-dependant RNA helicases, and plays a critical role in the initiation of cap-dependent eukaryotic protein translation as a component of the eIF4F translation initiation complex. eIF4A1 unwinds the secondary structure of RNA within the 5'-UTR of mRNA, a critical step necessary for the recruitment of the 43S preinitiation complex, and thus the translation of protein in eukaryotes. It was first characterized in 1982 by Grifo, et al., who purified it from rabbit reticulocyte lysate.

Eukaryotic translation initiation factor 4 G (eIF4G) is a protein involved in eukaryotic translation initiation and is a component of the eIF4F cap-binding complex. Orthologs of eIF4G have been studied in multiple species, including humans, yeast, and wheat. However, eIF4G is exclusively found in domain Eukarya, and not in domains Bacteria or Archaea, which do not have capped mRNA. As such, eIF4G structure and function may vary between species, although the human EIF4G1 has been the focus of extensive studies.

The eukaryotic initiation factor-4A (eIF4A) family consists of 3 closely related proteins EIF4A1, EIF4A2, and EIF4A3. These factors are required for the binding of mRNA to 40S ribosomal subunits. In addition these proteins are helicases that function to unwind double-stranded RNA.

Translational regulation refers to the control of the levels of protein synthesized from its mRNA. This regulation is vastly important to the cellular response to stressors, growth cues, and differentiation. In comparison to transcriptional regulation, it results in much more immediate cellular adjustment through direct regulation of protein concentration. The corresponding mechanisms are primarily targeted on the control of ribosome recruitment on the initiation codon, but can also involve modulation of peptide elongation, termination of protein synthesis, or ribosome biogenesis. While these general concepts are widely conserved, some of the finer details in this sort of regulation have been proven to differ between prokaryotic and eukaryotic organisms.

In molecular biology, the eukaryotic translation initiation factor 4E family (eIF-4E) is a family of proteins that bind to the cap structure of eukaryotic cellular mRNAs. Members of this family recognise and bind the 7-methyl-guanosine-containing (m7Gppp) cap during an early step in the initiation of protein synthesis and facilitate ribosome binding to an mRNA by inducing the unwinding of its secondary structures. A tryptophan in the central part of the sequence of human eIF-4E seems to be implicated in cap-binding.

Eukaryotic initiation factor 4F (eIF4F) is a heterotrimeric protein complex that binds the 5' cap of messenger RNAs (mRNAs) to promote eukaryotic translation initiation. The eIF4F complex is composed of three non-identical subunits: the DEAD-box RNA helicase eIF4A, the cap-binding protein eIF4E, and the large "scaffold" protein eIF4G. The mammalian eIF4F complex was first described in 1983, and has been a major area of study into the molecular mechanisms of cap-dependent translation initiation ever since.

References

1 2 Interlandi, Geneen (February 9, 2007). "FOCUS | February 9, 2007 | BIOCHEMISTRY: Small Synthetic Molecule Curbs Cancer Growth". Focus Magazine, Harvard University. Archived from the original on May 19, 2007.
↑ "Reactome: Cap-dependent Translation Initiation". Archived from the original on October 1, 2011.
↑ Moerke NJ; et al. (2007). "Small-Molecule Inhibition of the Interaction between the Translation Initiation Factors eIF4E and eIF4G". Cell. 128 (2): 257–67. doi: 10.1016/j.cell.2006.11.046 . PMID 17254965. S2CID 14963329.

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[Interlandi-1] 1 2 Interlandi, Geneen (February 9, 2007). "FOCUS | February 9, 2007 | BIOCHEMISTRY: Small Synthetic Molecule Curbs Cancer Growth". Focus Magazine, Harvard University. Archived from the original on May 19, 2007.

[2] "Reactome: Cap-dependent Translation Initiation". Archived from the original on October 1, 2011.

[3] Moerke NJ; et al. (2007). "Small-Molecule Inhibition of the Interaction between the Translation Initiation Factors eIF4E and eIF4G". Cell. 128 (2): 257–67. doi: 10.1016/j.cell.2006.11.046 . PMID 17254965. S2CID 14963329.

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