Fragments vs eIF4E: a chemical probe

Cancer cells are known for growing and multiplying quickly, and to do so they need to produce large amounts of protein. The rate determining step in protein translation happens early, when ribosomes are recruited to the 5’-end of mRNA by the eukaryotic initiation factor 4F (eIF4F) complex. This complex has long been a target for drug discovery, and in a recent open-access Nat. Comm. paper Paul Clarke, Andrew Woodhead, Caroline Richardson, and collaborators at Institute of Cancer Research and Astex describe a chemical probe. (Andrew spoke about this program last year at FBLD 2024.)

 

The eIF4F complex includes three core proteins, confusingly named eIF4E, eIF4G, and eIF4A. eIF4E binds to the 5’cap of mRNA and recruits eIF4G. Blocking the interaction of eIF4E either with mRNA or eIF4G could in principle shut down protein synthesis, but intensive efforts by multiple groups have struggled: the mRNA binding site is very polar, and disrupting protein-protein interactions is tough. Thus, the researchers took a fragment approach.

 

Developing a form of eIF4E suitable for fragment screening was itself a challenge because the protein mostly exists as part of a complex in cells and the native monomer is unstable. After making more than two dozen different constructs, the researchers developed a stable, soluble form that could be crystallized. This construct was screened against a library of 1371 fragments in pools of four, each at 500 µM, using CPMG NMR followed by crystallography, leading to 50 hits. A few bound at the mRNA cap-binding site but most bound to a previously unreported “site 2,” which is near where eIF4G binds.

 

One of these, compound 1, has a reasonable ligand efficiency despite its low affinity as assessed by ITC. The phenol appeared to be making no interactions and so was removed. Adding a fluorine usefully enforced the twisted biaryl conformation and filled a small dimple; fragment growing then led to mid micromolar compound 3. Further growing to pick up additional lipophilic and polar contacts eventually led to compound 4, with low nanomolar affinity. Understanding the importance of negative controls for chemical probes, the researchers also switched the stereochemistry at the benzylic carbon to produce compound 5, which has >30-fold lower affinity for eIF4E. 

 

Crystallography revealed that binding of compound 4 to eIF4E causes conformational changes that should impair binding of the protein to eIF4G. Experiments in cell lysates bore out this hypothesis. Moreover, compound 4 also inhibited protein translation in cell lysates at low micromolar concentrations, while compound 5 did not.

 

Unfortunately, these observations did not extend to intact cells. A cellular thermal shift assay (CETSA) demonstrated that compound 4 did stabilize eIF4E in cells with an EC₅₀ = 2 µM, consistent with binding. But it was much less effective at blocking the interaction with eIF4G in cells, even at high concentrations, and showed no inhibition of protein translation.

 

To understand why, the researchers conducted a series of targeted protein degradation and genetic rescue experiments that are beyond the scope of this blog post. The upshot is that eIF4G binds to several regions of eIF4E, and that while compound 4 disrupts binding to the “non-canonical binding site”, it does not block binding to the “canonical binding site,” and thereby does not block overall complex formation. Why there should be a difference between intact cells and cell lysates is not obvious to me, but perhaps the more dilute conditions of cell lysates play a role, as seen for a paper we discussed last year.

 

One interesting feature of this story is that the initial fragment makes no polar interactions with the protein; all of the polar interactions in compound 4 were added during optimization. This is quite the opposite of ASTX660, where all the polar interactions in the final clinical compound came from the initial fragment. Indeed, a 2021 analysis of fragment to lead successes found that fewer than one in ten retained no polar interaction from the initial fragment.

 

This paper also illustrates the gap that can occur between research and publication; a couple of the authors listed as affiliated with Astex left in 2017. But better late than never, and this study nicely integrates fragment-based lead discovery with elegant biology. Compound 4 should be a useful tool for further exploring the nuances of eIF4E.